Research Article Evaluation of Novel Design Strategies for ...downloads.hindawi.com/journals/btri/2014/970595.pdfResearch Article Evaluation of Novel Design Strategies for Developing

Research ArticleEvaluation of Novel Design Strategies for Developing ZincFinger Nucleases Tools for Treating Human Diseases

Christian Bach1 William Sherman2 Jani Pallis3 Prabir Patra1 and Hassan Bajwa4

1 University of Bridgeport Biomedical Engineering 221 University Avenue Bridgeport CT 06604 USA2 Physics Faculty BHSEC Queens 30-20 Thomson Avenue Long Island City NY 11101 USA3University of Bridgeport Mechanical Engineering 221 University Avenue Bridgeport CT 06604 USA4University of Bridgeport Electrical Engineering 221 University Avenue Bridgeport CT 06604 USA

Correspondence should be addressed to Christian Bach cbachbridgeportedu

Received 13 September 2013 Revised 2 January 2014 Accepted 2 January 2014 Published 6 April 2014

Academic Editor Michael Hust

Copyright copy 2014 Christian Bach et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations Thiscytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with highaffinity In general binding preferences of transcription factors are associated with significant degenerated diversity and complexitywhich convolutes the design and engineering of precise DNA binding domains Evolutionary success of natural zinc finger proteinshowever evinces that nature created specific evolutionary traits and strategies such as modularity and rank-specific recognition tocope with binding complexity that are critical for creating clinical viable tools to precisely modify the human genome Our findingsindicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-fingerbinding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger

1 Introduction

Indications of intense complexity [1] of DNA recognitionare manifested in many forms including observed diversityand equal functionality of secondary binding motifs [2]degeneracy modularity and ldquooverlap problemrdquo [3ndash5] cyto-toxicity [6] high failure rate [7] and dependencies of context[8 9] and condition [10] and DNA sequence deformability[9 11] Widespread use of zinc finger proteins in nature [212] however suggests that natural zinc finger domains havenature-given advantages and that those evolutionary traitsshould be replicated or reused to produce molecular toolssuch as zinc finger nucleases (ZFNs) Also to be considered isthat single zinc fingers which contain 28ndash30 amino acids aresimple structures with an unusual high degree of functionalflexibility and structural malleability to bind distinctively anytriplet depending on certain tissue condition protein contextand form of DNA sequence In addition they are used ina naturally occurring setting by nature to fulfill a variety ofdissimilar and exchangeable functions in the same or betweenorganisms in a modular fashion [1]

Exchangeability of small molecules protein structuresDNA sequences and entire functional units and systemsdenotes that the modularity principle is fundamentally usedby nature to manage life in an uncomplicated manner Anexample reported by [1] on gene regulatory regions showsexchange of four TFs and binding sequences to controlactivation and repression of genes in the same and betweenyeast species in which nature did not change nucleotides andamino acids to develop new units and functions but con-served the TF structures and binding sites to exchange entirefunctional units [1 page 69 Figure 2] Nature thereforeoperates via relocating functional units in the same organismand between species beyond the need of changing aminoacids and nucleotides to adapt to evolutionary pressureA strong argument for universal modularity is that it isa tool through which nature is primed to efficiently andeffectively manage instant changes This might lead to theassumption that nature had reasons to create and conservethe frameworks of zinc finger domains and use and reusethem over long evolutionary distances of time To utilizeparticular inherent evolutionary traits may turn out to be

Hindawi Publishing CorporationBiotechnology Research InternationalVolume 2014 Article ID 970595 27 pageshttpdxdoiorg1011552014970595

2 Biotechnology Research International

critical in the design of zinc finger domains to deal with theoverwhelming complexity of DNA binding

In an effort to reduce complexity and to develop solutionsin a timely fashion it might be realistic to use a naturalzinc finger binding domain and exchange amino acids inthe alpha helical region of one of the fingers to change thedomainsrsquo binding preferences [8 13] To test the feasibilityof changing the rank-ordered binding preferences the three-finger binding domain of SP1 is used to reduce complexityby focusing on exchanging amino acids in the alpha helicalregion of the 2nd finger

2 The Translational Case for Using SP1 toDesign a ZFN with Low Side Effects forSickle-Cell Anemia

Zinc fingers describe a class of DNA binding proteins witha modular design [5 14 15] in which single fingers can beassembled to form multifinger arrangements and recognizeany desired target sequence on the genome Each individualfinger binds preferentially to a specific DNA triplet ldquowithdefined three-base-specificityrdquo [3 page 1] Naturally occur-ring protein-binding domains typically contain three fingersthat bind to a DNA-binding site of a 9-base pair long DNAsequence (9-mers) The modularity of the fingers lends itselfnaturally to a broad variety of bioengineering applicationsProteinDNA hybrid structures have applications for exam-ple in the fabrication of nanoscale functional assemblies [16]Of course their primary application is as a versatile tool fordesigning DNA binding proteins for any target sequence onthe human genome [5 14] for the purpose of gene regulationand genome modification

Such designer zinc fingers have been successfully usedin curing genetic diseases for example for curing sickle-cellanemia [17] in disrupting the HIV CCR5 gene for example[18ndash20] in advanced stem cell therapies for example [21 22]cancer [23] and in other potential applications for example[24ndash26] as well as modifying plant and animal genomes[27 28] In addition a fast growing number of translationalapplications and test assays in biotechnology are reported infor example [20 29 30] However in all these cases ldquooff-targetrdquo binding is a problemwith unacceptable side effects [1431] for which the goal of this study is to show potentially novelways to significantly improve these emerging technologies byincreasing accuracy of binding to a single target site and thusreducing side effects

From the literature it is clear that practical applicationof engineered zinc fingers in humans is severely limited dueto cytotoxic side effects caused by ldquooff-targetrdquo binding siteactivities leading to cell death and apoptosis [32] To addto the challenge recent findings indicate discrepancies andinconsistencies of results produced by various in vitro andin vivo assays [3 7 33] This may be caused by evolutionaryplasticity [34] in which the binding capabilities of singlefingers vary significantly due to the high malleability oftheir 3-dimensional structure which leads to changes intheir binding preferences in various tissue conditions [35]Because of ldquoour limited understanding of even simple DNA

proteininteractionrdquo [36 page 2500] limited knowledge oftranscription factors (TF) functions [35 page 253] and lack ofprecise data to accurately predict binding recognition [37 38]page 144 progress is slow to systematically translate brillianttherapies from for instance animal models [17] into clinicalpractice

Therefore to progress the science it is critical to investi-gate the nature of ldquooff-targetrdquo binding to identify and elimi-nate the potential factors which prevent medical implemen-tation and to gain insights from diverse sources for directingfurther research efforts and technological advances Theseefforts will provide themeans to create critical knowledge andtechnological breakthroughswith broad research and societalimpact This is especially true since today molecular biologyenables us to modify the human genome to cure inheritedgenetic diseases and in the foreseeable future has the potentialto replace damaged and aging tissues and organs

This is due to the unprecedented advances in the biomed-ical sciences which provide the capability to induce thecreation of stem cells from our own ordinary skin cells andthen grow them in numbers to replace burned skin or entireorgans In the case of sickle-cell anemia [17] induced pluripo-tent stem (iPS) cells carrying the disease have been repairedby introducing a healthy HBB gene (Hb A) near the mutatedlocation of the diseased gene (Hb S) (Figures 1 and 2) Cuttingthe HBB gene at the specific location GTGGAG (Figure 1)using a nuclease and introducing a healthy donor genecompletes the correction Nucleases are proteins with theenzymatic capability to cut the genome at any locationIn order to introduce one specific cut at one location thenuclease is guided by a bespoken zinc finger protein designedto bind to the specific DNA sequence next to the HbSmutation (Figure 2)

Two nuclease domains are required at the same locationbut on opposite strands of the genomersquos DNA sequence toform a dimer (Fok I nuclease domains in orange in Figure 2)that can induce a cut at both strands [6] To get the twonuclease domains to the one desired location each domainis tethered to the binding domain of a zinc finger proteinthat specifically recognizes and attaches to its binding sitewhich is a nine-base pair long DNA string for exampleTCCTCAGTC in Figure 2 The hope of this strategy isthat through modular assembly of individual fingers zincfinger nucleases can be created that specifically bind to onedesired DNA sequence [3] In the HBB example symbolizedin Figure 2 the upper three-finger binding domain shouldrecognize exclusively the binding site TCCTCAGTC (lowerGGCAGACTT) where each finger binds to one nucleotidetriplet The two three-finger DNA binding domains com-bined should have the unique quality of bringing the twonuclease domains together at only the one specific target siteGGCAGACTT - - - - - - TCCTCAGTC

This technique called gene targeting has been success-fully applied to cure sickle-cell anemia in a mouse model[17] It has been suggested that statistically the two three-finger binding domains should enable the formation ofthe nuclease dimer only at the one desired location Anexact match search on the NCBI-HuRef genome (NationalCenter for Biotechnology Information) revealed that the

Biotechnology Research International 3

Figure 1 Normal HBB gene retrieved from NCBI website

Diseased HBB sequence in human hemoglobin (HbS)

Fok Inuclease domain

9-mer binding site

Normal HBB sequence in human hemoglobin (HbA)

GTG GAG TCC TCA GTC

GAG GAG TCC TCA GTC

GGC AGA CTT CAC CTC

GGC AGA CTT CTC CTC

Three finger binding domain

2-fi

nger

3-fi

nger

1-fi

nger

2-finger 3-finger1-finger

- - - - - - - - - - - - - -- - - - - - - - - --- - -

- - - -- - - - - - - - - -- - - - - - - - -- - - - -

Figure 2 Mutated HBB diseased gene Normal HbA targetsequence versus single point mutation of diseased HbS gene andtarget sequence of a three-finger binding domain

TCCTCAGTC (AGGAGTCAG) sequence occurs 18279times and the GGCAGACTT sequence 8676 times whereasthe GGCAGACTTGTGGAGAGGAGTCAG sequence wasfound exactly one time at the proper location in the HBBgene which provides some rational that this approach mightproduce clinically feasible products However despite the factthat the target sequence occurs just one time cytotoxicityis observed and attributed to the zinc finger nucleasersquosability to bind not only to the one desired target site butalso to numerous ldquooff-targetrdquo sites that induce deleteriousgenetic changes preventing cells from functioning properlyand causing cell death and apoptosis In addition the lackof technologies to precisely control genome modificationshampers human application [6 17 32 39ndash41] Concomitant

ldquooff-targetrdquo binding is tied into the observation that zincfingers typically bind degenerated motifs of hundreds ofsimilar sequences [2] connoting that three-base specificity [3page 1] does not signify that a single zinc finger only binds toone or few best triplets

In the last two decades the binding specificity of hun-dreds of artificial and natural zinc fingers has been character-ized Yet despite fast progress little is known about even sim-ple DNA-protein interactions [36] and computational toolsto design proteins and predict binding sites lack accuracy[37 38 42] Accompanying large scale studies have shownan unmanageable diversity of DNA recognition [2] where themassive amounts of data on transcription factor domains andbinding sites increased complexity to a point wheremore datacontribute little to gain vital understanding of DNA-proteininteractions

At this point it might be rational to reduce complexityand bring it onto a manageable level by using an exemplarycase that focuses on generating data about one finger togain insight before further proceeding SP1 one of the mostubiquitous transcription factors has been chosen with theintent to test which of the 64 putative triplets (Table 3) forits 2nd finger still allows the entire three-finger domain toform aDNA-protein complexThe focus on one finger and 64triplets as a first step appears to be reasonablymanageable andmore productive than testing the 262144 putative bindingsites of the entire three-finger domain

Referring to Lam et alrsquos report on general degeneracyit can be realistically expected that the outcome should befairly degenerated 64 three-base-specificity codes [3] thatcould provide guidance to develop concomitant core and sup-porting technologies to focus on further investigations andgenerate precise data on themechanisms ruling the reversible


formation and dissolution processes of amodel DNA-proteincomplex Among the many known and unknown factors wefocus in this paper on selected factors with the highest proba-bility of having practical relevance to advancing translationalresearch

3 Material and Methods

Expression of three-finger domain using plasmid pPacSpl-516c is provided by Tjianrsquos lab and purified by FPLC MonoS chromatography [13] The DNA binding capability of the2nd finger of SP1 and mutants has been assessed by incu-bating the 64 (Tables 2 and 3) P32-labeled double-strandedoligonucleotides (Figure 4(a)) by performing electrophoresismobility shift assays (EMSA) P32 counts of band shifts havebeen produced by Phosphor Imager Screening (MolecularDynamics) [13 43 44]

31 Oligonucleotides Oligonucleotides for site-directed mu-tagenesis and for electrophoretic mobility shift assays(EMSA) were synthesized on 380A Applied BiosystemsDNA Synthesizer The oligonucleotide 1892 used in EMSAcontains one SP1 binding site TTGGGGCGGGGCTT sur-rounded by cassette sequences which contain the appropriateprimer annealing sites for primer A and primer B For theEMSA analysis cassette oligonucleotide 3028 was gener-ated (Table 2) resulting in 51015840 GTCGGATCCTGTCTGAG-GTGAGTTGGGNNNGGGCTTGTCTTCCGACGTCGA-ATTCGCG31015840 Site-directed mutagenesis oligonucleotide2744 (AAGTCGTCTGCCCTAATTAGTCACAAACGT-ACACACACAGGTGAGAAG) and oligonucleotide 2745(GTGACTAATTAGGGCAGACGACTTTGTGAAGCG-TTTCCCACAGTATGA) were synthesized encoding lysine(K) at zinc finger position 15 serine (S) at position 17 alanine(A) at position 18 isoleucine (I) at position 20 and serine atposition 21 The oligonucleotides 393 (GTAAAACGACGG-CCAGTG) and 392 (AAACAGCTA TGACCATG) whichare universal primers of Bluescript plasmid (Stratagene) havebeen used together with the oligonucleotides 2744 and 2745in PCR mutagenesis Oligonucleotide 1956 (CAGCCCGGG-AGATCTGCCACCTGCA TGAC) introduces a BglII site atthe 31015840 end of the SP 1 fragment in pB-516c

32 Site-Directed Mutagenesis The BamHI-BglII fragmentderived from pPacSpl-516c encoding 3 zinc fingers of thehuman transcription factor SP1 was cloned into the BamHIsite of Bluescript (Stratagene) to yield pB-516c Two poly-merase chain reactions (PCR)were performed using oligonu-cleotide pairs 3932745 and 2744392 together with pB-516cgenerating SP1 fragments A and B Each fragment harborsthe introduced mutations at either the 31015840 or 51015840 site They wereisolated from a 6070 polyacrylamide gel The complete SP1fragment encoding the desiredmutations and a restored BglIIsite was generated by performing a secondPCRusing primers393 and 1956 on SP1 fragments A and BThePCRproduct wasextracted with phenolchloroform digested with BamHI andBglII gel-purified and cloned into pAR3039 to yield pAR-SP1

mutants Standard PCR conditions were applied Introducedmutations were verified by dideoxy sequence analysis

33 E coli Expression Mutated SPl protein was expressedand purified according to the procedure described for theanalogous wild type SP1 protein Mutated SP1 protein wasdiluted 1 10 in buffer A (8M urea 20mM MES pH 50and 2mM EDTA) subjected to FPLC Mono S chromatog-raphy and eluted with an increasing salt gradient of bufferB (1M NaCl 8M urea 20mM MES pH 50 and 2mMEDTA) Peak fractions were collected and analyzed togetherwith recently purified SP1 on 15 polyacrylamide-SDS gelFractions containing the mutated SP1 protein were pooledProtein concentrations were determined by the method ofBradford to be 05mgmL [43]

34 Electrophoretic Mobility Shift Assay (EMSA) Oligonu-cleotides for electrophoretic mobility shift assays (EMSA)were synthesized on 380A Applied Biosystems DNA Syn-thesizer Proteins CB1 MR14 MQ91 MQ135 and MQ151were incubated (15 ng) with 10 120583L labeled double-strandedoligonucleotide in a 30 120583L standard electrophoretic mobilityshift assay (EMSA) The reaction mixture consisted of 10 120583Lof 3x band shift buffer (15mM NaCl 150mM KCl 36mMHEPES pH 79 36 glycerol and 5mMMgCl 300 120583MZnCl)6 120583L H

20 3 120583L DTT (10mM) 10 120583L labeled oligonucleotides

(10000ndash20000 Cerenkov cpm) and 1 120583L protein (15 ng)Proteins were diluted by addition of H

2O The band shift

reactions were incubated for 30min at RT and loaded onto a6 polyacrylamide band shift gel (acrylamidebisacrylamide30 08) containing 100 120583M ZnCl and 025x Tris-borateelectrophoresis buffer (TBE) By performing EMSA analysisthe fragments of mutants present in plasmids were identifiedto bind to the majority of 64 possible triplets The bindingsites in the mutant plasmids were determined by dideoxysequence analysis [13]

4 Results and Discussion

The exchange of amino acids in the alpha helical region of the2nd finger of SP1 (Figure 3 colored in blue and underlined)produced the five mutants CB1 MR14 MQ91 MQ135 andMQ151 as displayed in Table 1The exchanged amino acids aredouble underlined

The EMSA assay results in Figures 4(a) and 4(b) showsignificant changes in the binding preferences of the 64triplets for the 2nd finger of SP1 CB1 MR14 MQ91 MQ135and MQ151

41 Malleability of Binding Preferences Variations of theSP1 binding domain have been created via site-directedmutagenesis of nonconserved positions in the alpha helicalregion of the 2nd finger of which CB1 MR14 MQ91 MQ135and MQ151 are listed in Table 1 and of which the bindingcapability has been tested using electrophoretic mobility shiftassay (EMSA) with P32 labeled oligonucleotides Remarkablysix binding patterns in Figure 4(a) with significant differences


C

CG

IQ

M

K

C

C

C

C

H

G

G

G G

G

K

K K

K

KK

VY

K T

T

T

T T

TT

I

S

S

S

H

H

H H

H

H

H

Zn

L

L

LR

R

R

R

R

R

RYS

A

A

W

E E

E

P

P

P

W

F

F

F

FD

ZnZn

M

SP1-binding domain (three fingers)

R

R

HK

SD

E

L

Q

Figure 3 Amino acid sequence and structure of the SP1 binding domain

Table 1 List of exchanged amino acids in 2nd finger of SP1

2nd finger Amino acids in alpha helical regionSP1 (wild type) R S D E L K R H K

Exchanged Amino AcidsCB1 H S S R L I R H EMR14 R S S T L I Q H K

MQ91 Q S S Y L I K H K

MQ135 Q S S H L I Q H K

MQ151 Q S S Y L T Q H K

have been obtained that show extraordinary diversity of bind-ing occurrences with distinct dissimilar binding preferenceswhich supports the notion of context dependency among thethree-domain fingers and beyond degeneracy the paper by[3] noted ldquounanticipated specificityrdquo [3 page 4683] and thatby [2] noted ldquorank-ordered listing of the (DNA binding site)preferencesrdquo amid millions of measurements of which onecan derive that the patterns in Figure 4(b) and the systematic1 to 64 ranking in Table 4 are specific rank-ordered listingsof binding preferences [2] in which the altered 2nd fingerchanges the rank of binding preferences of the entire domainInstead of assembling finger arrays frommodified Zif268 andSP1 fingers [3] our findings suggest the viability of a strategyto adjust the natural framework of a zinc finger domain byexchanging amino acids of one finger at a time to alter bindingpreferences of the entire domain In addition two three-finger domains in a 2 times 3 strategy [5] can be combined toform a six-finger domain binding an 18-base pair long DNAsequence that is unlikely to occur twice in the human genomeThis could be a way to sensitize the domain to a point thatallows producing clinical viable molecular tools to influencethe human genome

42 Rank-Specific Recognition of the 2nd Finger of SP1and Mutants The rank-specific recognition (RSR) code inTable 4 signifies the rank ordered stability of the DNA-protein complex in a certain condition in which complexstability denotes the degree of binding reversibility or inother words the time a zinc finger protein sticks to the

genome The rank denotes the sensitivity of the proteinto bind a specific DNA sequence in which the bindingis sensitized to the contextual influences the fingers exerton each other the environmental condition of tissues andorganisms and the shape of the DNA [2 3 11] The lower therank (higher number) in Table 4 is the less time a complexhas to form which is extremely important for zinc fingernucleases because the time factor is a crucial indicator toreduce cytotoxic behavior at off-target sites

Depending on the assay and measurement techniquedegeneracy of rank-specific recognition can be defined as(1) time period a DNAprotein complex holds together(visible spectroscopy) (2) complex reversibility (bindingenergy of formation and dissolution change induced byphysical parametermdashthermal ph UV etc) (3) complexstability (delta of binding energies of formation and dis-solution process) (4) binding sensibility (binding energyof initiation before formation) (5) influence on biologicalfunctionality and (6) condition-dependent shift of rank-specific recognition and functionality Following this notionthe rank order from 1 to 64 represents the (1) decrease ofthe time period a DNAprotein complex holds together (2)increase of complex reversibility (3) decrease of complexstability (4) increase of binding sensibilitysensitivity (5)control of biological functionality (eg gene expression anddouble-strand cleavage of ZFN) and (6) shift of rank-specificrecognition and functionality of the same zinc finger in adifferent environmental condition (tissue organism) Theobservation of Badis in which secondary binding motifs(2ndndash64th rank) potentially execute biological functionality(gene expression) to the full extent and ldquoindependent of theprimary motif rdquo (1st rank) [2 page 1723] denotes that therank of the ldquoDNA binding capability of zinc finger domainsrdquodoes not influence the quality of the biological functionality(gene expression) but that the rank represents the controlto which extent the biological functionality is executed bylimiting the time period a DNA-protein complexrsquos activityis active at a specific location on the genome in a specificcondition In other words nature is limiting the time perioda DNAprotein complex is functional by choosing ldquoalternaterecognition interfacesrdquo [2 page 1723] which in this casemeans a sequence of secondary binding preference In regard


A A A CC

C C C GG

G G G TT A C G T A C G T A C G T

T T T A A A CC

C C C GG


T T TA A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

AAA

A A CC

C C C GG


T T T AAA

A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

SP1-ANN SP1-CNN SP1-GNN SP1-TNN

CB1-ANN CB1-CNN CB1-GNN CB1-TNN

MQ91-ANN MQ91-CNN MQ91-GNN MQ91-TNN


MR14-ANN MR14-CNN MR14-GNN MR14-TNN


2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

(a)

Figure 4 Continued


0

1000

2000

3000

4000

5000

CGG

GCG CG

TG

GC

TTG

TGG

GAG CC

CTA

GCC

GAG

GG

GT

GAC

GTG

GG

AAG

TTA

ACA

GG

TCG

CT TAT

GTA

GAT

GCA AAT

CGC

TCT

TCG

TAC

CCT

CAA

GTT

GG

GCA

TTG

TTC

AG

CCTG

ATT

TCG

AAT

GA

AG ACT

CTG

ATA

AGA

TTA

TCC

CCA

TGC

TTC

AGC

ACA

ACG

CAC

ATT

CTA

ATC

CTT

GA

ACT

CAC

CA

ACA

AA

S1

0

10000

20000

30000

CCC

GAG GG

TAG

GG

GA

TAA

CAG

GTG

GG

GTG

GTG

TTA

TCA

TAT

GG

TTCG

TCG

GG

TC TAG

TCG

CAC

GA

AG

AC GTA

GCG

AGA

CGC

GG

CA

AG ACA

CCG

AAT

GAT

AGT

CAA

GCT

TGA

TCC

CCT

CGA

AA

ATT

GCC

ACT

GTC

ATA

CCT

AAT

TCT

TAG

CAC

TAT

ATT

AG

CC TTT

CTC

TCT

AAC AC

CAC

GAT

CG

CA TGC

TTC

0

10000

20000

30000

40000

GAG

GG

AG

TGG

AA

GG

GG

GT

GTA

AGG

GG

CG

TTTG

GCC

CAG

TCG

GG

ATTG

AA

AG GAC

GCG TG

CTT

GTA

GG

CA TTT

CGC

CGT

GTC

ATG

AGA

CAG

CCT

GCT AT

ACA

ACT

GTG

TCG

ACA

TCT

AA

ATCC

GCT

TTA

AAG

CTA

TAT

TAC

ACA

CAC

TCT

CAC

GTT

ACC

ATC

AAC

CG

CCA

AA

AAC TA

CTC

GTC

TAT

CTC

CTT

C

Rank-specific recognition code SP1

Rank-specific recognition code CB1

Rank-specific recognition code MR14

Rank-specific recognition code MQ91

0100002000030000400005000060000700008000090000

100000

AGG

CGG

GG

AG

AGA

AGG

GC

GTG

AGA

ATG

AAT

AA

AG

GT

CAG

GG

GG

ACG

AA

AGT

ACA

ATT

ACC

GCG GCT TA

GAT

AG

TACA

ACG

TTA

AG

TCTG

GG

CAG

CC CCC

CGA

GAT

ACT

TGA

CAT

CCG

ACG

GTT

AGC

CCT

CAC

CGC

CTA

TAC

AAC TC

GTA

TCC

ATG

CCT

TTC

TCT

GTC

ATC

CCT

CTG

TTT

GAT

CTT

TTT

ATT

C

(b)

Figure 4 Continued


0

10000

20000

30000

40000

50000

60000

70000

80000

GG

AG

ACG

AG TAA

GG

GG

GT

AGA

GAT

GTG AG

TG

GC

GTA

AGG

TGA

CGA

GCT

GTT

GCG AT

GG

CAAG

CTG

TCG

TG

AA

ATT

ACT

TCA

ACC

CGG

ACA

TGG

CAA

CCT

GCC AAT

AAG AT

ATT

TCG

CAC

GA

AA

TTA

AAC TA

GTT

CG

TC CAT

CCC

TAC

TGC

CAG

CAC

TTG

TAT

CTT

TCG

CCA

CCG

CTG

TCT

ATC

TCC

CTC

CTA

0

10000

20000

30000

40000

50000

AGG

GG

AA

AGG

AG GTA

AGT

TGA

GTG TT

GG

CAG

GG

GG

TCG

TTT

TG

CG GCT GAT

TGG

GTC AAT AT

TTC

TCG

GTG

TG

GC

TAG

TAT

ACT

AGA

TTC

CCC

GTT

AGC

ATA

TGC

TTA

TCC

AA

AAC

ACG

AG

ACA

AC ATG

ACC

CGC

GCC TC

ACT

TCT

GCC

GG

AA

TAA

TAC

CAA

CAG

CTA

ACG

CAT

CTC

CCA

CCT

CAC

TCG

ATC


(b)


Figure 4 (a) Band shifts (SP1 protein-DNA P32 oligonucleotide complex) (b) Results from Phosphor Imager Screening (MolecularDynamics) Results of complete recognition code of the 64 binding sites of the 2nd finger of SP1 (nonstandardized P32 count)

Table 2 P32-labeled double-stranded oligonucleotide 58-base pairs

51015840GTCGGATCCTGTCTGAGGTGAGTTGGGNNNGGGCTTGTCTTCCGACGTCGAATTCGCG31015840

to [45] observation of a relatively poor relationship betweensequence specificity in vitro and nuclease targeting capacityin vivo might indicate that degeneracy can be defined asa ldquoloss of functionalityrdquo [33] or ldquoloss of pioneer factorsrdquo[45 page 289] However considering the dependencies oncontext condition and DNA shape together with rank-specific recognition rather denotes that degeneracy can bedefined as the ldquoshift of functionalityrdquo to dissimilar bindingsites in a different condition

43 Rank-Specific Recognition of Altered SP1 Zinc Finger CB1MR14 MQ91 MQ135 and MQ151 The exchange of aminoacids in the 2nd finger of Sp1 induces a change in the domaincontext of the entire three-finger binding domain and a shiftto a distinctively different rank order of binding preferencesin which a zinc finger is able to execute biological functionsat dissimilar target sequences Rank-specific recognition thendenotes a ranking of locations on the genome where a zincfinger potentially induces a biological function rather than agradual loss of a functionrsquos quality In other words the rank

does not denote the quality of gene expression but rather theduration of gene expression Following this notion certainsequences in the rank in Table 4 might be associated witha certain biological functionality However a higher rank inTable 4 does not indicate improved functionality and the rankdoes not determine the type and strength of functionalityin the notion that weaker affinity does not result in lessfunctionality but rather retained functionality independent ofaffinity

Rank-specific recognition then means that dependenciesof context condition and DNA shape are consistent withthe general concept of modularity [3] and are applicable tosingle fingers as well as an entiremultifinger domain Becauseof context dependency in which each finger influences thebinding behavior of adjacent fingers and the entire bindingdomain [8] the modularity and binding character of theentire domain can be altered and adjusted to recognizeany DNA sequence This delivers a significant advantageover randomly altering single fingers of Zif268 and SP1 andassembling them to arrays with high affinity of uncontrollable


Table 3 The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A C G and T

ANN-series AAA AAC AAG AAT ACA ACC ACG ACT AGA AGC AGG AGT ATA ATC ATG ATTCNN-series CAA CAC CAG CAT CCA CCC CCG CCT CGA CGC CGG CGT CTA CTC CTG CTTGNN-series GAA GAC GAG GAT GCA GCC GCG GCT GGA GGC GGG GGT GTA GTC GTG GTTTNN-series TAA TAC TAG TAT TCA TCC TCG TCT TGA TGC TGG TGT TTA TTC TTG TTT

binding capability Following the notion of functionalitythe inference is that binding specificity is not degeneratedwhich means no loss or degradation of functional activitybut is rank-ordered degenerated time sensitivity at multipletarget sequences in which a module shifts its DNA bindingcapability to dissimilar DNA sequences and furthermoreretains the same or has new function in dissimilar context andconditions

44 DNA-Protein Interactions Because of condition depen-dency results derived from a single assay are tentative and aredisallowing generalizability but substantial inferences aboutthe influence of evolutionary traits on the malleability ofbinding preferences can be drawn that can lead research toa pragmatic direction to produce clinical viable molecularapproaches and tools Reportedly the binding domain of Sp1in its natural conditions within a large number of cellular andviral promoters for example [8] binds GC-rich boxes andespecially the second finger of the triplet GCG Looking at theRSR in Figure 4(a) and Table 4 under the unique (unnatural)EMSA conditions SP1 recognizes AT-rich triplets at ranks5 and 9 as well as AT-boxes at ranks 17 21 and 25 It canbe inferred that in the same condition the SP1 zinc fingerdomains potentially bind any triplets and that patterns ofshifting preferences of certain nucleotide positions in thetriplet emerge when comparing the six patterns The findingthat the 2nd fingerrsquos best binding site is CGG might be dueto the specific condition in EMSA however it has to be con-sidered that in vivo the observed preference to GCG is likelyFor MQ91 the ranks 60 61 62 and 64 (TTG TTT TTA andTTC) might show that at the third position G T A and C donot play a role and that the 2nd finger bindsGTT inwhich theoverlap mechanism that stabilizes the DNA-protein complexis disabled and cannot initiate complex formation

Despite the attempt to reduce the quantity of informationto one altered finger and six proteins the complexity of resultsalready exceeds full analysis and understanding However itshows the possibilities from a full data set of 262144 DNAsequences towhich a three-finger protein can bind importantinferences can be drawn regarding the clinical viability of adomain With microarrays there is the capability to producedata sets of the entire range of 262144 nine-base pair bindingsites It remains open if in vitro data can be triangulated within vivo data to generate a clearer picture of specific DNA-protein interactions A more pragmatic approach is to mea-sure the formation and dissolution of DNA-protein complex

45 Electrophoretic Mobility Shift Assay (EMSA) The elec-trophoretic mobility shift assay (EMSA) band shifts inFigure 4(a) and computational results in Figure 4(b) show

context dependency The electrophoretic mobility shift assay(EMSA) bandshifts show context dependency in that he 2ndfinger influence the binding ability of the 1st and 3rd fingervia three-dimensional-malleability of the domain structurewhich results in the six distinctly different binding patternsshown in Figures 3 and 4 The binding ability of the 1st and3rd fingers via three-dimensional malleability of the domainstructure This might be interpreted as degeneracy in thatthe domain binds a significant number of related individualsequences [3 page 1] However the pattern does not indicatethat the domain either binds or not (onoff binding) butrather shows subtle differences of specific binding represent-ing a decreasing gradient of complex stability

In Figure 5 the band shifts in the upper portion of thepictures represent the stable DNA-protein complexes of eachof the 64 assays Relative comparison of the band shifts withthe unbound P32-labeled DNA oligonucleotides in the lowerportion of the pictures using a Phosphor Imager infers thatthe complex stabilities in a specific condition systematicallydecrease

In Table 5 the columns list the 64 (9-mer) GGGNNN-GGG (P32) are the Phosphor Imager Screening counts and(loc) is the number of locations the 9-mer string occurs asexact matches in the human genome using the NCBI-HuRefdatabase

Table 5 and Figure 6 contains the exact number of loca-tions of the 64NNNnucleotide combinations (Table 3) of the9-mer DNA strings GGGNNNGGG in the human genome(NCBI-HuRef) which might represent potential ldquooff-targetrdquolocations

Of the 26 highest P32counts the SP1 binding domainrecognizes around 70 of (1826) GC-rich triplets of which27 (626) are GC-triplets In addition of noticeable impor-tance is the observation that 30 (826) are AT-rich tripletsof which 10 (326) are AT-triplets which in turn signifiesthat the 2nd finger sufficiently influences the formation of aDNA-protein complex to create a distinguished recognitionpattern The sorting of the Phosphor Imager readings fromthe highest to the lowest P32count shows gradually decreasingformation of 26 DNA-protein complexes (band shifts) withP32 counts above 500 and 35 below 500 The three tripletsAAA AAC and ACC did not yield detectable measureshowever the binding ability of a transcription factor canchange with conditions [3] thus it can be assumed thatcomplex formation is possible under altered circumstancesIn general the outcome confirms that the SP1 domain not onlypreferably binds GC-rich triplets but also has the ability tobind AT-rich sequences



A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

Figure 5 Band shifts of the SP1 protein-DNA P32 oligonucleotide complex

0

1000

2000

3000

4000

5000

(1)16697

(2)19345

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10

)(11

)47

(12

)(13

)(14

)(15

)(16

)26799

(17

)(18

)34437

(19

)(20

)(21

)(22

)(23

)(24

)(25

)17749

(26

)(27

)(28

)(29

)(30

)(31

)15503

(32

)(33

)(34

)14458

(35

)(36

)(37

)(38

)(39

)(40

)(41

)58

(42

)36175

(43

)16928

(44

) (45

)7850

(46

)38448

(47

)(48

)(49

)(50

)(51

)(52

)6704

(53

)26050

(54

)6423

(55

)3769

(56

)2

(57

)(58

)1919

(59

)(60

)(61

)(62

)3170

(63

)2925

(64

)29730

Figure 6 Results from Phosphor Imager Screening (Molecular Dynamics) of the complete binding spectrum of the 64 binding triplets ofthe 2nd finger of SP1 and number of exact GGGNNNGGG (Table 3) matches in the human genome

The findings are consistent with evidence that emergedover the last few years and in particular highlights thechallenges to produce clinical viable molecular zinc fingertools Research on transcription factors has advanced rapidlyand data and knowledge have created a multifaceted pic-ture with an overwhelming abundance of aspects Extensivereviews for example [1 3 5 11] and detailed discoveries forexample [2 3 7 35 46] paint a picture of an increasinglycomplex situation regarding the DNA binding properties oftranscription factors

The goal addressed here in particular is to investigate thefeasibility to produce clinical viable tools to securely modifythe human genome with the current state of knowledgeand technical capabilities Zinc finger proteins seem to beinteresting candidates despite the correct assessment of [3]presenting a complex collection of challenges to the notion of

modularity and that one finger binds to one triplet thus cast-ing doubt on the feasibility of producing zinc finger domainsthat allow precise modifications of the human genome [3]Nonetheless with the complexity and doubts at hand zincfingers are the right candidates primarily because nature usesthem extensively because they are the most important forgene regulation have a reasonably small structure (bindingdomain) and seem to have evolutionary traits that mightbe of practical importance in the design and function ofmolecular tools to safely influence genomes

From the start there was the hope that a single fingerthat consists of 28ndash30 amino acids is a simple enoughstructure that can easily be studied in detail and assembledinto bespoken multifinger domains for any desired DNAsequence thus specifically reaching any location in the humangenome However the efforts of the last two decades resulted


Table 4 Listed rank-specific recognition codes for SP1 CB1 MR14MQ91 MQ135 and MQ151

SP1 CB1 MR14 MQ91 MQ135 MQ151(1) CGG CCC GAG AGG GGA AGG(2) GCG GAG GGA CGG GAC GGA(3) CGT GGT GTG GGA GAG AAG(4) GGC AGG GAA GAG TAA GAG(5) TTG GGA GGG AAG GGG GTA(6) TGG TAA GGT GGC GGT AGT(7) GAG CAG GTA GTG AGA TGA(8) CCC GTG AGG AGA GAT GTG(9) TAG GGG GGC ATG GTG TTG(10) CCG TGG GTT AAT AGT GCA(11) AGG TGT TGG AAA GGC GGG(12) GGT TAT CCC GGT GTA GGT(13) GAC CAT AGT CAG AGG CGT(14) GTG ATG CGG GGG TGA TTT(15) GGA GTT GAT GAC CGA GCG(16) AGT CGT TGA GAA GCT GCT(17) TAA CGG AAG AGT GTT GAT(18) CAG GTC GAC ACA GCG TGG(19) GTC TAG GCG ATT ATG GTC(20) GCT TCG TGC ACC GCA AAT(21) TAT CAC TTG GCG AGC ATT(22) GTA GAA TAG GCT TGT TCT(23) GAT GAC GCA TAG CGT CGG(24) GCA GTA TTT ATA GAA TGT(25) AAT GCG CGC GTA ATT GGC(26) CGC AGA CGT CAA ACT TAG(27) TCT CGC GTC CGT TCA TAT(28) TCG GGC ATG TAA ACC ACT(29) TAC AAG AGA GTC CGG AGA(30) CCT ACA CAG TGG ACA TTC(31) CAA CCG CCT GCA TGG CCC(32) GTT AAT GCT GCC CAA GTT(33) GGG GAT ATA CCC CCT AGC(34) CAT AGT CAA CGA GCC ATA(35) TGT CAA CTG GAT AAT TGC(36) TCA GCT TGT ACT AAG TTA(37) GCC TGA CGA TGA ATA TCC(38) TGA TCC CAT CAT TTT AAA(39) TTT CCT CTA CCG CGC ACA(40) CGA CGA AAT ACG ACG CGA(41) ATG AAA CCG GTT AAA GAC(42) AAG TTG CTT AGC TTA AAC(43) ACT CCA TAA CCT AAC ATG(44) CTG CTG AGC CAC TAG ACC(45) ATA TCA TAT CGC TTC CGC(46) AGA TAC ATT CTA GTC GCC(47) TTA CTA ACA TAC CAT TCA(48) TCC ATT CAC AAC CCC CTT

Table 4 Continued

SP1 CB1 MR14 MQ91 MQ135 MQ151(49) CCA CTT ACT TCG TAC CTG(50) TGC AGC CTC TAT TGC CCG(51) TTC ACT ACG CCA CAG GAA(52) AGC ATA TTA TGC CAC TAA(53) ACA TTA CCA CTT TTG TAC(54) ACG GCC TCA TCT TAT CAA(55) CAC TTT ACC CTG CTT CAG(56) ATT CTC GCC TCA TCG CTA(57) CTA TCT AAA TCC CCA ACG(58) ATC AAC AAC CTC CCG CAT(59) CTT ACC TAC TGT CTG CTC(60) GAA ACG TCG TTG TCT CCA(61) CTC ATC TCT ATC ATC CCT(62) ACC GCA ATC TTT TCC CAC(63) AAC TGC TCC TTA CTC TCG(64) AAA TTC TTC TTC CTA ATC

in high failure rates of modular assembled zinc fingerarrays [7 page 374] and cytotoxicity which is thought tobe caused by cleavage at ldquooff-targetrdquo sites [6 39 40] whenused in zinc finger nucleases In addition despite the factthat several quantitative methods have been developed tomodel DNA-protein interactions with specific focus on theC2H2zinc finger proteins the overall predictive accuracy

of current computational tools is still limited [37] Tompaet al concluded earlier that sequence variability among thebinding sites of a given transcription factor and the nature ofvariability itself are not well understood (page 137) and thatthe accuracy of prediction of computational tools cannot beaccomplished because ldquowe do not understand the full truthabout transcription factor binding sites [38 page 144]rdquo In amore recent study [36] uncovered some surprising resultshighlighting ldquoour limited understanding of even simpleprotein-DNA interactions [36 page 2500]rdquo When looking atthe number of 1261301 exact locations for the 64 considered9-mers in Table 5 which are just 64 combinations out of262144 (64 times 64 times 64 or 49) possible combinations of 9-mers (a multiplier of 4100) the following question ariseshow nature ensures evolutionary success and functionalityof natural three-finger domains One answer might be thattranscription factors are part of a regulatory network systemand are controlled by factors that are absent using artificiallycreated zinc finger arrays However this would not explainwhy nature would create and extensively use three-fingerdomains that can interfere with millions of exact locationswithout any evolutionary purpose and sustainable biologicalfunctionality

46 Observations Relevant to Understand Cytotoxicity Theextraordinary evolutionary success of C

2H2binding proteins

has been attributed to the modularity and three-base speci-ficity of single zinc fingers (Figure 3) and the ability to chainthem together to form a multifinger domain that possesses


Table 5 (a) Visible band shifts with value ranging from 4570 to 500 counts 1ndash26 (40) (b) Lessno visible band shifts with value lt500counts 27ndash64 (38 60)

(a)

9-mers P32 loc 9-mers P32 loc 9-mers P32 loc 9-mers P32 loc 9-mers P32 loc(1) CGG 4570 16697 (2) GCG 3450 22741 (4) GGC 2850 16087 (8) CCC 1900 6153 (19) GTC 950 6246

(3) CGT 3125 6508 (5) TTG 2320 28372 (9) TAG 1850 14922 (20) GCT 800 42489(6) TGG 2300 55636 (10) CCG 1850 8662 (21) TAT 660 7319(7) GAG 2180 58721 (11) AGG 1750 41257 (22) GTA 630 15656

(12) GGT 1440 38721 (23) GAT 560 23908(13) GAC 1215 5109 (24) GCA 555 40076(14) GTG 1200 69427 (25) AAT 550 17749(15) GGA 1175 50364 (26) CGC 500 4098(16) AGT 1150 26799(17) TAA 1100 9037(18) CAG 1010 34437

(b)

9-mers P32 loc 9-mers P32 loc 9-mers P32 loc(27) TCT 475 16364 (40) CGA 225 5420 (53) ACA 63 26050(28) TCG 450 7161 (41) ATG 188 28958 (54) ACG 62 6423(29) TAC 380 1172 (42) AAG 188 36175 (55) CAC 50 3769(30) CCT 300 27004 (43) ACT 187 16928 (56) ATT 50 11132(31) CAA 300 15503 (44) CTG 150 38542 (57) CTA 40 9049(32) GTT 280 24314 (45) ATA 124 7850 (58) ATC 30 1919(33) GGG 260 18460 (46) AGA 123 38448 (59) CTT 20 15023(34) CAT 250 14458 (47) TTA 110 10717 (60) GAA 10 36693(35) TGT 245 20901 (48) TCC 110 2733 (61) CTC 10 4500(36) TCA 238 23485 (49) CCA 80 22765 (62) ACC 0 3170(37) GCC 235 12890 (50) TGC 75 4411 (63) AAC 0 2925(38) TGA 230 24010 (51) TTC 73 1854 (64) AAA 0 29730(39) TTT 225 16500 (52) AGC 65 6704 Total 1261301

the binding specificity to only recognize one primary DNAtarget sequence at which it exerts biological activity [5]This is an indispensable requirement to ensure genomemodifications occur at only one desired location to preventdamaging changes in the human genome that could interferewith cell functions and lead to cell death and apoptosis [6]However reported degeneracy and the overlap problem [3page 2] as well as supporting observations in Tables 6 and 7have complicated the straightforward approach of one fingerbinding to one primary triplet

This section selectively discusses observations that mightmost evidently determine and regulate the reversible natureof the DNA-protein complex in particular its stability andformation and dissolution mechanisms Particularly consid-ered are the genetic and functional conservation on one handand universality on the other hand that defines evolutionarysuccess of TFs the DNA-protein complex stability andthe role of single fingers Finally evolutionary issues areconsidered These observations together seem to providethe pivotal insights of naturersquos success that may lead to adistinguished research strategy and clinical success

47 Genetic and Functional Conservation and Universality ofTFs Degeneracy is themost recognizable challenge since theprecise clinical use of zinc finger nucleases requires three-finger C

2H2domains having a binding preference to only a

single 9-base DNA sequence on the entire human genome [3page 7] Consequently this requirement should be applicableto a single finger as well and the observed recognition patternin Figures 4(a) and 5 that at first glance seems to be a seriousthreat for its clinical use and first of all would certainlyexplain the abundant binding occurrences at ldquooff-targetrdquo sitesas observed with engineered zinc finger domains

Similarly the natural zinc finger SP1 should to someextent bind at undesired locations as well however thereis no evidence that SP1 introduces deleterious genomemodifications or displays other side effects which in turnindicates that the observations in Figure 3 do not justshow degenerated binding at multiple triplets but that themore accurate interpretation would be what [3] specified asldquounanticipated specificityrdquo [3 page 7] Furthermore it hasbeen well documented that degeneracy is common amongtranscription factors and it is discussed that the flexibility


Table 6 Evolutionary success of C2H2 binding proteins Relevant observations concerning evolutionary success of C2H2 binding proteins

Observations References and comments

Degeneracy

(i) Engineered ZFAs typically yielded degenerate motifs binding dozens to hundreds of related individualsequences [3](ii) Observed clear secondary DNA binding preferences and the secondary motifs were bound nearly aswell as the primary motifs [2](iii) The secondary motif can recruit genomic loci independently of the primary motif [2](iv) Beyond simply providing a DNA binding site motif these data provide rank-ordered listing of thepreference of a protein [2](v) Observed ldquosecondary motif rdquo phenomenon had not been described before and it has importantimplications for understanding how proteins interact with their DNA binding sites [2]

High failure rates The modular assembly method of engineering zinc finger arrays has an unexpectedly higher failure rate[7]

Evolutionary plasticity

(i) The dramatic expansion of the number of C2H2-ZFs in mammals appears to be a recent evolutionary

event [3](ii) Evolutionary plasticity [34 47](iii) Conserved expression without conserved regulatory sequence the more things change the more theystay the same [1]

Complexity(i) Half of the proteins each recognized multiple distinctly different sequence motifs [2](ii) 10605 combinations for a 1000 bp long gene [48](iii) The dramatic expansion of the number of C

2H2-ZFs in mammals appears to be a recent evolutionary

event [3]

Simplicity (i) Origami structure [49ndash53](ii) Fractal organization [54ndash56]

Directional evolution (i) Expression of ftz changed at least three times during arthropod evolution [47](ii) The complexity robustness and evolvability of regulatory systems [1]

Evolutionary traits(i) ldquoThe contribution of finger 1 to the DNA binding affinity of SP1 is smaller than that of fingers 2 and 3but the presence of finger 1 is still essential for the high DNA binding affinity These unique features havenever been detected in other zinc fingers [8]

CytotoxicityCell death and apoptosis associated with ZFN expression are most likely the result of excessive cleavage atoff-target sites which in turn suggests imperfect target-site recognition by the ZF DNA-bindingdomains [6 39 40]

to bind dissimilar sequences and the capacity of functioningat different binding regulatory sequences could be beneficialin the evolutionary process for establishing new regulatorysystems [2 59] Especially the interesting finding of [1]demonstrates that fully conserved promoter sequences canbe replaced in a gene and fully conserved proteins take overthe functionality in the new regulatory system For thisnature does not rely on single-base pair mutations alonebut can rearrange DNA sequences of any length on thehuman genome while at the same time preserving themWith this in mind the observation by [2] of ldquorank-orderedlisting of DNA binding site preferencesrdquo for a wide range oftranscription factors might help to explain the significantlyhigh number of DNA-triplets withwhich the 64 triplets of the2nd finger of SP1 form a noticeable complex [2 page 1720]Carrying the rank-ordered thought forward Figure 3 showsthat the binding capability of the 2nd finger of SP1 is notreduced to one or a few triplets but that the DNA-proteincomplex can possess any degree of stability in which thebinding site specificity and affinity primarily determine thestability of the complex Lam noted that degeneracy actuallyis specific binding leading to the conclusion that the pattern

in Figure 3 is actually a rank-specific recognition (RSR)code

More importantly beyond specificity and affinity ranksthe well-documented condition dependency ultimately con-notes that the RSR code primarily depends on the conditionof a specific environment (tissue organism) that determinesspecificity and affinity Condition dependency has beenobserved by [60] who reported a relatively poor relationshipbetween sequence specificity in vitro and nuclease targetingcapacity in vivo [60] and [2] who reported that secondarybinding motifs do bind in vivo and that the secondary motifsare used independently of the primary motif [2] Conditiondependency also is likely to be responsible for the highfailure rate of zinc finger arrays because the intended targetbinding site is not the preferred binding site in a specifictest condition [7 33] This might be of importance becauseit could indicate that degeneracy and condition dependencyare vital evolutionary traits that allow TFs to conserve theamino acid sequence but do not exclude its use for executingdifferent functionalities which explains the widespread useof TFs in nature [47] This allows conjecture that the 2ndfinger has inherited the potential of binding any triplet


Table 7 Evolutionary traits to be considered or reused in the design of zinc finger domains

Evolutionary trait References and comments

Binding spectra A single zinc finger binds many triplets[2 3] related to Lamrsquos degeneracy

Condition dependencyBinding of dissimilar sequence in various conditions [1ndash3 10 11] and high failure ratesof intended target site [7]

Context dependency The interdependence between all fingers in a domain through the overlap loci [3 8]

Sequence dependency Form of DNA determines the binding sequence a domain can recognize [11] p 242

Dynamic biological 3D malleabilityOf the three-dimensional structure of single fingers and entire domains and the formand deformability of DNA structure [8 11 57]

ReversibilityFormation and dissolution of DNA-protein complex is an indispensable property of afunctional regulatory network system

Evolutionary dualismsDuality of conserving and changing gene and protein sequences [1] and reversibility ofbiological processes and functions [1 2 11 57]

4th base overlap loci Complex stabilization and dissolution loci to control the reversibility process [3]

Binding initiation 1st finger of SP1

The 1st finger has unique evolutionary traits never detected in other fingers as well asrelaxed binding specificity and affinity and therefore is likely to initiate complexformation [8]The 1st fingerrsquos condition dependency is unlike other fingers [58]

under certain circumstances and that when circumstanceschange so does the order of the rank-specific recognitioncode Considering the number of exact matches (1261301)found in the human genome of the 64 possible 9-mers inTable 5 to be clinically useful only one of them should berecognized leading to the conclusion that nature must havethe ability to make small incremental changes in the proteinstructure that might be induced by changes in the conditionwhich among other factors make TFs only bind at one ora few very specific locations The RSR code together withcondition dependency demonstrates the challenge to copewith potentiallymillions of putative ldquooff-targetrdquo binding loca-tions and highlights an increased complexity in coping withcytotoxicity

48 DNA-Protein Complex Stability Role of Single FingersTo find ways to better investigate the molecular mechanismsthrough which nature might use rank-specific recognitionand condition dependency for example [10] [11] persua-sively argue that the three-dimensional structures of boththe DNA and the protein change when forming a DNA-protein complex and subsequently both the DNA and theprotein are able to morph their three-dimensional structureto adapt to altering conditions [11 57] Because of the factthat the nucleotide and amino acid sequences do adapttheir structures to each other the rank-specific recognition(RSR) code in Figures 4(a) and 4(b) shows a coordinatedanalog pattern of decreasing recognition where the stabilityof the DNA-protein complex decreases in small incrementaldegrees It has to be noted that the recognition pattern ishighly complex in that even the 2nd finger prefers GC-richtriplets (1826 in Table 5) the remaining 5 AT-rich and 3

AT-triplets seem to indicate that the 2nd finger adjustedthe structure of the entire domain to fit AT-triplets by alsoutilizing DNA deformability in specific conditions in whichAT-rich sequences can take forms that allow the formationof a complex The RSR thus supports [57] observation thatboth the DNA and protein have structural malleability thatprovides an evolutionary advantage which is more efficientthan building new biological systems components andfunction from scratch via Darwinian randomness to adaptto evolutionary demandsThe three-dimensionalmalleability(3Dmalleability) however significantly raises the complexityfor designing simple zinc finger based tools for clinicalapplications In particular both three-dimensional structures(2 times 3D malleability) can change in many ways and quiteinconsistently under various conditions which severely chal-lenges our ability for predicting recognition and biologicalfunctionality

49 Evolutionary Dualism and Reversibility of DNA-ProteinComplex One of the fundamental underpinning principlesis that evolution is a process in which nature needs toaccomplish the duality of conserving and changing gene andprotein sequences as well as structures and biological func-tionalities [1 2 9 11 12 34 47 57] The evolutionary dualismsignificantly increases the chances of having straightforwardways of dealing with complexity change and conservationand time Nature after all needs to have pragmatic ways tocope with the extraordinary complexity to adapt in a timelymanner to required modifications In addition evolutionarydualism has not received particular attention regarding whattraits zinc fingers need to make a multi-finger domain viablefor clinical application


For TFs in general evolutionary dualism entailsldquoreversibilityrdquo of the DNA-protein complex formation Itinvolves a fundamental mechanism that nature employsto control biological functionality and prevent undesiredactivities Nature thus has to create the means throughwhich it can control stability to achieve balanced reversibilityin which specificity and affinity are important to arrangebinding at the right location but in a way to allow reversibilityof binding High affinity in this regard would result in ahighly stable complex with a low ability of controllingreversibility High specificity however does not necessarilyresult in diminished reversibility which then would lead tothe conclusion that zinc finger domains with high specificityand low affinity are preferable and could be designed withthe ability to avoid cytotoxicity Testing zinc finger arraysfor high affinity sequences then may result in arrays withless favorable binding occurrences for the intended targetsite especially because a substantial number of bindingoccurrences could occur at undesired locations For clinicaltools that are employed to fight genetic diseases it is howeverdesirable to have extensive affinity to form irreversible orcovalent binding to deter the growth of microorganismssuch as for example HIV by disabling the CCR5 gene witha high affinity zinc finger array

410 DNA-Protein Complex Stability and Overlap To regu-late reversibility nature may have ways beyond specificityand affinity to influence the formation and dissolution of aDNA-protein complex An indication for this may be theability of the 2nd finger to distinguish specific DNA tripletsThis demonstrates that three-base modularity in general isplausible but specifically to further use themodular characterfor designing zinc finger arrays it has to be taken into accountthat the binding domains of the 2nd and 3rd fingers reach the4th converse nucleotide of the binding triplet of the adjacentfinger (see Figure 7) (usually referred to as ldquotarget site overlapproblemrdquo [3 61]) However the overlap should not be seen asa problembut rather as an evolutionary trait exerting a certainbiological function The specific binding preferences seen intheRSR code indicate that the overlap has no adverse effect ongeneral three-base modularity and it might be in the range ofpossibility that nature uses the overlap as part of a reversibilityapparatus The RSR code for that provides strong indicationsthat in order to distinguish between potentially millionsof target binding sites in Table 5 by means of inducingincremental differences in cell conditions the reversibilityapparatus needs to include highly sophisticated and delicatemechanisms of which one of them is the ability of a finger tobind to the 4th converse nucleotide That it is the conversenucleotide might be a purposeful feature in that the locationof the converse position is accessible to potential factors thatcan incept a mechanism to form and dissolve a complexFollowing this notion natural zinc fingers with high affinitythat are part of a regulatory network system can be regulatedthrough factors that can initiate a dissolution process at the4th converse nucleotide This is not possible for artificialzinc finger arrays that are not part of a regulatory networksystem

It is relatively evident that binding to a fourth nucleotideincreases stability of the DNA-protein complex withoutnecessarily increasing affinity of a zinc finger domain [3 page2] With this feature nature added the capability to delicatelyadjust the reversibility apparatus to form and dissolve acomplex in small degrees Behind the term ldquooverlaprdquo thereforeseems to be the larger issue of a ldquocomplex stabilizationand dissolutionrdquo mechanism that is part of the reversibilityapparatus in which the 4th base converse nucleotides assistas complex dissolution points for potential factors in aregulatory network system (see Figure 7)

The overlap with the two loci connecting 1st2nd and2nd3rd fingers (see Figure 7) strongly indicates generalcontext dependency of the entire three-finger domain thatallows transcription factors to have the capacity to recognizeldquosecondary binding sitesrdquo [11 page 235] or secondary motifs[2] There is a complex blending between general modularityof single fingers and overall context dependency of anentire domain Especially in regard to condition dependency(tissues organisms and genomes) the recognition of a singlefinger and the whole domain can shift to a dissimilar bindingsite In other words the specific binding capability seemsto be influenced by complex relations in the context ofadjacent fingers (context dependency) as well as to specificenvironmental conditions (condition dependency) allowinga finger to change binding preferences at any incrementaldegree to recognize secondary binding sites [2 3] Thisinterconnection between the three fingersmight indicate that3D malleability could affect the three fingers simultaneouslywhich makes it a powerful tool for effective and sensitivereversibility However in static conditions 3D malleabilitymay not occur [8 page 16034] These evolutionary traitsintegral to the reversibility apparatus significantly increasethe complexity of specific modularity in the sense that subtlechanges in the environment can lead to instant subtle changesin the context of the whole domain This may considerablycomplicate design of single fingers and the predictability ofwhich the triplet might be recognized in various conditions

411 Binding Initiation and the Role of the 1st Finger ofSP1 Oka reported on previous studies which found uniquefeatures in the DNA recognition mode of the 1st finger thatldquohave never been detected in other zinc fingersrdquo [8 page16027] According to those accounts the 1st finger has a morerelaxed sequence and site specificity than other Cys

2His2zinc

fingers in general Because of this relaxed base recognition offinger 1 Sp1-(530-623) can bind more various sequences thanother multi-C2H2-type zinc fingers and such a propertymaybe required for the ubiquitous transcription factor Sp1 whichactivates transcription of many genes [8 page 16034] The 1stfinger contributes less to the DNA binding affinity of SP1 butldquothe presence of finger 1 is still essential for the high DNAbinding affinityrdquo of the entire domain [8 page 16027] [62]This is a strong attestation for the pivotal context influencethe 1st finger exerts instantly on the entire three-fingerdomain Eventually nature has created a delicate system ofcontext dependencies among the three fingers inwhich the 1stfinger was given a key role for establishing and maintaining


GG GGG G GG CC C

1 2 3

9

3

- - - - - - -

Figure 7 Evolutionary traits of SP1

a functional domain The main functions of the 1st fingerare binding initiation and the timing of the formation anddissolution of a DNA-protein complex to correctly maneuverreversibility Notably [58] found that the 1st finger peptide ofSP1 is not stable in acidic solution as are other finger peptides[58] This may demonstrate that the 1st finger does notfunction in the same way as other fingers and is able to exertquite different functions in the same condition that can bedistinctively different from the functions of the other fingersin the same domain The inference that can be drawn is thateach finger in a domainmay have different evolutionary traitsand exercise distinct functions at its defined location whichmight limitmodularity in away that a fingerrsquos functional traitshave to be considered at its original location Thenceforththe uniqueness of features is the evolutionary trait of the 1stfinger that needs to be replicated or preserved in the designof a clinical viable zinc finger domainThe evolutionary traitsare of crucial clinical relevance to utilize the evolutionarymechanisms that control the formation and dissolution of aDNA-protein complex Because of the potential millions ofputative binding sites a three-finger domain can recognizewith a number that is actually significantly greater than thatfor the 2nd finger in Table 5 one of the unique evolutionaryfunctions of the 1st finger is to initiate the binding processThis is mainly due to its relaxed specificity and affinity andthe fact that it does not engage in 4th base overlap binding[8] The relaxed nature of the 1st finger emphasizes theimportance of this feature in that it allows the 1st fingerto touch at many positions on the human genome withoutinitiating binding in which specificity and affinity are not thevital features in a ldquobinding initiation mechanismrdquo The typeof measures that can be employed for testing clinical viabilitymight come from [9]who reported that ldquothe sequence contextof a binding site significantly influences binding energeticsrdquoand that the binding energy provides the ldquofull contextualinformationrdquo about a complex [9 page 4544]

412 Binding Energy as a Key for Binding Initiation andComplex Formation With the full contextual informationldquobinding energyrdquo [9 page 4547]might provide a complex andconsidering the context dependency [8] of a finger domain aswell as sequence dependency ofDNAstructure [11 pages 246-247] [9] there might be the possibility to assign ldquopotentialbinding energyrdquo to a protein domain and a DNA sequenceand consider them in various conditions in computationalmodels Because both the DNA and protein can change theirconformation to initiate stabilize andor enhance proteinbinding [11 page 247] this change in the 3D structure

might be measurable via a change in the binding energiesof both Now with three data sets of the binding energiesof the protein domain the DNA sequence and the DNA-protein complex we might be able to understand and predictthe 3D malleability of protein and DNA in various clinicalconditions Accurate measurement of binding energy mightbe a fast and efficient way to design and test clinical viablezinc finger proteins and improve their binding recognitioncapability to the point that only one location has the conditionfor forming a complexThe changes in binding energies in thebinding initiation phase are probably the most delicate andimportant and together with the observation that ldquoflankingsequence influences binding properties to an unexpecteddegreerdquo [9] thus influencing binding energy as well they area further property that can be used to pinpoint the location aDNA-protein complex can form via compatibility of bindingenergies of the protein and the DNA sequence In this waywhen the 1st finger touches a DNA sequence at the rightposition both 3D structures change and so do their respectivebinding energies Complex formation then is only initiatedif there is compatibility of structure and binding energy ofboth DNA and protein Designing zinc finger domains bymeasuring binding energies that can be confirmedwith struc-tural insight at a later stage might be a more pragmatic andmanageable way for fast success of producing clinical viableproteins In conclusion if the binding energy of the entirethree-finger domains is compatible with the binding energyof the DNA sequence including the influence of the flankingsequences the 1st finger will initiate the binding process andwill utilize the two overlap loci to stabilize the complex

The evolutionary advantage however demands that theprocess must be reversible for which again the relaxed natureand the overlap locus between the 1st and 2nd finger mightplay key roles in that the protein and the DNA sequence canchange binding energies by deforming the 1st finger slightlyto trigger the dissolution process The deformation could beinitiated either at the overlap locus between the 1st and 2ndfingers by a factor docking at the overlap locus at the conversenucleotide or by changing the conformation of the DNA via achange in the condition [11] The overlap mechanism has theevolutionary trait of stabilizing and destabilizing the DNA-protein complex of the regulatory binding mechanism thatimportantly does not interfere with general modularity ofzinc finger design

Reversibility of the DNA-protein complex appears to beof essential significance in the design of viable clinical zincfinger proteins Employing different technologies to measurethe complex formation and dissolution properties in various


conditions might be a manageable way to create a criticalmass of data and knowledge to build cytotoxicity-free zincfinger domains With accurate and clinical relevant datasets it is possible to establish a reversibility index for eachDNA-protein complex that can assist in ensuring the clinicalfeasibility of the zinc finger domain

5 Evolutionary Issues

51 Evolutionary Traits The evolutionary traits listed inTable 7 seem to be planned and purposeful products of naturewhich provide vital mechanisms that might be utilized inthe design of zinc finger domains to cope with the pervasivecomplexity

These evolutionary traits that are part of the variousreversibility processes engaged in regulating the formationand dissolution processes of a DNA-protein complex deter-mine the functionality of engineered zinc finger domains Forthis high affinity makes a complex less reversible to the pointwhere tools like zinc finger nucleases stick for an extendedperiod of time on the genome interferingwith cell function orcausing damaging effects Looking at Table 7 with potentiallymillions of putative exact locations for a three-finger domain(which represents just a small sample of 64 out of the 262144combinations of 9-mers) it becomes clear that there is anabundance of possibilities to form a stable and enduringcomplex on the genome

52 Complexity The immense complexity resulting fromprevious findings listed in Table 7 still appears to have deeperroots Observations describe SP1 as regulating transcriptionldquothrough synergistic effects with other transcription factorsrdquo[12 page 36] [63 64] and supporting cofactors [47] ingene regulatory systems [2] The role of transcription factorstherefore is part of a delicate network which has to emergeentirely and simultaneously in an already existing organismin order to survive the evolutionary selection process Theability alone to create and place all components entirelyand simultaneously seems to have to overcome profoundcomplexity that requires consideration of more fundamentalissues Perhaps the most striking findings are the staggeringcomplexity and diversity of DNA binding observed in [2] thefact that transcription factors encode a significant portion ofthe genome for example [5 35] and that nature has devel-oped gene regulatory networks in a rather short evolutionaryperiod of time [2] Considering the binding to secondarymotifs which bind equivalently and independently to theprimary motif [2] and the observations presented hereof rank-specific recognition (Figure 4) together with thenumber of locations in Table 5 might lead to the seriousquestion of how nature manages to produce viable regulatorysystems and what possible ways nature might have taken toproduce them Considerations of these fundamental issuesmight help to exclude ways that cannot succeed in handlingcomplexity and prepare for taking into account that new andunconventional ideas and approaches from a broad inter-disciplinary perspective are needed for producing clinicallyrelevant outcomes

Inferences from the probabilities of a hypothetical sim-plistic gene regulatory network that might contain (1) onetarget binding site of 9-mers in the promoter region (2) one28-amino-acid long zinc finger and (3) a small 1000-basepair long gene that would deliver the following numbers(a) 9-mers randomly appear every 700000 years [46] (b)there are 2736 different amino acid combinations for onefinger and (c) 10605 possible combinations are to arrange onethousand nucleotides [65] which represents ldquoa complexityfor which we have no imaginationrdquo especially in comparisonto the fact that ldquoonly 10108 hydrogen atoms would fit intothe whole universe with a radius of 1010light-yearsrdquo [48]Notably this setting still would require a functional organismwhich is not considered This might lead to the conclusionthat the practical success of nature to establish ad hoc suchan oversimplified regulatory network is so remote in anyevolutionary distance of time that naturemore likely employsa strategy of underlying simplicity and modularity wherecomplexity results from a reductionist scientific approachthat produces detailed but fragmented pieces of data fromwhich the whole of biological functionalities cannot bededuced

53 Simplicity The evolutionary success of TFs has to dowith adapting quickly to environmental changes and to doso necessitates flexibility to change the components of aregulatory network system on a genetic and functional levelGoing through an unsystematic process of trial and error tofind simultaneously the one amino acid sequence out of 2736and the one nucleotide sequence out of 10605 possible com-binations strongly indicates that nature utilizes underlyingsimplistic rules to produce modular structures with a highdegree of flexiblemalleability that can be turned into differentfunctional units via minute structural and genetic changesExisting concepts that might serve as examples for producingcomplexity out of underlying simplicity can be found in asimple fractal equationrsquos ability to grow structures that areever more complex and origami where one plain plane sheetof paper can be folded in unlimited ways to form endlessforms

It has been considered that the human genome has anunderlying fractal structure that repeats itself in a modularfashion for example [54ndash56] and that with repeated foldingand unfolding processes in origami for example [49ndash53]limitless information might be reversibly used archivedand revitalized in dynamic information processing cycleswhich are the tools of evolution to directly produce andchange biological functionalities Such directional evolutionis capable of directly fabricating a selection of modules inwhich minute structural differences in the modules can beproduced via changes in the microconditions for executingdissimilar functions The evolutionary selection process thendetermines the success of the closely related modules fromwhich the capability arises to adapt to evolutionary pressurefrom changes in the environmental macrocondition Modu-larity thus is an evolutionary trait that is extensively used bynature to cope with complexity In terms of building proteinand DNA structures modularity is the repeated use of simple


elementary information processing modules that determinethe functionality of the protein and the specific amino acidsequence In other words the underlying simplicity consistsof information processing modules that pinpoint out ofthe 2736 and 10605 combinations the exact amino acid andnucleotide sequence In this way it is not the amino acidor nucleotide sequence that determines what informationis contained but the underlying intrinsic information of awhole system defines what sequence is needed In this waya zinc finger protein as a whole evolves like a landscape outof a simple information fractal or a repeated elementary foldin origami whence building an infinite manifold of things inwhich the amino acid sequence is the resulting representationof the underlying information Important in this notion isthat the 3D structure and function of the protein are notonly determined by the amino acid sequence but also byyet unknown information-related properties that lie outsidethe observable scope of science Following the thought ofsimplicity the human genome then seems to have an underly-ing information fabric from which nature forms appropriateconfigurations

Nature is able to fold and unfold information in thehuman genome in limitless ways which provides the abilityto create endless forms and expresses them via the geneexpression path to timely adapt to environmental changeswithout the need of going through an unmethodical evo-lutionary selection process In this way positive selectivepressures guide the information unfolding and componentforming mechanisms Reflecting on what is said it is clearthat beyond complexity rules simplicity and it might bereasonable to see the human genome not merely in a reducedview as a string of three billion nucleotides with a rather fixedstatic structure encoding only the information for buildingproteins but holistically as a dynamic Gestalt that is not thesum of its parts but always in its totality is an informationsingularity that has no parts that would encode less thanan infinite amount of inseparable information [66] In sucha Gestalt form the human genome rather functions likean organism with the ability of expressing an interminablevariability of forms and systems thus being capable of unde-viating dynamic formability under purposeful evolutionarypressures of directional evolution

54 Evolutionary Plasticity The dynamic Gestalt form ofthe human genome then explains the high evolvability andextraordinary evolutionary plasticity needed to react tochanges while minimizing the risk of failure as well as havingthe flexibility to allow minor variation of a sequence andstructure that drives expression in a given tissue withoutotherwise altering the regulatory properties of a gene [1 page71] With the extraordinary evolutionary plasticity nature isable to address the evolutionary dualism of conserving andchanging life in an organized fashion

6 Discussion

The most important finding is that the exchange of aminoacids in one finger alters the binding preference of the

entire domain (context dependency) which has significantimplications for strategies to produce clinically viable zincfinger domains inwhich each finger can be gradually adjustedto find a sensible complex for a specific DNA sequence whichmight produce better molecular tools to achieve successfulclinical outcomes

From a historical perspective since we know that aminoacid alteration of natural fingers results in bondage to newDNA target sites [13] it should have become feasible veryearly on to pursue the creation of libraries of altered domainsinstead of focusing on single fingers

Producing precise measures of DNA-protein interactionsunder one condition does not provide relevant clinicalknowledge Thus to further reduce complexity there shouldbe a focus on the one condition of iPS cells A morerealistic way to go about this is to think along the lines ofcomparison of rank-specific recognition codes within largedata sets in one condition Rules can then be deduced thatgovern certain evolutionary traits that are simple enough tobe directly used and modified to designer domains If onereduces complexity to a point where new discoveries havethe most clinical relevance it is reasonable to argue thatthe condition of iPS cells among individuals is identical andthus genomemodifications are accomplished under standardand repeatable conditions before being differentiated intodissimilar cell types Most importantly proper technologiesneed to be developed that allow continuous measurement ofgene expression Such functional nanobiology would provideextremely valuable insight about clinical behavior of zincfinger based molecular tools

Thus to guarantee clinical success it is crucial to focusthe development of technologies on delivering the two mainingredients producing precise data in one condition andmodifying the human genome at one location In a first stepassays need to be developed to make data comparable amongthe different zinc finger domains The most practical way toproduce precise and repeatablemeasures is the formation anddissolution process of the DNA-protein complex in variousconditions which at a later stage can be complimented withprecise data regarding DNA-protein interaction

61 Cytotoxicity and Proposition for a Solution Consideringthe above findings the inference that can be made onthe nature of cytotoxicity is that engineered zinc fingernucleases bind specifically to an unpredictably high numberof locations that are determined by the rank-specific recog-nition of each of the fingers and the binding domain as awhole In particular the problem is compounded becausezinc finger domains usually have been selected for highaffinity and specificity High affinity causes the complexto remain too long at undesirable locations which causeuncontrollable genome breaches and cell death Because oflack of evolutionary traits that could control the biologicalactivity of artificial zinc finger nucleases it is indeed chal-lenging to build cytotoxicity-free ZFNs in a straightforwardway by assembling high specificity and affinity fingers intomultifinger domains More needs are to be understood aboutthe reversible nature of the DNA-protein complex beyond


specificity and affinity To cope with cytotoxicity a reasonableapproach however would be that the 1st finger would havehigh specificity without having any affinity to a triplet untilthe 1st overlap locus supports complex formation in whichevent the affinity of the 1st finger should switch to a balancedaffinity to stabilize the complex The life span of the complexshould be just long enough to regulate a gene and shortenough to get dislodged before inducing any irregularitiesThis precise balance is naturersquos key for achieving evolutionarysuccess which needs to be replicated to build clinically viablebinding domains

62 Evolutionary Traits and Aspects of SP1 A practical way toachieve clinical solutions is to modify the natural frameworkof SP1 by leaving each finger at its evolutionary location Inthis way there might be the opportunity to retain knownand unknown evolutionary traits of SP1 and utilize themfor binding new target sequences that might give enoughcontrol for successfully using them in clinical applicationsStrong support for SP1 as a candidate is the finding that SP1is both highly conserved throughout evolution and used inmany organisms tissues and stages during development [12page 39] [1 page 70] The key question is how nature canuse the highly conserved SP1 binding domain for fulfillinga variety of different functions in different conditions andthe most meaningful answer is via the malleability of its3D structure of the binding domain without changing theamino acid sequence The flexibility that provides SP1 withthe universality to be used throughout nature is a result ofits inherent evolutionary traits of which two are illustrated inFigure 7

Considering the narrowly defined purpose of this studyto produce clinically viable tools in an ethically meaningfultime frame and manageable way the discussed observa-tions (listed in Table 7) indicate a potential way to suc-ceed without gaining full understanding of all componentsOf practical importance for a manageable approach arereversibility the rank-specific recognition code the 1st fin-ger and the overlap loci which can be influenced anddesigned in a way to create a clinical viable domain Thecomplexity following most evolutionary traits in Table 7might be beyond the practical capabilities of direct mea-surement and influence however they are indirectly beingaccounted for when studying the reversibility mechanisms ofthe formation and dissolution processes of the DNA-proteincomplex

The 1st finger of SP1 has the unique evolutionary traitof initiating binding which makes it the first and foremosttool for controlling the formation of a DNA-protein complexOf practical importance then is the sensibility of the 1stfinger to contact many locations without initiating complexformation through which control of cleavages at off-targetsites can be implemented When using zinc finger nucleasehowever the sensibility needs to be particularly refined andthe 1st finger particularly sensitized because of a lack of aregulatory network system that controls binding initiationand reversibility of the complex To avoid cytotoxicity thecomplex should contact the target location just briefly enough

to allow the nuclease dimer to make one cleavage whichrequires high specificity and particularly low affinity of thethree-finger domain This is in particular significant to avoidinducing cleavage at off-target locations where the domainmight bind but with such low intensity that the initiationof complex formation is diverted by the sensitized 1st fingerIn order to avert off target binding both the 1st finger andthe three-finger domain should have high specificity andlow affinity in which ideally the complex should only beheld in place at the overlap loci in order to easily releasethe contact but just long enough to induce cleavage at onelocation In addition high specificity of the domain canbe achieved with the influence of flanking sequences [9]next to the binding site that might deter or encourage theformation of a complex Specifically the careful design ofthe 1st finger will improve binding accuracy of a sensitizeddomain by determining the three-dimensional fit to the targetsequence in many ways that influence the 3D malleabilityof both the protein domain and DNA sequence The three-dimensional fit between a protein domain and a DNAsequence can be determined when producing measures ofthe complex formation and dissolution by detecting changesfor example in the binding energies thermal differences andoptical absorption In particular the potential behavior of acomplex can be drawn by characterizing structural changesassociated with on- and off-target zinc finger binding as wellas their thermal and pH dependence via circular dichro-ism spectroscopy ultravioletvisible absorption spectroscopydynamic light scattering and colocalization confocal fluo-rescence microscopy In combination the resulting accuratedata sets will eventually provide the much needed clinicalrelevant information to select and verify constructs in variouscombinations To ensure single location modification (SLM)further supporting technologies are essential to fully controlinsertion of geneticmaterial at a single location For thisDNAtagging technologies can be considered to tag the genome ata single location for controlling site-directed modification inwhich for verificationmicroscopymight be used to detect andverify modifications at the right location

63 Managing Cytotoxicity via Mutated and Sensitized SP1Domains The SP1 binding domain has unique evolutionarytraits which are not found in other fingers andwhich are quiteclearly responsible for its universal employment throughoutnature see for instance [12] In particular the widespreadappearance of C

2H2zinc fingers in mammals as a recent evo-

lutionary event [3] indicates that simplistic underlying rulesand procedures keep the observed complexity manageablethrough inherent evolutionary traits of which 3Dmalleabilityallows in general the targeted adjustment of each finger andthe context of a particular domain to fulfill a distinct functionin various conditions Of particular interest are both thecomplex stabilization and dissolution points and the bindinginitiating capacity of the 1st finger that allows the design ofeither high or low stabilization (affinity) or high and lowdissolution properties as part of the reversibility apparatusThe complexity of the possible combinations that cannotrationally be tested in reasonable evolutionary time suggests


Table 8 Sensitizing the SP1 framework Potential design strategiesfor sensitizing versions of the SP1 framework

1 times 1st finger 3212 times 1st finger 32112 times 3 design 3213213f + 4f design 32132112 times 4f design 32113211 (to lower affinity and higher specificity)

that simple underlying rules do let the right combinationemerge at a particular time and such rules might be revealedby studying in depth a natural zinc finger domain and itsmodificationsThus it is a prudent approach to take advantageof inherited evolutionary traits to improve binding accuracyIt is reasonable to assume that each SP1 finger can bemodifiedby substituting amino acids in zinc fingers that result inaltered DNA binding recognition [8 12 13 67] and it mightbe possible to utilize some of naturersquos evolutionary traitsDepending on the form of the DNA [11 page 242] aminoacids can be replaced in the fingers of SP1 to recognize AT-rich boxes Indication for this can be seen in the RSR code asthe occurrence of AT-rich boxes with high P32 counts TTC(the 5th highest) TAG (9th) and AT-boxes TAA (17th) TAT(21st) and AAT (25th)

For the HBB example several strategies might improveaccuracy of binding to significantly reduce cytotoxicity Thekernel of several potential strategies listed in Table 8 is theuse of the 1st finger and the SP1 framework as a whole tocreate combinations out of the two components to increasesensibility and specificity in order to obtain clinical viabledomains (1) Strategy 1 incorporates the exchange of aminoacids in the alpha helical region of SP1 to create mutants witha different rank-specific recognition code (2) in strategy 2it might be of use to add a second 1st finger to increase thesensibility and specificity of the initial contact (3) strategy3 follows the Klug reviewed approach to thread togethertwo three-finger domains to obtain a six-finger domain withhigher domain specificity (4) strategy 4 adds a second 1stfinger to create a seven-finger domain and (5) strategy 5 isan eight-finger domain which includes four 1st fingers

It remains to be seen which strategy is more practicaland manageable to produce viable outcomes To discussthe various features the eight-finger domain of strategy 5has been drawn in Figure 8 and might have an enrichedsensibility to the point of clinical relevance

The strategy illustrated in Figure 8 is to use the SP1 frame-work as awhole to fully utilize the different evolutionary traitsand functions of each finger The entire binding domain iscomposed of two SP1 subdomains each enhanced with anadditional 1st finger The 1st finger of SP1 that initiates thebinding process is of significant importance for preventingthe two domains from binding at off-target sites and havingtwo 1st fingers in each subdomain allows successively placingthe fingers resulting in the first subdomain to complete halfof the complex formation starting with 1101584010158401015840-Finger which hasthe function of initiating the binding process It requires thatthe 1101584010158401015840-Finger needs to have a slightly higher affinity thanall the other fingers in the domain It is crucial that only

the 1101584010158401015840-Finger initiates binding because if any of the otherfingers binds before the domain as whole cannot be sensitizedand the frequency of off-target binding occurrences would beuncontrollable The binding sequence should follow a zipperpattern starting with the 1101584010158401015840-Finger and concluding with the31015840-Finger After forming a DNA-protein complex with thefirst subdomain the second crucial point to sensitize thedomain is the 11015840-Finger in the second subdomain to preventthe complex formation of the entire domain if the complexof the first subdomain is at an off-target location It is notablethat the affinities of all the fingers are the lowest possible justat the point to form a DNA-protein complex (lower rank inthe RSR code) The first finger might be able to be designedby substituting amino acids to be sensible to certain triplets inthe sense of having low affinity and high specificity to a tripletConsidering the 1261301 exact 9-mer locations in the humangenome it is of importance to eliminate as many of those9-mer locations as possible by making the 3-mer initiationbinding occurrence as sensible as possible To design themostsensitive binding the 1st finger needs to be adjusted to the celltype environment and context to the other fingers and thenucleotide sequence of the target site that is highly flexibledue to deformability a feature that is used by proteins torecognize specific DNA sequences rephrase [11 page 242]

64 A Practical Approach Interdisciplinary Innovation andNew Technologies The complexity of the matter at handseems to coerce a clinical solution consisting of an allianceof scientific and managerial skills and the concerted effortof genuine collaborators For medical and social purposesgenuine collaborative environmentsmust be formed to createan ethical value which cannot be created by individuals orinstitutions alone It is of ethical urgency to make therapiesthat have been successfully developed in animal modelsavailable to cure patients In the case of sickle-cell anemia thisrequires a full understanding of the nature and mechanismsof ldquooff-targetrdquo binding The purpose of ethical research isto enable concerted collaborative efforts to reduce sufferingby developing end-point therapies in an accelerated andmanageable way Because of the complexity at hand the goalof understanding protein-DNA interactions remains elusiveuntil the underlying simplistic rules can be determined Tomanage technical progress in the short term complexityneeds to be reduced to a point where accurate and repeatabledata can be produced and fully understood in the exemplarycase of the three-finger SP1 domain and each of its fingersAlso technologies which can be applied on a broad scalemust be developed While most of the research efforts arededicated to detect binding sites and identifying TFs ongenomes little has been done to understand the biologicalfunctions [35] The general lack of understanding of TFs[36] promotes the idea to reduce complexity and developcore technologies that delve into the very details of DNA-protein interactions complex formation and dissolution andevolutionary fundamentals [35] To bridge this gapwhich sig-nificantly hinders scientific progress of gene regulation andgenome modification research needs to address issues aboutthe fundamental aspects hereThis should include three parts


- - - - - - - - - - - -- - - - - -CA CC T

GT GG A CG TCT C AT C T A C T GAAA C CCTG G

A C - -C G

G

C

G

First subdomainSecond subdomain

Complex stabilizationdissolution overlap loci Binding-initiation

domainFok I

nuclease domain3-finger

3-finger

2-finger 1-finger 1998400-finger

1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger

Binding initiation site2 times 12-mer binding sites

SP1 sensitized designer domain

Figure 8 Sensitizing the SP1 frameworkPotential design strategies for sensitizing versions of the SP1 framework for HBB gene target

(1) in vitro and in vivo cell-based assays (2) customized highprecision detection instruments (3) functional nanobiologyfor example to measure continuous gene expression and (4)computational tools to capture process analyze and reusedata In this focusing on the 64 binding sites for each fingerof SP1 reduces complexity to a point where it might be man-ageable to generate precise and repeatable data with a varietyof instruments and assays that can be used to develop accuratecomputational tools to predict complex formation in variousconditions

In order to escape cytotoxicity however the core chal-lenge is to fully control the introduction of genetic materialat a single location in the human genome which is for sickle-cell anemia the cleavage of the genome and introduction ofthe healthy donor via homologous recombination at the exactHBB location signified in Figures 1 and 2 Most importantlythese technologies are applicable to introducing the factorsfor creating induced pluripotent stem (iPS) cells at the properlocations in the human genome To further ensure singlelocation modification (SLM) supporting technologies suchas DNA tagging at a single location are essential to fullycontrol and verify insertion of genetic material at a clinicallyrelevant single location Especially clinically relevant aretechnologies that measure the formation and dissolution ofa DNA-protein complex which can provide feedback on thesensitivity and reversible behavior of a binding domainWiththis in mind on the technical side we have supplementedour experimental capabilities by taking advantage of thebroad selection of tools available in the Soft and BiologicalNanomaterials Section of the Center for Functional Nano-materials in Brookhaven National Laboratory We will becharacterizing structural changes associated with on- andoff-target zinc finger binding as well as their thermal andpH dependence via circular dichroism spectroscopy ultra-violetvisible absorption spectroscopy dynamic light scatter-ing and colocalization confocal fluorescence microscopy Incombination the resulting accurate data sets will eventuallyprovide the much needed understanding of the functionalbiology of the binding mechanisms

When those data sets and constructs are availabletwo major technological and scientific achievements havebeen accomplished (1) a scientific base for clinical viableconstructs and (2) the technological base to examine theactual DNA-protein interactions and behavior in various

conditions Furthermore integration of data sets from exist-ing assays such as DNA affinity precipitation assay dual-luciferase promoter activity assay SP1-knockout mice [12]microarrays [2] and a variety of other methods [6] mightcomplement the overall effort The core technologies alsoprovide the ability to study DNA-binding properties oftranscription activator-like effectors (TALEs) that can bedeveloped into robust tools for controlling the introductionof genetic material for instance [68]

7 Limitations

The reduction of complexity brought about the valuableinsight of rank-specific recognition However many aspectsremain to be discovered For example of interest is todetermine the number of exact matches in Table 5 thatoccur in promoter regions of genes to define more preciselyhow many matches should be regarded as ldquooff-targetrdquo Inparticular because of the strong influence of conditiondependency single assay results remain tentative For eachclinical condition a rank-specific recognition code needsto be established together with more precise assays thatmake cytotoxicity reversibility and genotoxicity preciselyquantifiable

8 Contributions

81 Overall Contribution The overall contribution of thisstudy is that we persuasively argue that there are no generalrules for affinity and specificity of DNA binding of zincfinger domains because of condition dependency of bindingRefinements of existing as well as additional definitions areprovided

811 The Existing Literature Appears to Describe Affinity inConsensus as the Strength of Noncovalent Temporary Bindingof a Zinc Finger Domain to a DNA Sequence Howeverstrength of binding to DNA is not the only translationalimportant and clinical relevant measure of affinity Refineddefinitions of affinity should include the circumstance thatpreferably one zinc finger domain should bind to only onesingle location in the human genomeThis wouldmake it safe


for clinical application tomodify one diseased location [17] inthe human genome

We contend that the three-finger domain of the zincfinger protein (ZFP) SP1 significantly increases its affinity toa specific DNA 9-mer sequence by ldquolocking inrdquo binding bymeans of a 4th base overlap mechanism of its 2nd and 3rdfingersThismechanism locks and stabilizes theDNAproteincomplex and enables the complex to induce a functional effector biological activity Consequently we contend that there aretwo types of affinities regulated and unregulated affinity forregulated affinity nature employs a reversibility apparatus toregulate affinity of three-finger domains where it controls theformation and dissolution of the DNAprotein complex butnot for unregulated affinity

This makes DNA binding well planned and reversible Azinc finger domain has to be ldquolocked inrdquo in order to inducean effect In contrast unregulated affinity allows uncontrolledbinding at many locations in the human genome which mayinduce severe clinical side effects Natural zinc finger proteinsdo not display side effects because unregulated binding ata location does not induce a functional effect or biologicalactivity

Wemay be able to replicate or preserve naturersquos reversibil-ity apparatus by carefully modifying natural domains tobind novel intended target sites as has been previouslydemonstrated [13 69]

To distinguish between the two affinities we define reg-ulated affinity as adherence of zinc finger domains to a singlelocation in the human genomeThe zinc finger domain formsa complex only at particular locations in the human genomeand because of condition dependency of binding the DNAtarget site at the different locations can be dissimilar

812 Sequence Specificity Is the Selective Binding of a ZincFinger Domain to Preferably Only One Specific DNA SequenceOur own as well as other previous findings show that a 9-merDNA sequence to which a three finger zinc finger domainbinds occurs thousands of times in the human genome Thisdegeneration of sequence specificity for example [2 3] wherethere is more than one DNA sequence that a zinc fingerdomain binds to requires further refinement and additionaldefinitions of specificity

In refinement we contend that there is no generalsequence specificity of a zinc finger domain to specific DNAtarget sites but that targeted specificity is accomplished bya cell-type specific reversibility apparatus of which the 4thbase overlap mechanism is an important factor to accomplishtargeted specificity at specific locations

Consequently we argue for an additional definition oflocation specificity (in contrast to sequence specificity) inwhich natural zinc finger proteins form a DNAproteincomplex at particular locations in the human genome TheDNA sequences can be dissimilar at the different locationsbecause of the condition dependency of forming a biologicalactive complex

82 Translational Research Reversibility and Adherence Wepersuasively argue that translational research on reversibility

and adherence that takes condition dependency into accountshould result in identifying and consequently developingnovel strategies for reducing side effects in which the ldquogoalfor optimal zinc finger design is to generate high affinityto the intended target with low affinity to additional sitesin the genome [14 page 3] [70]rdquo and that this might beaccomplishable by using evolutionary traits to sensitize athree-finger domain (making a domain sensible to only bindto one location) to the point that a zinc finger nuclease (ZFN)only induces a functional effect at the intended target site butnot at additional locations it binds to in the genome

In summary our own as well as other previous findingsindicate that there are three translational factors that regulatebiological activity of natural C

2H2zinc finger domains

reversibility adherence and specificity [40] and to a lesserextend unregulated affinity We suspect that high unregulatedaffinity is associated with elevated toxicity and side effects

Based on our own and previous findings we concludewith the following definitions that have the potential offostering advancements of translational research

83 Difference of Complex Formation and Zinc Finger Binding

DNAProtein Complex Definition Active DNAprotein com-plex that has the authority to induce a functional effect orbiological activity with regulated affinity (adherence) by acondition-dependent reversibility apparatus

Comments(i) Regulated binding by a largely unknown reversibility

apparatus(ii) 1st finger initiating binding(iii) 4th base overlap loci ldquolocking inrdquo to form the

DNAprotein complex that allows the protein beingactive to perform its function

ZFPDNABindingDefinition Binding of natural and artificialzinc finger proteins (ZFPs) or their binding domains to manylocations in the human genomewithout inducing a biologicalactivity or having a functional effect (no formation of aDNAprotein complex) in contrast artificial zinc fingerdomainswith high unregulated affinity can establish bindingsthat allow unregulated functional effects (eg the nuclease ofa ZFN tool that induces side effects)

Comments(i) Unregulated binding of artificial zinc finger domains

to locations on the human genome causes side effects(ii) Unregulated binding of natural zinc finger domains

does not induce biological activity or functionaleffect

9 Definitions Arrived at and Used inThis Paper

(1) Functional Adherence (Regulated Affinity) DefinitionFunctional adherence is regulated affinity that is defined as


adhesion or binding (attachment) that lasts for a specificallycontrolled time frame a DNAprotein complex is functionallyactive to induce a functional effect or biological activityThe attachment is regulated by a cell-specific reversibilityapparatus Part of a reversibility apparatus is the 4th baseoverlap mechanism that increases the strength of noncovalentbonds

Comments

(i) Artificial designer zinc finger domains are not regu-lated by a reversibility apparatus

(ii) Artificial designer zinc finger domains are able toform a functionally active binding In zinc fingernucleases (ZFNs) the nuclease can execute its func-tion of cutting a single strand of DNA at manylocations on the human genomewhich results in toxicside effects

(iii) Modifying natural zinc fingerrsquos specificity withoutchanging its framework [13 69] might still be regu-lated by a specific cellrsquos reversibility apparatus

(2) Unregulated Affinity Definition Unregulated affinity isdefined as noncovalent temporary and uncontrolled adhe-sion or binding (attachment) that lasts for a randomtime frame Unregulated bindings of natural DNA-bindingproteins do not induce a functional effect or biologicalactivity

Adhesion or binding of artificial DNA-binding proteinsand especially zinc finger nucleases (ZFNs)with high affinityto a condition-dependent thus unspecifiable number of DNAsequences lasts longer than a certain nonfunctional timeframe with the ability to induce a functional effect that canlead to clinical side effects

Comments

(i) Atomic forces are condition dependent(ii) General rules for zinc finger domains for binding

the same target site for all conditions cannot beestablished

(iii) ZFP might bind to a specific DNA sequence in onecondition (cell type) but to another DNA sequence inanother condition (cell type)

(iv) Nuclease of artificial zinc finger nucleases (ZFNs)seems to be causing damage at casual ZFPDNAbinding locations on the human genome

(v) If no time sensitive regulation occurs via the 4th basemechanism a zinc finger domain binds unregulatedtomany locations inducing a functional effect causingside-effects

(3) Specificity Definitions (1) Sequence specificity is thebinding of zinc finger domain and DNA-binding factors topreferably only one specific DNA sequence (2) Locationspecificity is the binding to preferably only one location in thehuman genome

Comments

(i) The longer the time the higher the specificity(ii) if the time is too short there is no formation of a

DNAprotein complex so(iii) the longer the time the higher the probability of

forming a DNAprotein complex(iv) the time a ZFP is attached at a specific location in

the human genomewhere induces a clinically relevantactivity

(4) Functional Reversibility of DNA-Binding Complex Defini-tion Functional reversibility is the regulatory mechanism thatgoverns attachment of an active DNA-binding complex at aspecific location in the human genome It is the time frameof activity during which a DNAprotein complex can exert afunctional effect or biological activity at specific locations inthe human genome

Comments

(i) Binding regulated by reversibility apparatus(ii) Induced and timed biological activity and artificial

functional effect(iii) Regulation of binding accomplished using the 4th

base overlap loci that lie at the opposite site of theDNAprotein binding grooves

(iv) Binding initiated by the 1st finger enhances selectivityand decreases affinity Binding sites that would havehigh affinity but low specificity to a domain do notundergo binding-initiation by the 1st finger

(5) Nonfunctional Reversibility of DNA Binding DefinitionNonfunctional reversibility of DNA binding of for exampleunregulated zinc finger protein (ZFP) binding an engineeredzinc finger nuclease (ZFN) tool affects and changes thegenome uncontrollably producing clinical side effects

Comments

(i) Binding of natural zinc finger proteins does notinduce a functional effect or biological activity Bind-ing of artificial zinc finger domains with high affinityis not released in a timelymanner causing side effects

(ii) Artificial zinc finger domains can forman unregulatedDNAprotein complex of ZFNs causing clinical sideeffects because the ldquolock-inrdquo situation initiated by the4th base overlap remains intact unregulated

Our own and previous findings support the idea that it isnecessary to shift the research focus of translational researchfrom specificity and affinity to reversibility adherence andspecificity of a DNAprotein complex and to a lesser extentto unregulated affinity of a zinc finger domain We see the4th base overlap of the 2nd and 3rd fingers of SP1 as a ldquolock-inrdquo mechanism that stabilizes a DNAprotein complex thatallows a natural zinc finger protein to induce its intended


High affinity + high specificity rarr medium reversibilityrarrmediumtoxicityhigh affinity + low specificityrarr low reversibilityrarr high toxicitylow affinity + medium specificityrarrmedium reversibilityrarrmediumtoxicity

Box 1

High location specificity + high adherencerarr high reversibilityrarrmanageable side effectslowndashvery low toxicity

Box 2

natural biological activity or artificial functional effect that isreversible and well planned

Our recommendation is that a three-finger domain withhigh location specificity high adherence and high reversibil-ity and low unregulated affinity will show the lowest toxicityand clinical side effects We contend that unregulated affinityof artificial zinc finger domains is the problem while trans-lational researchers tend to consider that adherence inducedby the 4th base overlap mechanism of the 2nd and 3rd fingersof SP1 stabilizes the DNAprotein complex Adherence occurswhen the 4th base overlap of the 2nd and 3rd fingers ofSP1 ldquolocks inrdquo The consequence is that the ldquolock-inrdquo of theDNAprotein complex allows the protein to fulfill its uniquefunctionThe ldquolock inrdquo function is associated with a ldquolock-outrdquofunction It allows nature to control DNAprotein complexbinding at a single location in the genome with the sameor different target DNA sequences at different locations bychanging the conditions

10 Assessments

Assessment of toxicity of artificially created three-fingerdomainswith unregulated binding affinity is according to ourand previous findings displayed in Box 1

Natural three-finger frameworks of natural zinc fingerdomains that are carefully modified to alter their bindingspecificity that keeps their reversible regulated binding affin-ity intactwould presumably have lowor no toxicity thatmightprove successful in personalized therapies see Box 2

11 Conclusion

Cytotoxicity is the outcome of deleterious genetic changesin the human genome which are not well understood andbeyond the control of present technology Observation ofcell death and apoptosis is widely associated with excessivecleavage at ldquooff-targetrdquo sites which has been attributed toimperfect target site recognition by a zinc finger bindingdomain [6 39 40] In order to meet the ethical requirementsof bringing cures to patients in an uncompromised safe aswell as morally fastest way a concerted interdisciplinary

research effort needs to be organized to uncover the ldquobiologi-cal truthrdquo [38 page 141] and ldquounderlying biology of regulatorymechanisms (which) is very incomplete understoodrdquo [38page 140] The rank-specific recognition code of a singlefinger sheds light on the nature and scope of ldquooff-targetrdquobinding and associated cell death and apoptosis [6] A simpletable of all triplets as has been deemed ldquoextremely usefulrdquo[3 page 9] for each finger of SP1 would be particularlyhelpful in estimating the level of cytotoxicity that mightbe associated with a three-finger domain The known andutilizable evolutionary traits of overlap specificity condi-tion dependency and context dependency together mightbe a viable way to produce cytotoxicity-free zinc fingerdomains Combined with data from RSR various in vitroand in vivo assays with computational analytic tools thebinding accuracy of a binding domain can be significantlyincreased

Dealing with three rank-specific recognition codes ofthe three fingers of the SP1 domain and considering theinterdependency among the adjacent C

2H2fingers while

distinguishing between relevant and nonrelevant 9-mersunder certain conditions are an immense computationaltask that needs to be done in order to use the technologyin clinical settings This can help to identify the biologicalactive 9-mers out of a pool of 262144 putative 9-mersThis number of combinations cannot be lab-tested evenwith high throughput testing In addition data sets fromone assay alone will not supply sufficient information tobuild accurate computational tools to design novel proteinsfor any location on the human genome and predict target-binding sites To bring research onto a manageable level thefocus on the three fingers and the framework of SP1 as anexemplary case to gain full understanding should supplyknowledge on how to approach other venues of researchFor this standards and reproducible methods need to beestablished Such a task needs an unprecedented concertedcollaborative interdisciplinary effort as well as organizationaland managerial tasks Clinical endpoints so to speak mightbe pursued by an interdisciplinary approach including thespecific disciplines of biology biomedical engineering nan-otechnology bioinformatics computational protein foldingfractal and origami to generate accurate data sets to yield


molecular tools comprehensive knowledge and collabora-tion that forms the basis for a branch of ethical research tocure unprofitable diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank R Tjian for supplyingthe plasmid pPacSP1-516c as well as IBM and Intel forgraciously supplying computational infrastructure They areespecially thankful for the support of Paul Kontogiorgis atIBM and Brian Lenaghan at Intel and H-J Thiesen for hiscontinuous support They also thank members of the Centerof FunctionalNanotechnology at BNL for providing scientificinsights and technological support Research was carriedout in part at the Center for Functional NanomaterialsProposal no 30097 Brookhaven National Laboratory whichis supported by theUS Department of Energy Office of BasicEnergy Sciences under Contract no DE-AC02-98CH10886Special thanks are due to M Bishop and C Gajdusek forreviewing the work

References

[1] M T Weirauch and T R Hughes ldquoConserved expression with-out conserved regulatory sequence themore things change themore they stay the samerdquo Trends in Genetics vol 26 no 2 pp66ndash74 2010

[2] G Badis M F Berger A A Philippakis et al ldquoDiversityand complexity in DNA recognition by transcription factorsrdquoScience vol 324 no 5935 pp 1720ndash1723 2009

[3] K N Lam H van Bakel A G Cote A van der Ven and T RHughes ldquoSequence specificity is obtained from the majority ofmodular C

2H2zinc-finger arraysrdquo Nucleic Acids Research vol

39 no 11 pp 4680ndash4690 2011[4] S Myers R Bowden A Tumian et al ldquoDrive against hotspot

motifs in primates implicates the PRDM9 gene in meioticrecombinationrdquo Science vol 327 no 5967 pp 876ndash879 2010

[5] A Klug ldquoThe discovery of zinc fingers and their applications ingene regulation and genome manipulationrdquo Annual Review ofBiochemistry vol 79 pp 213ndash231 2010

[6] T Cathomen and J Keith Joung ldquoZinc-finger nucleases thenext generation emergesrdquo Molecular Therapy vol 16 no 7 pp1200ndash1207 2008

[7] C L Ramirez J E FoleyDAWright et al ldquoUnexpected failurerates for modular assembly of engineered zinc fingersrdquo NatureMethods vol 5 no 5 pp 374ndash375 2008

[8] S Oka Y Shiraishi T Yoshida T Ohkubo Y Sugiura and YKobayashi ldquoNMR structure of transcription factor Sp1 DNAbinding domainrdquo Biochemistry vol 43 no 51 pp 16027ndash160352004

[9] C D Carlson C L Warren K E Hauschild et al ldquoSpecificitylandscapes of DNA binding molecules elucidate biological

functionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 10 pp 4544ndash4549 2010

[10] M-S Kim G Stybayeva J Y Lee A Revzin and D J Segal ldquoAzinc finger protein array for the visual detection of specificDNAsequences for diagnostic applicationsrdquo Nucleic Acids Researchvol 39 no 5 article e29 2011

[11] R Rohs X Jin S M West R Joshi B Honig and R S MannldquoOrigins of specificity in protein-DNA recognitionrdquo AnnualReview of Biochemistry vol 79 pp 233ndash269 2010

[12] C-J Lin T-H Hsiao Y-S Chung et al ldquoZebrafish Sp1-likeprotein is structurally and functionally comparable to humanSp1rdquo Protein Expression and Purification vol 76 no 1 pp 36ndash43 2011

[13] H-J Thiesen and C Bach ldquoDetermination of DNA bindingspecificities of mutated zinc finger domainsrdquo FEBS Letters vol283 no 1 pp 23ndash26 1991

[14] D Davis and D Stokoe ldquoZinc Finger Nucleases as tools tounderstand and treat human diseasesrdquo BMC Medicine vol 8no 42 pp 1ndash11 2010

[15] P Perez-Pinera DGOusterout andCAGersbach ldquoAdvancesin targeted genome editingrdquo Current Opinion in ChemicalBiology vol 16 no 3-4 pp 268ndash277 2012

[16] C M Niemeyer ldquoSemisynthetic DNA-protein conjugates forbiosensing and nanofabricationrdquo Angewandte Chemie vol 49no 7 pp 1200ndash1216 2010

[17] J Hanna M Wernig S Markoulaki et al ldquoTreatment ofsickle cell anemia mouse model with iPS cells generated fromautologous skinrdquo Science vol 318 no 5858 pp 1920ndash1923 2007

[18] J Wang G Friedman Y Doyon et al ldquoTargeted gene additionto a predetermined site in the human genome using a ZFN-based nicking enzymerdquo Genome Research vol 22 no 7 pp1316ndash1326 2012

[19] R van Rensburg I Beyer X-Y Yao et al ldquoChromatin structureof two genomic sites for targeted transgene integration ininduced pluripotent stem cells and hematopoietic stem cellsrdquoGene Therapy vol 20 no 2 pp 201ndash214 2012

[20] F Buchholz and J Hauber ldquoEngineered DNA modifyingenzymes components of a future strategy to cure HIVAIDSrdquoAntiviral Research vol 97 no 2 pp 211ndash217 2013

[21] A Trounson R GThakar G Lomax andD Gibbons ldquoClinicaltrials for stem cell therapiesrdquo BMCMedicine vol 9 no 1 article52 2011

[22] L-T Cheng L-T Sun andT Tada ldquoGenome editing in inducedpluripotent stem cellsrdquoGenes to Cells vol 17 no 6 pp 431ndash4382012

[23] C Yan and P J Higgins ldquoDrugging the undruggable transcrip-tion therapy for cancerrdquoBiochimica et Biophysica Acta vol 1835no 1 pp 76ndash85 2013

[24] N-H Zschemisch S Glage D Wedekind et al ldquoZinc-fingernuclease mediated disruption of Rag1 in the LEWZtm ratrdquoBMC Immunology vol 13 no 1 article 60 2012

[25] M Lusser C Parisi D Plan andE Rodrıguez-Cerezo ldquoDeploy-ment of new biotechnologies in plant breedingrdquoNature Biotech-nology vol 30 no 3 pp 231ndash239 2012

[26] M J Ortuno A R Susperregui N Artigas J L Rosa andF Ventura ldquoOsterix induces Col1a1 gene expression throughbinding to Sp1 sites in the bone enhancer and proximalpromoter regionsrdquo Bone vol 52 no 2 pp 548ndash556 2013


[27] V K Shukla Y Doyon J C Miller et al ldquoPrecise genomemodification in the crop species Zea mays using zinc-fingernucleasesrdquo Nature vol 459 no 7245 pp 437ndash441 2009

[28] J A Townsend D A Wright R J Winfrey et al ldquoHigh-frequency modification of plant genes using engineered zinc-finger nucleasesrdquo Nature vol 459 no 7245 pp 442ndash445 2009

[29] D Mittelman C Moye J Morton et al ldquoZinc-finger directeddouble-strand breaks within CAG repeat tracts promote repeatinstability in human cellsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 24 pp9607ndash9612 2009

[30] P Li Y Xiao Z Liu and P Liu ldquoUsing mouse models to studyfunction of transcriptional factors in T cell developmentrdquo CellRegeneration vol 1 no 1 article 8 2012

[31] A Gupta R G Christensen A L Rayla A Lakshmanan GD Stormo and S A Wolfe ldquoAn optimized two-finger archivefor ZFN-mediated gene targetingrdquo Nature Methods vol 9 pp588ndash590 2012

[32] T Cathomen ldquoZinc-finger nucleases finding the balancebetween activity and toxicityrdquoHumanGeneTherapy vol 20 no11 p 1356 2009

[33] J-S Kim H J Lee and D Carroll ldquoGenome editing withmodularly assembled zinc-finger nucleasesrdquo Nature Methodsvol 7 no 2 p 91 2010

[34] S J Maerkl and S R Quake ldquoExperimental determination ofthe evolvability of a transcription factorrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 44 pp 18650ndash18655 2009

[35] JM Vaquerizas S K Kummerfeld S A Teichmann andNMLuscombe ldquoA census of human transcription factors functionexpression and evolutionrdquo Nature Reviews Genetics vol 10 no4 pp 252ndash263 2009

[36] T A Desai D A Rodionov M S Gelfand E J Alm and CV Rao ldquoEngineering transcription factors with novel DNA-binding specificity using comparative genomicsrdquo Nucleic AcidsResearch vol 37 no 8 pp 2493ndash2503 2009

[37] J Liu and G D Stormo ldquoContext-dependent DNA recognitioncode for C

2H2zinc-finger transcription factorsrdquo Bioinformatics

vol 24 no 17 pp 1850ndash1857 2008[38] M Tompa N Li T L Bailey et al ldquoAssessing computational

tools for the discovery of transcription factor binding sitesrdquoNature Biotechnology vol 23 no 1 pp 137ndash144 2005

[39] D Carroll ldquoProgress and prospects zinc-finger nucleases asgene therapy agentsrdquo Gene Therapy vol 15 no 22 pp 1463ndash1468 2008

[40] T I Cornu S Thibodeau-Beganny E Guhl et al ldquoDNA-binding specificity is a major determinant of the activity andtoxicity of zinc-finger nucleasesrdquoMolecularTherapy vol 16 no2 pp 352ndash358 2008

[41] J C Miller M C Holmes J Wang et al ldquoAn improved zinc-finger nuclease architecture for highly specific genome editingrdquoNature Biotechnology vol 25 no 7 pp 778ndash785 2007

[42] A V Persikov R Osada and M Singh ldquoPredicting DNArecognition by Cys2His2 zinc finger proteinsrdquo Bioinformaticsvol 25 no 1 pp 22ndash29 2009

[43] H-J Thiesen and C Bach ldquoTarget detection assay (TDA)a versatile procedure to determine DNA binding sites asdemonstrated on SP1 proteinrdquo Nucleic Acids Research vol 18no 11 pp 3203ndash3209 1990

[44] H-J Thiesen and C Bach ldquoDNA recognition of C2H2zinc-

finger proteins Evidence for a zinc-finger-specific DNA recog-nition coderdquo Annals of the New York Academy of Sciences vol684 pp 246ndash249 1993

[45] K Kim A Doi B Wen et al ldquoEpigenetic memory in inducedpluripotent stem cellsrdquo Nature vol 467 no 7313 pp 285ndash2902010

[46] S Behrens and M Vingron ldquoStudying the evolution of pro-moter sequences a waiting time problemrdquo Journal of Compu-tational Biology vol 17 no 12 pp 1591ndash1606 2010

[47] A Heffer J W Shultz and L Pick ldquoSurprising flexibility in aconserved Hox transcription factor over 550 million years ofevolutionrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 107 no 42 pp 18040ndash180452010

[48] M Eigen ldquoThe origin of genetic information viruses as mod-elsrdquo Gene vol 135 no 1-2 pp 37ndash47 1993

[49] P Yin H M T Choi C R Calvert and N A PierceldquoProgramming biomolecular self-assembly pathwaysrdquo Naturevol 451 no 7176 pp 318ndash322 2008

[50] E S AndersenM DongMM Nielsen et al ldquoSelf-assembly ofa nanoscale DNA box with a controllable lidrdquo Nature vol 459no 7243 pp 73ndash76 2009

[51] C E Castro F Kilchherr D-N Kim et al ldquoA primer toscaffolded DNA origamirdquoNatureMethods vol 8 no 3 pp 221ndash229 2011

[52] P W K Rothemund ldquoFolding DNA to create nanoscale shapesand patternsrdquo Nature vol 440 no 7082 pp 297ndash302 2006

[53] Y He T Ye M Su et al ldquoHierarchical self-assembly of DNAinto symmetric supramolecular polyhedrardquoNature vol 452 no7184 pp 198ndash201 2008

[54] B V S Iyer M Kenward and G Arya ldquoHierarchies ineukaryotic genome organization insights from polymer theoryand simulationsrdquo BMC Biophysics vol 4 no 1 article 8 2011

[55] E Lander ldquoInitial sequencing and analysis of the humangenomerdquo Nature vol 409 no 6822 pp 860ndash921 2001

[56] E S Lander ldquoInitial impact of the sequencing of the humangenomerdquo Nature vol 470 no 7333 pp 187ndash197 2011

[57] R Rohs SMWest P Liu andBHonig ldquoNuance in the double-helix and its role in protein-DNA recognitionrdquoCurrent Opinionin Structural Biology vol 19 no 2 pp 171ndash177 2009

[58] V A Narayan R W Kriwacki and J P Caradonna ldquoStructuresof zinc finger domains from transcription factor Sp1 Insightsinto sequence-specific protein-DNA recognitionrdquo Journal ofBiological Chemistry vol 272 no 12 pp 7801ndash7809 1997

[59] X Meng S Thibodeau-Beganny T Jiang J K Joung and S AWolfe ldquoProfiling the DNA-binding specificities of engineeredCys2His2 zinc finger domains using a rapid cell-basedmethodrdquoNucleic Acids Research vol 35 no 11 article e81 2007

[60] H J Kim H J Lee H Kim S W Cho and J-S Kim ldquoTargetedgenome editing in human cells with zinc finger nucleasesconstructed via modular assemblyrdquo Genome Research vol 19no 7 pp 1279ndash1288 2009

[61] R R Beerli D J Segal B Dreier and C F Barbas III ldquoTowardcontrolling gene expression at will specific regulation of theerbB-2HER-2 promoter by using polydactyl zinc finger pro-teins constructed frommodular building blocksrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 95 no 25 pp 14628ndash14633 1998


[62] M Yokono N Saegusa K Matsushita and Y Sugiura ldquoUniqueDNA binding mode of the N-terminal zinc finger of transcrip-tion factor Sp1rdquo Biochemistry vol 37 no 19 pp 6824ndash68321998

[63] E Pascal and R Tjian ldquoDifferent activation domains of Sp1govern formation of multimers and mediate transcriptionalsynergismrdquoGenes andDevelopment vol 5 no 9 pp 1646ndash16561991

[64] KThomas J Wu D Y Sung et al ldquoSP1 transcription factors inmale germ cell development and differentiationrdquoMolecular andCellular Endocrinology vol 270 no 1-2 pp 1ndash7 2007

[65] M Eigen and P Schuster ldquoStages of emerging lifemdashfive princi-ples of early organizationrdquo Journal of Molecular Evolution vol19 no 1 pp 47ndash61 1982

[66] C Bach and S Belardo The General Theory of InformationOrigin of Truth andHope AmazonNorthCharleston SCUSA2012

[67] P Bouwman and S Philipsen ldquoRegulation of the activityof Sp1-related transcription factorsrdquo Molecular and CellularEndocrinology vol 195 no 1-2 pp 27ndash38 2002

[68] F Zhang L Cong S Lodato S Kosuri G M Church andP Arlotta ldquoEfficient construction of sequence-specific TALeffectors for modulating mammalian transcriptionrdquo NatureBiotechnology vol 29 no 2 pp 149ndash154 2011

[69] J R Desjarlais and J M Berg ldquoUse of a zinc-finger consensussequence framework and specificity rules to design specificDNA binding proteinsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 90 no 6 pp 2256ndash2260 1993

[70] SM Pruett-Miller J P ConnellyM LMaeder J K Joung andM H Porteus ldquoComparison of zinc finger nucleases for use ingene targeting inMammalian CellsrdquoMolecularTherapy vol 16no 4 pp 707ndash717 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

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Zoology


Molecular Biology International

GenomicsInternational Journal of


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BioinformaticsAdvances in

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Evolutionary BiologyInternational Journal of



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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


critical in the design of zinc finger domains to deal with theoverwhelming complexity of DNA binding

In an effort to reduce complexity and to develop solutionsin a timely fashion it might be realistic to use a naturalzinc finger binding domain and exchange amino acids inthe alpha helical region of one of the fingers to change thedomainsrsquo binding preferences [8 13] To test the feasibilityof changing the rank-ordered binding preferences the three-finger binding domain of SP1 is used to reduce complexityby focusing on exchanging amino acids in the alpha helicalregion of the 2nd finger

2 The Translational Case for Using SP1 toDesign a ZFN with Low Side Effects forSickle-Cell Anemia

Zinc fingers describe a class of DNA binding proteins witha modular design [5 14 15] in which single fingers can beassembled to form multifinger arrangements and recognizeany desired target sequence on the genome Each individualfinger binds preferentially to a specific DNA triplet ldquowithdefined three-base-specificityrdquo [3 page 1] Naturally occur-ring protein-binding domains typically contain three fingersthat bind to a DNA-binding site of a 9-base pair long DNAsequence (9-mers) The modularity of the fingers lends itselfnaturally to a broad variety of bioengineering applicationsProteinDNA hybrid structures have applications for exam-ple in the fabrication of nanoscale functional assemblies [16]Of course their primary application is as a versatile tool fordesigning DNA binding proteins for any target sequence onthe human genome [5 14] for the purpose of gene regulationand genome modification

Such designer zinc fingers have been successfully usedin curing genetic diseases for example for curing sickle-cellanemia [17] in disrupting the HIV CCR5 gene for example[18ndash20] in advanced stem cell therapies for example [21 22]cancer [23] and in other potential applications for example[24ndash26] as well as modifying plant and animal genomes[27 28] In addition a fast growing number of translationalapplications and test assays in biotechnology are reported infor example [20 29 30] However in all these cases ldquooff-targetrdquo binding is a problemwith unacceptable side effects [1431] for which the goal of this study is to show potentially novelways to significantly improve these emerging technologies byincreasing accuracy of binding to a single target site and thusreducing side effects

From the literature it is clear that practical applicationof engineered zinc fingers in humans is severely limited dueto cytotoxic side effects caused by ldquooff-targetrdquo binding siteactivities leading to cell death and apoptosis [32] To addto the challenge recent findings indicate discrepancies andinconsistencies of results produced by various in vitro andin vivo assays [3 7 33] This may be caused by evolutionaryplasticity [34] in which the binding capabilities of singlefingers vary significantly due to the high malleability oftheir 3-dimensional structure which leads to changes intheir binding preferences in various tissue conditions [35]Because of ldquoour limited understanding of even simple DNA

proteininteractionrdquo [36 page 2500] limited knowledge oftranscription factors (TF) functions [35 page 253] and lack ofprecise data to accurately predict binding recognition [37 38]page 144 progress is slow to systematically translate brillianttherapies from for instance animal models [17] into clinicalpractice

Therefore to progress the science it is critical to investi-gate the nature of ldquooff-targetrdquo binding to identify and elimi-nate the potential factors which prevent medical implemen-tation and to gain insights from diverse sources for directingfurther research efforts and technological advances Theseefforts will provide themeans to create critical knowledge andtechnological breakthroughswith broad research and societalimpact This is especially true since today molecular biologyenables us to modify the human genome to cure inheritedgenetic diseases and in the foreseeable future has the potentialto replace damaged and aging tissues and organs

This is due to the unprecedented advances in the biomed-ical sciences which provide the capability to induce thecreation of stem cells from our own ordinary skin cells andthen grow them in numbers to replace burned skin or entireorgans In the case of sickle-cell anemia [17] induced pluripo-tent stem (iPS) cells carrying the disease have been repairedby introducing a healthy HBB gene (Hb A) near the mutatedlocation of the diseased gene (Hb S) (Figures 1 and 2) Cuttingthe HBB gene at the specific location GTGGAG (Figure 1)using a nuclease and introducing a healthy donor genecompletes the correction Nucleases are proteins with theenzymatic capability to cut the genome at any locationIn order to introduce one specific cut at one location thenuclease is guided by a bespoken zinc finger protein designedto bind to the specific DNA sequence next to the HbSmutation (Figure 2)

Two nuclease domains are required at the same locationbut on opposite strands of the genomersquos DNA sequence toform a dimer (Fok I nuclease domains in orange in Figure 2)that can induce a cut at both strands [6] To get the twonuclease domains to the one desired location each domainis tethered to the binding domain of a zinc finger proteinthat specifically recognizes and attaches to its binding sitewhich is a nine-base pair long DNA string for exampleTCCTCAGTC in Figure 2 The hope of this strategy isthat through modular assembly of individual fingers zincfinger nucleases can be created that specifically bind to onedesired DNA sequence [3] In the HBB example symbolizedin Figure 2 the upper three-finger binding domain shouldrecognize exclusively the binding site TCCTCAGTC (lowerGGCAGACTT) where each finger binds to one nucleotidetriplet The two three-finger DNA binding domains com-bined should have the unique quality of bringing the twonuclease domains together at only the one specific target siteGGCAGACTT - - - - - - TCCTCAGTC

This technique called gene targeting has been success-fully applied to cure sickle-cell anemia in a mouse model[17] It has been suggested that statistically the two three-finger binding domains should enable the formation ofthe nuclease dimer only at the one desired location Anexact match search on the NCBI-HuRef genome (NationalCenter for Biotechnology Information) revealed that the





9-mer binding site


GTG GAG TCC TCA GTC

GAG GAG TCC TCA GTC

GGC AGA CTT CAC CTC

GGC AGA CTT CTC CTC


2-fi

nger

3-fi

nger

1-fi

nger


- - - - - - - - - - - - - -- - - - - - - - - --- - -

- - - -- - - - - - - - - -- - - - - - - - -- - - - -


















2O The band shift







C

CG

IQ

M

K

C

C

C

C

H

G

G

G G

G

K

K K

K

KK

VY

K T

T

T

T T

TT

I

S

S

S

H

H

H H

H

H

H

Zn

L

L

LR

R

R

R

R

R

RYS

A

A

W

E E

E

P

P

P

W

F

F

F

FD

ZnZn

M


R

R

HK

SD

E

L

Q













A A A CC

C C C GG


T T T A A A CC

C C C GG


T T TA A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

AAA

A A CC

C C C GG


T T T AAA

A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T







2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

(a)

Figure 4 Continued


0

1000

2000

3000

4000

5000

CGG

GCG CG

TG

GC

TTG

TGG

GAG CC

CTA

GCC

GAG

GG

GT

GAC

GTG

GG

AAG

TTA

ACA

GG

TCG

CT TAT

GTA

GAT

GCA AAT

CGC

TCT

TCG

TAC

CCT

CAA

GTT

GG

GCA

TTG

TTC

AG

CCTG

ATT

TCG

AAT

GA

AG ACT

CTG

ATA

AGA

TTA

TCC

CCA

TGC

TTC

AGC

ACA

ACG

CAC

ATT

CTA

ATC

CTT

GA

ACT

CAC

CA

ACA

AA

S1

0

10000

20000

30000

CCC

GAG GG

TAG

GG

GA

TAA

CAG

GTG

GG

GTG

GTG

TTA

TCA

TAT

GG

TTCG

TCG

GG

TC TAG

TCG

CAC

GA

AG

AC GTA

GCG

AGA

CGC

GG

CA

AG ACA

CCG

AAT

GAT

AGT

CAA

GCT

TGA

TCC

CCT

CGA

AA

ATT

GCC

ACT

GTC

ATA

CCT

AAT

TCT

TAG

CAC

TAT

ATT

AG

CC TTT

CTC

TCT

AAC AC

CAC

GAT

CG

CA TGC

TTC

0

10000

20000

30000

40000

GAG

GG

AG

TGG

AA

GG

GG

GT

GTA

AGG

GG

CG

TTTG

GCC

CAG

TCG

GG

ATTG

AA

AG GAC

GCG TG

CTT

GTA

GG

CA TTT

CGC

CGT

GTC

ATG

AGA

CAG

CCT

GCT AT

ACA

ACT

GTG

TCG

ACA

TCT

AA

ATCC

GCT

TTA

AAG

CTA

TAT

TAC

ACA

CAC

TCT

CAC

GTT

ACC

ATC

AAC

CG

CCA

AA

AAC TA

CTC

GTC

TAT

CTC

CTT

C





0100002000030000400005000060000700008000090000

100000

AGG

CGG

GG

AG

AGA

AGG

GC

GTG

AGA

ATG

AAT

AA

AG

GT

CAG

GG

GG

ACG

AA

AGT

ACA

ATT

ACC

GCG GCT TA

GAT

AG

TACA

ACG

TTA

AG

TCTG

GG

CAG

CC CCC

CGA

GAT

ACT

TGA

CAT

CCG

ACG

GTT

AGC

CCT

CAC

CGC

CTA

TAC

AAC TC

GTA

TCC

ATG

CCT

TTC

TCT

GTC

ATC

CCT

CTG

TTT

GAT

CTT

TTT

ATT

C

(b)

Figure 4 Continued


0

10000

20000

30000

40000

50000

60000

70000

80000

GG

AG

ACG

AG TAA

GG

GG

GT

AGA

GAT

GTG AG

TG

GC

GTA

AGG

TGA

CGA

GCT

GTT

GCG AT

GG

CAAG

CTG

TCG

TG

AA

ATT

ACT

TCA

ACC

CGG

ACA

TGG

CAA

CCT

GCC AAT

AAG AT

ATT

TCG

CAC

GA

AA

TTA

AAC TA

GTT

CG

TC CAT

CCC

TAC

TGC

CAG

CAC

TTG

TAT

CTT

TCG

CCA

CCG

CTG

TCT

ATC

TCC

CTC

CTA

0

10000

20000

30000

40000

50000

AGG

GG

AA

AGG

AG GTA

AGT

TGA

GTG TT

GG

CAG

GG

GG

TCG

TTT

TG

CG GCT GAT

TGG

GTC AAT AT

TTC

TCG

GTG

TG

GC

TAG

TAT

ACT

AGA

TTC

CCC

GTT

AGC

ATA

TGC

TTA

TCC

AA

AAC

ACG

AG

ACA

AC ATG

ACC

CGC

GCC TC

ACT

TCT

GCC

GG

AA

TAA

TAC

CAA

CAG

CTA

ACG

CAT

CTC

CCA

CCT

CAC

TCG

ATC


(b)























A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A


0

1000

2000

3000

4000

5000

(1)16697

(2)19345

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10

)(11

)47

(12

)(13

)(14

)(15

)(16

)26799

(17

)(18

)34437

(19

)(20

)(21

)(22

)(23

)(24

)(25

)17749

(26

)(27

)(28

)(29

)(30

)(31

)15503

(32

)(33

)(34

)14458

(35

)(36

)(37

)(38

)(39

)(40

)(41

)58

(42

)36175

(43

)16928

(44

) (45

)7850

(46

)38448

(47

)(48

)(49

)(50

)(51

)(52

)6704

(53

)26050

(54

)6423

(55

)3769

(56

)2

(57

)(58

)1919

(59

)(60

)(61

)(62

)3170

(63

)2925

(64

)29730









Table 4 Continued





2H2binding proteins




(a)




(b)











Degeneracy








event [3]































2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





9-mer binding site


GTG GAG TCC TCA GTC

GAG GAG TCC TCA GTC

GGC AGA CTT CAC CTC

GGC AGA CTT CTC CTC


2-fi

nger

3-fi

nger

1-fi

nger


- - - - - - - - - - - - - -- - - - - - - - - --- - -

- - - -- - - - - - - - - -- - - - - - - - -- - - - -


















2O The band shift







C

CG

IQ

M

K

C

C

C

C

H

G

G

G G

G

K

K K

K

KK

VY

K T

T

T

T T

TT

I

S

S

S

H

H

H H

H

H

H

Zn

L

L

LR

R

R

R

R

R

RYS

A

A

W

E E

E

P

P

P

W

F

F

F

FD

ZnZn

M


R

R

HK

SD

E

L

Q













A A A CC

C C C GG


T T T A A A CC

C C C GG


T T TA A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

AAA

A A CC

C C C GG


T T T AAA

A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T







2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

(a)

Figure 4 Continued


0

1000

2000

3000

4000

5000

CGG

GCG CG

TG

GC

TTG

TGG

GAG CC

CTA

GCC

GAG

GG

GT

GAC

GTG

GG

AAG

TTA

ACA

GG

TCG

CT TAT

GTA

GAT

GCA AAT

CGC

TCT

TCG

TAC

CCT

CAA

GTT

GG

GCA

TTG

TTC

AG

CCTG

ATT

TCG

AAT

GA

AG ACT

CTG

ATA

AGA

TTA

TCC

CCA

TGC

TTC

AGC

ACA

ACG

CAC

ATT

CTA

ATC

CTT

GA

ACT

CAC

CA

ACA

AA

S1

0

10000

20000

30000

CCC

GAG GG

TAG

GG

GA

TAA

CAG

GTG

GG

GTG

GTG

TTA

TCA

TAT

GG

TTCG

TCG

GG

TC TAG

TCG

CAC

GA

AG

AC GTA

GCG

AGA

CGC

GG

CA

AG ACA

CCG

AAT

GAT

AGT

CAA

GCT

TGA

TCC

CCT

CGA

AA

ATT

GCC

ACT

GTC

ATA

CCT

AAT

TCT

TAG

CAC

TAT

ATT

AG

CC TTT

CTC

TCT

AAC AC

CAC

GAT

CG

CA TGC

TTC

0

10000

20000

30000

40000

GAG

GG

AG

TGG

AA

GG

GG

GT

GTA

AGG

GG

CG

TTTG

GCC

CAG

TCG

GG

ATTG

AA

AG GAC

GCG TG

CTT

GTA

GG

CA TTT

CGC

CGT

GTC

ATG

AGA

CAG

CCT

GCT AT

ACA

ACT

GTG

TCG

ACA

TCT

AA

ATCC

GCT

TTA

AAG

CTA

TAT

TAC

ACA

CAC

TCT

CAC

GTT

ACC

ATC

AAC

CG

CCA

AA

AAC TA

CTC

GTC

TAT

CTC

CTT

C





0100002000030000400005000060000700008000090000

100000

AGG

CGG

GG

AG

AGA

AGG

GC

GTG

AGA

ATG

AAT

AA

AG

GT

CAG

GG

GG

ACG

AA

AGT

ACA

ATT

ACC

GCG GCT TA

GAT

AG

TACA

ACG

TTA

AG

TCTG

GG

CAG

CC CCC

CGA

GAT

ACT

TGA

CAT

CCG

ACG

GTT

AGC

CCT

CAC

CGC

CTA

TAC

AAC TC

GTA

TCC

ATG

CCT

TTC

TCT

GTC

ATC

CCT

CTG

TTT

GAT

CTT

TTT

ATT

C

(b)

Figure 4 Continued


0

10000

20000

30000

40000

50000

60000

70000

80000

GG

AG

ACG

AG TAA

GG

GG

GT

AGA

GAT

GTG AG

TG

GC

GTA

AGG

TGA

CGA

GCT

GTT

GCG AT

GG

CAAG

CTG

TCG

TG

AA

ATT

ACT

TCA

ACC

CGG

ACA

TGG

CAA

CCT

GCC AAT

AAG AT

ATT

TCG

CAC

GA

AA

TTA

AAC TA

GTT

CG

TC CAT

CCC

TAC

TGC

CAG

CAC

TTG

TAT

CTT

TCG

CCA

CCG

CTG

TCT

ATC

TCC

CTC

CTA

0

10000

20000

30000

40000

50000

AGG

GG

AA

AGG

AG GTA

AGT

TGA

GTG TT

GG

CAG

GG

GG

TCG

TTT

TG

CG GCT GAT

TGG

GTC AAT AT

TTC

TCG

GTG

TG

GC

TAG

TAT

ACT

AGA

TTC

CCC

GTT

AGC

ATA

TGC

TTA

TCC

AA

AAC

ACG

AG

ACA

AC ATG

ACC

CGC

GCC TC

ACT

TCT

GCC

GG

AA

TAA

TAC

CAA

CAG

CTA

ACG

CAT

CTC

CCA

CCT

CAC

TCG

ATC


(b)























A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A


0

1000

2000

3000

4000

5000

(1)16697

(2)19345

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10

)(11

)47

(12

)(13

)(14

)(15

)(16

)26799

(17

)(18

)34437

(19

)(20

)(21

)(22

)(23

)(24

)(25

)17749

(26

)(27

)(28

)(29

)(30

)(31

)15503

(32

)(33

)(34

)14458

(35

)(36

)(37

)(38

)(39

)(40

)(41

)58

(42

)36175

(43

)16928

(44

) (45

)7850

(46

)38448

(47

)(48

)(49

)(50

)(51

)(52

)6704

(53

)26050

(54

)6423

(55

)3769

(56

)2

(57

)(58

)1919

(59

)(60

)(61

)(62

)3170

(63

)2925

(64

)29730









Table 4 Continued





2H2binding proteins




(a)




(b)











Degeneracy








event [3]































2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












2O The band shift







C

CG

IQ

M

K

C

C

C

C

H

G

G

G G

G

K

K K

K

KK

VY

K T

T

T

T T

TT

I

S

S

S

H

H

H H

H

H

H

Zn

L

L

LR

R

R

R

R

R

RYS

A

A

W

E E

E

P

P

P

W

F

F

F

FD

ZnZn

M


R

R

HK

SD

E

L

Q













A A A CC

C C C GG


T T T A A A CC

C C C GG


T T TA A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

AAA

A A CC

C C C GG


T T T AAA

A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T







2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

(a)

Figure 4 Continued


0

1000

2000

3000

4000

5000

CGG

GCG CG

TG

GC

TTG

TGG

GAG CC

CTA

GCC

GAG

GG

GT

GAC

GTG

GG

AAG

TTA

ACA

GG

TCG

CT TAT

GTA

GAT

GCA AAT

CGC

TCT

TCG

TAC

CCT

CAA

GTT

GG

GCA

TTG

TTC

AG

CCTG

ATT

TCG

AAT

GA

AG ACT

CTG

ATA

AGA

TTA

TCC

CCA

TGC

TTC

AGC

ACA

ACG

CAC

ATT

CTA

ATC

CTT

GA

ACT

CAC

CA

ACA

AA

S1

0

10000

20000

30000

CCC

GAG GG

TAG

GG

GA

TAA

CAG

GTG

GG

GTG

GTG

TTA

TCA

TAT

GG

TTCG

TCG

GG

TC TAG

TCG

CAC

GA

AG

AC GTA

GCG

AGA

CGC

GG

CA

AG ACA

CCG

AAT

GAT

AGT

CAA

GCT

TGA

TCC

CCT

CGA

AA

ATT

GCC

ACT

GTC

ATA

CCT

AAT

TCT

TAG

CAC

TAT

ATT

AG

CC TTT

CTC

TCT

AAC AC

CAC

GAT

CG

CA TGC

TTC

0

10000

20000

30000

40000

GAG

GG

AG

TGG

AA

GG

GG

GT

GTA

AGG

GG

CG

TTTG

GCC

CAG

TCG

GG

ATTG

AA

AG GAC

GCG TG

CTT

GTA

GG

CA TTT

CGC

CGT

GTC

ATG

AGA

CAG

CCT

GCT AT

ACA

ACT

GTG

TCG

ACA

TCT

AA

ATCC

GCT

TTA

AAG

CTA

TAT

TAC

ACA

CAC

TCT

CAC

GTT

ACC

ATC

AAC

CG

CCA

AA

AAC TA

CTC

GTC

TAT

CTC

CTT

C





0100002000030000400005000060000700008000090000

100000

AGG

CGG

GG

AG

AGA

AGG

GC

GTG

AGA

ATG

AAT

AA

AG

GT

CAG

GG

GG

ACG

AA

AGT

ACA

ATT

ACC

GCG GCT TA

GAT

AG

TACA

ACG

TTA

AG

TCTG

GG

CAG

CC CCC

CGA

GAT

ACT

TGA

CAT

CCG

ACG

GTT

AGC

CCT

CAC

CGC

CTA

TAC

AAC TC

GTA

TCC

ATG

CCT

TTC

TCT

GTC

ATC

CCT

CTG

TTT

GAT

CTT

TTT

ATT

C

(b)

Figure 4 Continued


0

10000

20000

30000

40000

50000

60000

70000

80000

GG

AG

ACG

AG TAA

GG

GG

GT

AGA

GAT

GTG AG

TG

GC

GTA

AGG

TGA

CGA

GCT

GTT

GCG AT

GG

CAAG

CTG

TCG

TG

AA

ATT

ACT

TCA

ACC

CGG

ACA

TGG

CAA

CCT

GCC AAT

AAG AT

ATT

TCG

CAC

GA

AA

TTA

AAC TA

GTT

CG

TC CAT

CCC

TAC

TGC

CAG

CAC

TTG

TAT

CTT

TCG

CCA

CCG

CTG

TCT

ATC

TCC

CTC

CTA

0

10000

20000

30000

40000

50000

AGG

GG

AA

AGG

AG GTA

AGT

TGA

GTG TT

GG

CAG

GG

GG

TCG

TTT

TG

CG GCT GAT

TGG

GTC AAT AT

TTC

TCG

GTG

TG

GC

TAG

TAT

ACT

AGA

TTC

CCC

GTT

AGC

ATA

TGC

TTA

TCC

AA

AAC

ACG

AG

ACA

AC ATG

ACC

CGC

GCC TC

ACT

TCT

GCC

GG

AA

TAA

TAC

CAA

CAG

CTA

ACG

CAT

CTC

CCA

CCT

CAC

TCG

ATC


(b)























A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A


0

1000

2000

3000

4000

5000

(1)16697

(2)19345

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10

)(11

)47

(12

)(13

)(14

)(15

)(16

)26799

(17

)(18

)34437

(19

)(20

)(21

)(22

)(23

)(24

)(25

)17749

(26

)(27

)(28

)(29

)(30

)(31

)15503

(32

)(33

)(34

)14458

(35

)(36

)(37

)(38

)(39

)(40

)(41

)58

(42

)36175

(43

)16928

(44

) (45

)7850

(46

)38448

(47

)(48

)(49

)(50

)(51

)(52

)6704

(53

)26050

(54

)6423

(55

)3769

(56

)2

(57

)(58

)1919

(59

)(60

)(61

)(62

)3170

(63

)2925

(64

)29730









Table 4 Continued





2H2binding proteins




(a)




(b)











Degeneracy








event [3]































2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


C

CG

IQ

M

K

C

C

C

C

H

G

G

G G

G

K

K K

K

KK

VY

K T

T

T

T T

TT

I

S

S

S

H

H

H H

H

H

H

Zn

L

L

LR

R

R

R

R

R

RYS

A

A

W

E E

E

P

P

P

W

F

F

F

FD

ZnZn

M


R

R

HK

SD

E

L

Q













A A A CC

C C C GG


T T T A A A CC

C C C GG


T T TA A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

AAA

A A CC

C C C GG


T T T AAA

A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T







2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

(a)

Figure 4 Continued


0

1000

2000

3000

4000

5000

CGG

GCG CG

TG

GC

TTG

TGG

GAG CC

CTA

GCC

GAG

GG

GT

GAC

GTG

GG

AAG

TTA

ACA

GG

TCG

CT TAT

GTA

GAT

GCA AAT

CGC

TCT

TCG

TAC

CCT

CAA

GTT

GG

GCA

TTG

TTC

AG

CCTG

ATT

TCG

AAT

GA

AG ACT

CTG

ATA

AGA

TTA

TCC

CCA

TGC

TTC

AGC

ACA

ACG

CAC

ATT

CTA

ATC

CTT

GA

ACT

CAC

CA

ACA

AA

S1

0

10000

20000

30000

CCC

GAG GG

TAG

GG

GA

TAA

CAG

GTG

GG

GTG

GTG

TTA

TCA

TAT

GG

TTCG

TCG

GG

TC TAG

TCG

CAC

GA

AG

AC GTA

GCG

AGA

CGC

GG

CA

AG ACA

CCG

AAT

GAT

AGT

CAA

GCT

TGA

TCC

CCT

CGA

AA

ATT

GCC

ACT

GTC

ATA

CCT

AAT

TCT

TAG

CAC

TAT

ATT

AG

CC TTT

CTC

TCT

AAC AC

CAC

GAT

CG

CA TGC

TTC

0

10000

20000

30000

40000

GAG

GG

AG

TGG

AA

GG

GG

GT

GTA

AGG

GG

CG

TTTG

GCC

CAG

TCG

GG

ATTG

AA

AG GAC

GCG TG

CTT

GTA

GG

CA TTT

CGC

CGT

GTC

ATG

AGA

CAG

CCT

GCT AT

ACA

ACT

GTG

TCG

ACA

TCT

AA

ATCC

GCT

TTA

AAG

CTA

TAT

TAC

ACA

CAC

TCT

CAC

GTT

ACC

ATC

AAC

CG

CCA

AA

AAC TA

CTC

GTC

TAT

CTC

CTT

C





0100002000030000400005000060000700008000090000

100000

AGG

CGG

GG

AG

AGA

AGG

GC

GTG

AGA

ATG

AAT

AA

AG

GT

CAG

GG

GG

ACG

AA

AGT

ACA

ATT

ACC

GCG GCT TA

GAT

AG

TACA

ACG

TTA

AG

TCTG

GG

CAG

CC CCC

CGA

GAT

ACT

TGA

CAT

CCG

ACG

GTT

AGC

CCT

CAC

CGC

CTA

TAC

AAC TC

GTA

TCC

ATG

CCT

TTC

TCT

GTC

ATC

CCT

CTG

TTT

GAT

CTT

TTT

ATT

C

(b)

Figure 4 Continued


0

10000

20000

30000

40000

50000

60000

70000

80000

GG

AG

ACG

AG TAA

GG

GG

GT

AGA

GAT

GTG AG

TG

GC

GTA

AGG

TGA

CGA

GCT

GTT

GCG AT

GG

CAAG

CTG

TCG

TG

AA

ATT

ACT

TCA

ACC

CGG

ACA

TGG

CAA

CCT

GCC AAT

AAG AT

ATT

TCG

CAC

GA

AA

TTA

AAC TA

GTT

CG

TC CAT

CCC

TAC

TGC

CAG

CAC

TTG

TAT

CTT

TCG

CCA

CCG

CTG

TCT

ATC

TCC

CTC

CTA

0

10000

20000

30000

40000

50000

AGG

GG

AA

AGG

AG GTA

AGT

TGA

GTG TT

GG

CAG

GG

GG

TCG

TTT

TG

CG GCT GAT

TGG

GTC AAT AT

TTC

TCG

GTG

TG

GC

TAG

TAT

ACT

AGA

TTC

CCC

GTT

AGC

ATA

TGC

TTA

TCC

AA

AAC

ACG

AG

ACA

AC ATG

ACC

CGC

GCC TC

ACT

TCT

GCC

GG

AA

TAA

TAC

CAA

CAG

CTA

ACG

CAT

CTC

CCA

CCT

CAC

TCG

ATC


(b)























A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A


0

1000

2000

3000

4000

5000

(1)16697

(2)19345

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10

)(11

)47

(12

)(13

)(14

)(15

)(16

)26799

(17

)(18

)34437

(19

)(20

)(21

)(22

)(23

)(24

)(25

)17749

(26

)(27

)(28

)(29

)(30

)(31

)15503

(32

)(33

)(34

)14458

(35

)(36

)(37

)(38

)(39

)(40

)(41

)58

(42

)36175

(43

)16928

(44

) (45

)7850

(46

)38448

(47

)(48

)(49

)(50

)(51

)(52

)6704

(53

)26050

(54

)6423

(55

)3769

(56

)2

(57

)(58

)1919

(59

)(60

)(61

)(62

)3170

(63

)2925

(64

)29730









Table 4 Continued





2H2binding proteins




(a)




(b)











Degeneracy








event [3]































2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


A A A CC

C C C GG


T T T A A A CC

C C C GG


T T TA A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

AAA

A A CC

C C C GG


T T T AAA

A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T

A A A CC

C C C GG


T T T







2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

(a)

Figure 4 Continued


0

1000

2000

3000

4000

5000

CGG

GCG CG

TG

GC

TTG

TGG

GAG CC

CTA

GCC

GAG

GG

GT

GAC

GTG

GG

AAG

TTA

ACA

GG

TCG

CT TAT

GTA

GAT

GCA AAT

CGC

TCT

TCG

TAC

CCT

CAA

GTT

GG

GCA

TTG

TTC

AG

CCTG

ATT

TCG

AAT

GA

AG ACT

CTG

ATA

AGA

TTA

TCC

CCA

TGC

TTC

AGC

ACA

ACG

CAC

ATT

CTA

ATC

CTT

GA

ACT

CAC

CA

ACA

AA

S1

0

10000

20000

30000

CCC

GAG GG

TAG

GG

GA

TAA

CAG

GTG

GG

GTG

GTG

TTA

TCA

TAT

GG

TTCG

TCG

GG

TC TAG

TCG

CAC

GA

AG

AC GTA

GCG

AGA

CGC

GG

CA

AG ACA

CCG

AAT

GAT

AGT

CAA

GCT

TGA

TCC

CCT

CGA

AA

ATT

GCC

ACT

GTC

ATA

CCT

AAT

TCT

TAG

CAC

TAT

ATT

AG

CC TTT

CTC

TCT

AAC AC

CAC

GAT

CG

CA TGC

TTC

0

10000

20000

30000

40000

GAG

GG

AG

TGG

AA

GG

GG

GT

GTA

AGG

GG

CG

TTTG

GCC

CAG

TCG

GG

ATTG

AA

AG GAC

GCG TG

CTT

GTA

GG

CA TTT

CGC

CGT

GTC

ATG

AGA

CAG

CCT

GCT AT

ACA

ACT

GTG

TCG

ACA

TCT

AA

ATCC

GCT

TTA

AAG

CTA

TAT

TAC

ACA

CAC

TCT

CAC

GTT

ACC

ATC

AAC

CG

CCA

AA

AAC TA

CTC

GTC

TAT

CTC

CTT

C





0100002000030000400005000060000700008000090000

100000

AGG

CGG

GG

AG

AGA

AGG

GC

GTG

AGA

ATG

AAT

AA

AG

GT

CAG

GG

GG

ACG

AA

AGT

ACA

ATT

ACC

GCG GCT TA

GAT

AG

TACA

ACG

TTA

AG

TCTG

GG

CAG

CC CCC

CGA

GAT

ACT

TGA

CAT

CCG

ACG

GTT

AGC

CCT

CAC

CGC

CTA

TAC

AAC TC

GTA

TCC

ATG

CCT

TTC

TCT

GTC

ATC

CCT

CTG

TTT

GAT

CTT

TTT

ATT

C

(b)

Figure 4 Continued


0

10000

20000

30000

40000

50000

60000

70000

80000

GG

AG

ACG

AG TAA

GG

GG

GT

AGA

GAT

GTG AG

TG

GC

GTA

AGG

TGA

CGA

GCT

GTT

GCG AT

GG

CAAG

CTG

TCG

TG

AA

ATT

ACT

TCA

ACC

CGG

ACA

TGG

CAA

CCT

GCC AAT

AAG AT

ATT

TCG

CAC

GA

AA

TTA

AAC TA

GTT

CG

TC CAT

CCC

TAC

TGC

CAG

CAC

TTG

TAT

CTT

TCG

CCA

CCG

CTG

TCT

ATC

TCC

CTC

CTA

0

10000

20000

30000

40000

50000

AGG

GG

AA

AGG

AG GTA

AGT

TGA

GTG TT

GG

CAG

GG

GG

TCG

TTT

TG

CG GCT GAT

TGG

GTC AAT AT

TTC

TCG

GTG

TG

GC

TAG

TAT

ACT

AGA

TTC

CCC

GTT

AGC

ATA

TGC

TTA

TCC

AA

AAC

ACG

AG

ACA

AC ATG

ACC

CGC

GCC TC

ACT

TCT

GCC

GG

AA

TAA

TAC

CAA

CAG

CTA

ACG

CAT

CTC

CCA

CCT

CAC

TCG

ATC


(b)























A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A


0

1000

2000

3000

4000

5000

(1)16697

(2)19345

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10

)(11

)47

(12

)(13

)(14

)(15

)(16

)26799

(17

)(18

)34437

(19

)(20

)(21

)(22

)(23

)(24

)(25

)17749

(26

)(27

)(28

)(29

)(30

)(31

)15503

(32

)(33

)(34

)14458

(35

)(36

)(37

)(38

)(39

)(40

)(41

)58

(42

)36175

(43

)16928

(44

) (45

)7850

(46

)38448

(47

)(48

)(49

)(50

)(51

)(52

)6704

(53

)26050

(54

)6423

(55

)3769

(56

)2

(57

)(58

)1919

(59

)(60

)(61

)(62

)3170

(63

)2925

(64

)29730









Table 4 Continued





2H2binding proteins




(a)




(b)











Degeneracy








event [3]































2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

1000

2000

3000

4000

5000

CGG

GCG CG

TG

GC

TTG

TGG

GAG CC

CTA

GCC

GAG

GG

GT

GAC

GTG

GG

AAG

TTA

ACA

GG

TCG

CT TAT

GTA

GAT

GCA AAT

CGC

TCT

TCG

TAC

CCT

CAA

GTT

GG

GCA

TTG

TTC

AG

CCTG

ATT

TCG

AAT

GA

AG ACT

CTG

ATA

AGA

TTA

TCC

CCA

TGC

TTC

AGC

ACA

ACG

CAC

ATT

CTA

ATC

CTT

GA

ACT

CAC

CA

ACA

AA

S1

0

10000

20000

30000

CCC

GAG GG

TAG

GG

GA

TAA

CAG

GTG

GG

GTG

GTG

TTA

TCA

TAT

GG

TTCG

TCG

GG

TC TAG

TCG

CAC

GA

AG

AC GTA

GCG

AGA

CGC

GG

CA

AG ACA

CCG

AAT

GAT

AGT

CAA

GCT

TGA

TCC

CCT

CGA

AA

ATT

GCC

ACT

GTC

ATA

CCT

AAT

TCT

TAG

CAC

TAT

ATT

AG

CC TTT

CTC

TCT

AAC AC

CAC

GAT

CG

CA TGC

TTC

0

10000

20000

30000

40000

GAG

GG

AG

TGG

AA

GG

GG

GT

GTA

AGG

GG

CG

TTTG

GCC

CAG

TCG

GG

ATTG

AA

AG GAC

GCG TG

CTT

GTA

GG

CA TTT

CGC

CGT

GTC

ATG

AGA

CAG

CCT

GCT AT

ACA

ACT

GTG

TCG

ACA

TCT

AA

ATCC

GCT

TTA

AAG

CTA

TAT

TAC

ACA

CAC

TCT

CAC

GTT

ACC

ATC

AAC

CG

CCA

AA

AAC TA

CTC

GTC

TAT

CTC

CTT

C





0100002000030000400005000060000700008000090000

100000

AGG

CGG

GG

AG

AGA

AGG

GC

GTG

AGA

ATG

AAT

AA

AG

GT

CAG

GG

GG

ACG

AA

AGT

ACA

ATT

ACC

GCG GCT TA

GAT

AG

TACA

ACG

TTA

AG

TCTG

GG

CAG

CC CCC

CGA

GAT

ACT

TGA

CAT

CCG

ACG

GTT

AGC

CCT

CAC

CGC

CTA

TAC

AAC TC

GTA

TCC

ATG

CCT

TTC

TCT

GTC

ATC

CCT

CTG

TTT

GAT

CTT

TTT

ATT

C

(b)

Figure 4 Continued


0

10000

20000

30000

40000

50000

60000

70000

80000

GG

AG

ACG

AG TAA

GG

GG

GT

AGA

GAT

GTG AG

TG

GC

GTA

AGG

TGA

CGA

GCT

GTT

GCG AT

GG

CAAG

CTG

TCG

TG

AA

ATT

ACT

TCA

ACC

CGG

ACA

TGG

CAA

CCT

GCC AAT

AAG AT

ATT

TCG

CAC

GA

AA

TTA

AAC TA

GTT

CG

TC CAT

CCC

TAC

TGC

CAG

CAC

TTG

TAT

CTT

TCG

CCA

CCG

CTG

TCT

ATC

TCC

CTC

CTA

0

10000

20000

30000

40000

50000

AGG

GG

AA

AGG

AG GTA

AGT

TGA

GTG TT

GG

CAG

GG

GG

TCG

TTT

TG

CG GCT GAT

TGG

GTC AAT AT

TTC

TCG

GTG

TG

GC

TAG

TAT

ACT

AGA

TTC

CCC

GTT

AGC

ATA

TGC

TTA

TCC

AA

AAC

ACG

AG

ACA

AC ATG

ACC

CGC

GCC TC

ACT

TCT

GCC

GG

AA

TAA

TAC

CAA

CAG

CTA

ACG

CAT

CTC

CCA

CCT

CAC

TCG

ATC


(b)























A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A


0

1000

2000

3000

4000

5000

(1)16697

(2)19345

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10

)(11

)47

(12

)(13

)(14

)(15

)(16

)26799

(17

)(18

)34437

(19

)(20

)(21

)(22

)(23

)(24

)(25

)17749

(26

)(27

)(28

)(29

)(30

)(31

)15503

(32

)(33

)(34

)14458

(35

)(36

)(37

)(38

)(39

)(40

)(41

)58

(42

)36175

(43

)16928

(44

) (45

)7850

(46

)38448

(47

)(48

)(49

)(50

)(51

)(52

)6704

(53

)26050

(54

)6423

(55

)3769

(56

)2

(57

)(58

)1919

(59

)(60

)(61

)(62

)3170

(63

)2925

(64

)29730









Table 4 Continued





2H2binding proteins




(a)




(b)











Degeneracy








event [3]































2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

10000

20000

30000

40000

50000

60000

70000

80000

GG

AG

ACG

AG TAA

GG

GG

GT

AGA

GAT

GTG AG

TG

GC

GTA

AGG

TGA

CGA

GCT

GTT

GCG AT

GG

CAAG

CTG

TCG

TG

AA

ATT

ACT

TCA

ACC

CGG

ACA

TGG

CAA

CCT

GCC AAT

AAG AT

ATT

TCG

CAC

GA

AA

TTA

AAC TA

GTT

CG

TC CAT

CCC

TAC

TGC

CAG

CAC

TTG

TAT

CTT

TCG

CCA

CCG

CTG

TCT

ATC

TCC

CTC

CTA

0

10000

20000

30000

40000

50000

AGG

GG

AA

AGG

AG GTA

AGT

TGA

GTG TT

GG

CAG

GG

GG

TCG

TTT

TG

CG GCT GAT

TGG

GTC AAT AT

TTC

TCG

GTG

TG

GC

TAG

TAT

ACT

AGA

TTC

CCC

GTT

AGC

ATA

TGC

TTA

TCC

AA

AAC

ACG

AG

ACA

AC ATG

ACC

CGC

GCC TC

ACT

TCT

GCC

GG

AA

TAA

TAC

CAA

CAG

CTA

ACG

CAT

CTC

CCA

CCT

CAC

TCG

ATC


(b)























A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A


0

1000

2000

3000

4000

5000

(1)16697

(2)19345

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10

)(11

)47

(12

)(13

)(14

)(15

)(16

)26799

(17

)(18

)34437

(19

)(20

)(21

)(22

)(23

)(24

)(25

)17749

(26

)(27

)(28

)(29

)(30

)(31

)15503

(32

)(33

)(34

)14458

(35

)(36

)(37

)(38

)(39

)(40

)(41

)58

(42

)36175

(43

)16928

(44

) (45

)7850

(46

)38448

(47

)(48

)(49

)(50

)(51

)(52

)6704

(53

)26050

(54

)6423

(55

)3769

(56

)2

(57

)(58

)1919

(59

)(60

)(61

)(62

)3170

(63

)2925

(64

)29730









Table 4 Continued





2H2binding proteins




(a)




(b)











Degeneracy








event [3]































2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A


0

1000

2000

3000

4000

5000

(1)16697

(2)19345

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10

)(11

)47

(12

)(13

)(14

)(15

)(16

)26799

(17

)(18

)34437

(19

)(20

)(21

)(22

)(23

)(24

)(25

)17749

(26

)(27

)(28

)(29

)(30

)(31

)15503

(32

)(33

)(34

)14458

(35

)(36

)(37

)(38

)(39

)(40

)(41

)58

(42

)36175

(43

)16928

(44

) (45

)7850

(46

)38448

(47

)(48

)(49

)(50

)(51

)(52

)6704

(53

)26050

(54

)6423

(55

)3769

(56

)2

(57

)(58

)1919

(59

)(60

)(61

)(62

)3170

(63

)2925

(64

)29730









Table 4 Continued





2H2binding proteins




(a)




(b)











Degeneracy








event [3]































2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T A A A CC

C C C GG


T T T2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A

2998400 A3998400 A


0

1000

2000

3000

4000

5000

(1)16697

(2)19345

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10

)(11

)47

(12

)(13

)(14

)(15

)(16

)26799

(17

)(18

)34437

(19

)(20

)(21

)(22

)(23

)(24

)(25

)17749

(26

)(27

)(28

)(29

)(30

)(31

)15503

(32

)(33

)(34

)14458

(35

)(36

)(37

)(38

)(39

)(40

)(41

)58

(42

)36175

(43

)16928

(44

) (45

)7850

(46

)38448

(47

)(48

)(49

)(50

)(51

)(52

)6704

(53

)26050

(54

)6423

(55

)3769

(56

)2

(57

)(58

)1919

(59

)(60

)(61

)(62

)3170

(63

)2925

(64

)29730









Table 4 Continued





2H2binding proteins




(a)




(b)











Degeneracy








event [3]































2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Table 4 Continued





2H2binding proteins




(a)




(b)











Degeneracy








event [3]































2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a)




(b)











Degeneracy








event [3]































2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Degeneracy








event [3]































2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







2His2zinc



GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


GG GGG G GG CC C

1 2 3

9

3

- - - - - - -




















6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





6 Discussion


























- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


- - - - - - - - - - - -- - - - - -CA CC T


A C - -C G

G

C

G



domainFok I


3-finger


1998400-finger

2998400-finger3

998400-finger 1998400998400998400-finger

1998400998400998400-finger

1998400998400-finger








7 Limitations


8 Contributions































Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Comments






Comments







Comments






Comments






Comments






Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Box 1


Box 2



10 Assessments



11 Conclusion




2H2fingers while






Acknowledgments


References






















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Evaluation of Novel Design Strategies for ...downloads.hindawi.com/journals/btri/2014/970595.pdfResearch Article Evaluation of Novel Design Strategies for Developing