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CHEMICAL BIOLOGY BIOLOGICAL CHEMISTRY

Chimia 55 (2001) 295-301copy Schweizerische Chemische Gesellschaft

ISSN 0009-4293

Design and Evaluation ofOligonucleotide Analogues

Christian J Leumann

295CHIMIA 200155 NO4

Abstract This article contains a short account of the chemical and biophysical properties ofthe oligonucleotideanalogues bicyclo-DNA tricyclo-DNA [321]-DNA [321]amide-DNA and the PNA analogue OPA recentlydeveloped in our laboratory Emphasis is given to various aspects of conformational restriction as a designertool for enhancing the performance of oligonucleotide analogues and for exploring the supramolecularchemistry of DNA A short summary of current problems in the field of DNA triple-helix chemistry as well ascontributions from our laboratory in this area concludes this communication

Keywords Conformational restriction DNAmiddot Oligonucleotide analoguesmiddot RNAmiddot Triple helix

Introduction

More than two decades ago in two semi-nal papers Stephenson and Zamecnik[1][2] disclosed the successful use of oli-gonucleotides to inhibit the expression ofspecific genes at the level of translationin vitro thus paving the way for what isnow more generally called the antisenseapproach in human therapy This thera-peutic concept is based on the interactionof an oligonucleotide analogue (antisenseoligonucleotide) with a complementarymRNA to inhibit translation of the mes-sage into a protein The attractiveness ofthis concept compared to traditional pro-tein targeting with small molecules liesin the fact that only minimal structuralknowledge about the target namely itsspecific gene sequence is required in or-der to rationally design a highly selectiveinhibitor A major effort in the search ofoligonucleotide analogues with favorablechemical and biological properties wasthe result A drug acting against cytome-galovirus (CMV) induced retinitis (Vit-ravenetrade) entirely based on an oligonu-cleotide analogue recently came to mar-ket and demonstrates the feasibility of the

Correspondence Prof Dr CJ LeumannDepartment of Chemistry amp BiochemistryUniversity of BernFreiestrasse 3CH-3012 BernTel +41316314355Fax +41 31 631 3422E-Mail leumanniocunibech

therapeutic antisense concept On theother hand rapid progress in molecularbiology and molecular medicine whichhas recently culminated in the sequencedetermination of the human genome de-mands the continuous development ofnew DNA-based analytic and diagnosticmethods such as chip technology [3] forhigh-throughput sequencing of DNAfast screening of gene function or realtime fluorescence detection of geneticmutations A further interest in oligonu-cleotide chemistry has arisen from the al-most unique opportunity for constructingwell-defined DNA-based molecular ar-chitectures including rods pseudoknotsborromean rings and two- and three di-mensional networks [4][5] by oligonu-cleotide sequence design and their even-tual use in nanotechnology [6][7] All ofthis however would not have been pos-sible were powerful methods for solidphase synthesis of oligonucleotides un-available [8]

In this realm our own research activi-ties focus on various supramolecular as-pects of DNARNA association and fold-ing With computer-aided design andmodeling the tools of organic synthesisbasic molecular biology and biophysicalanalysis we evaluate DNA-analogueswith the goal of determining the details ofthe interrelation between the structure ofthe monomeric constituents and the prop-erties of the corresponding oligomersThis is pivotal in order to be able to pro-duce oligonucleotide analogues withtailor-made properties for use in theabove-mentioned areas Besides this

such studies will almost certainly lead tonew insight into the basic non-covalentfactors that determine biomolecular or-ganization in general While we have ad-equate knowledge to predict conforma-tional preferences of a (bio)moleculeamongst other properties we are still farfrom being able to rationalize and thuspredict the exact effect of hydrophobicforces stacking interactions and solva-tion on structure In the following an ac-count of current research projects withspecial emphasis on conformational andnon-covalent aspects of DNA associationis given

Conformationally RestrictedOligonucleotide Analogues

Inspired by the chemical properties ofhomo-DNA [9] we started more than tenyears ago with the relatively simple ideato adapt the concept of structural preor-ganization [10] to oligonucleotides Byconstraining the conformational statesof a single-stranded oligonucleotide tomatch the geometry in its duplexed forman entropic benefit for complexation isto be expected which should result in ahigher thermodynamic stability of theduplex With this background the oligo-nucleotide analogues bicyclo-DNA andtricyclo-DNA showing conformationalrestriction in their sugar units were pre-pared and evaluated (Fig 1) The conceptas such has now been widely appliedby a number of research groups and wascovered in a recent review [11]s

ource httpsdoiorg107892boris68589 | downloaded 1732020

CHEMICAL BIOLOGY BIOLOGICAL CHEMISTRY 296CHIMIA 200155 No4

Fig 1 Chemical structures of a selection of oligonucleotide analogues built from conformation-ally constrained nucleoside analogues

Fig 2 The prinCipal differences in the preferred recognition mode of complementary strandsbetween oligopurine sequences of bicyclo-DNA and DNA (top) as a consequence of thedifference in the preferred conformation around torsion angle r (bottom)

H~ n H~~n J minor

~_ ~ --X ~ (N y--- ~N-f ~N W~SOn-CrickY ~N~ WI on-Cn

--0 N=lt mInor --0 N= )Ory

DNA

ning to investigate the [321]amide-DNA

We found the [321]DNA systemto be a competent base-pairing systemforming duplexes with natural DNA andRNA with thermal stability that is infe-rior by ca I-3degC in Tm per base-paircompared to natural DNA [14] Only anti-parallel and no-parallel duplexes wereformed with both natural DNA and itselfas the comp1exing partner which pointstowards a remarkable remote stereocon-trol of the sugar-phosphate backbone indetermining the polarity of strand associ-ation [15] Furthermore the Watson-Crick pairing mode is maintained as in-ferred from CD-spectroscopic analysisBase-mismatch discrimination is similarcompared to natural DNA These factscorroborate our current view that a cyclicunit between base and backbone is not anecessary requirement for obtaining acompetent selective and complementarybase-pairing system provided that thebackbone is structurally preorganized

Io)~JBase

IBicyclo-DNA

0 0 0

O~Base0

o~ O~Base ~BaseII II

-0--0 II

-O-PW 0 Base -O-P-O1

~B-O-P-O

-O~Base ~~0

~Base0

0 0

DNA homo-DNA bicyclo-DNA tricyclo-DNA

In a different design project we havereplaced the cyclic furanose unit of adeoxynucleoside by a linear linker unitwhile maintaining the conformational re-striction of the bonds directly involved inthe repeating unit of the sugar phosphatebackbone Thus we synthesized and eval-uated the two analogues shown in Fig 4While the number of bonds between thebackbone unit and the base is equal tothat of DNA in [32I]DNA it is longerby one bond in [321]amide-DNA Withthis design we tried to determine whethera cyclic sugar unit is a necessary require-ment for obtaining a complementarybase-recognition system and whetherthe three bond rule for the distance be-tween base and backbone must be strictlyobeyed At the outset this was not at allclear since there did not exist a function-al phosphodiester-based DNA analoguewith acyclic connection of the nucleobas-es to the backbone While we have stud-ied a number of oligonucleotides in thecase of [321]DNA we are only begin-

From detailed analysis of a seriesof different bicyc1o-DNA and tricyc1o-DNA oligonucleotide sequences 6-12nucleotide units in length we found thatin contrast to natural DNA homopurinehomopyrimidine sequences of these ana-logues preferentially form duplexes ofthe Hoogsteen and reversed Hoogsteentype with strongly enhanced thermal sta-bility [12][13] This unexpected changein the base-pairing preferences was foundto be mainly the consequence of a geo-metric change of one of the six torsionangles (torsion angle y) defining thebackbone geometry of DNA from thegauche to the antiperiplanar conforma-tion (Fig 2) Tricyclo-DNA behaves sim-ilarly and leads to a very strong Hoogs-teen base-pairing system Denaturationtemperatures (Tm) of homoadenine- andhomothymine- nonamer duplexes of tri-cyclo-DNA can be higher by almost 50degCcompared to the parent natural (Watson-Crick) base-paired duplex

The repertoire of secondary structuralmotifs of RNA is generally much largerthan that of genomic DNA because of thelack of a complement Typically ribo-somal RNA mRNA catalytic RNA andsingle stranded DNA-aptamer sequencestherefore fold intramolecularly to formcomplex three-dimensional structureswith unusual base-base arrangementshairpin-loops and bulges To improve theperformance of catalytic RNA or DNA-aptarners it is sometimes desirable to sta-bilize such secondary structural motifsFig 3 shows a self-complementary DNAdodecamer which occurs as an equilibri-um mixture between a bimolecular duplexand a monomolecular hairpin in solutionDoping the loop-region of the oligonu-cleotide with individual tricyclo-DNAunits leads to distinct differences in theequilibrium mixture and the hairpin sta-bility Exchanging the 5-thymidine resi-due by a tricyc10thymidine unit eg leadsto an increase in Tm of the hairpin by ap-proximately 20degC (red curve) and a com-plete shift of the equilibrium towardsthe hairpin structure as can be inferredqualitatively from the correspondingUV-melting curves Exchanging the 3-de-oxyadenosine residue by a tricyc1o-aden-osine unit (cyan curve) shows the oppo-site effect namely a stabilization andequilibrium shift towards the bimolecularduplex structure Thus besides being astructural probe for local loop conforma-tions such rigid nuc1eosides with alteredbackbone geometry may prove usefulfor enhancing the binding efficiency ofDNA-aptamers or the catalytic efficiencyof ribozymes


Fig 3 Schematic view of the equilibrium be-tween the biomolecular duplex and the tetra-loop hairpin structure of the indicated DNA-dodecamer (left) and corresponding UV-melt-ing curves (right) The black curve correspondsto the melting profile of the unmodified DNAdodecamer while the others correspond tothat ofthe single tricyclo-DNA mutant sequenc-es (indicated with small letters at the appropri-ate position) Some of the UV-melting curvesdisplay biphasic transitions (black blue or-ange) indicating the presence of both hairpin(transition at higher temperature) and bimo-lecular duplex (transition at lower temperature)in solution The cyan curve is reminiscent of asingle bimolecular duplex to single-strand tran-sition while the red curve corresponds to amonomolecular hairpin to single-strand transi-tion

10 20 30 40 50 60 70 80 90

Temp [C)

18middot

16

14middot

12

I 10

8

= 6middot

4middot

2middot

Omiddot

-2middotiJ

OHun

a t =

T T CGCG-3a A A GCGC-5

a

5-CGCGAATTCGCGGCGCTTAAGCGC-5

il

~Base

Io

1oIo=P-o

3

IoIO=F-O

C 36

I9o=P-o)

H 4NYBase

o

Fig 4 Chemical structures of DNA [321]-DNA and [321]amide-DNA Structural ele-ments in red correspond to the backbone ele-ments and those in blue to the base-linkerelements While the number of bonds for thebackbone element is the same in all threesystems that of the base-linker element islonger by one bond in [321]amide-DNA Thiselongation is expressed in the cartoon of thedouble helix by a wider helix

[321]DNA DNA [321 ]amide-DNA

While we do not yet have a detailedpicture of the properties of [321]amide-DNA we have been able to show thata corresponding homoadenine decamerforms a duplex with the natural DNA-complement as well as with a homothy-midine decamer of the same backbonetype of similar stability as natural DNA[16] This is interesting in view of the in-creased distance between base and back-bone Preliminary investigations by CD-spectroscopy revealed an almost normalWatson-Crick duplex structure for theDNA[321]amide-DNA hybrid duplexbut almost a perfect mirror image for thepure [321]amide-DNA decamer duplexindicating a left-handed Watson-Crickdouble helical structure This unexpectedproperty suggests a potential degeneracyin enantioselection of the [321]amide-DNA system Indeed we have shownvery recently that the [321]amide-DNA

homoadenine decamer not only binds toa (natural) D-RNA-complement but alsoto the enantiomeric L-RNA with similaraffinity forming enantiomorphic Wat-son-Crick double helices (Fig 5) Thus[321]amide-DNA is the first example ofa chiral non-chiroselective base-pairingsystem The structural basis for this de-generacy as well as the eventual conse-quences with respect to the molecularevolution of homochirality on earth arecurrently being investigated

A completely different aspect of con-formational restriction was the basis forour design of the PNA (polyamide nucle-ic acid) analogue OPA (Fig 6) PNA isan achiral amide-based nucleic acid ana-logue which only has the nucleobases incommon with DNA Although structural-ly completely different than DNA it dis-plays very similar functional propertiesand forms stable duplex and triplex struc-

tures with RNA and DNA The propertiesof PNA have recently been reviewed ex-tensively [17]

Structural investigations of PNADNA and PNARNA complexes re-vealed that the tertiary amide functionconnecting the base-linker with the back-bone is unidirectionally aligned with thecarbonyl oxygen pointing in the directionof the C-terminus of the chain In thePNA monomers however a mixture ofboth rotameric forms occurs in solutionWith the knowledge from the X-raystructure that the carbonyl oxygen is notinvolved in any direct hydrogen bondingit was appealing to ask whether this in-trinsically conformationally labile amideunit could be replaced by an isostructuralconfigurationally fixed C=C doublebond Such a replacement would not onlyallow us to separately address the two(E and Z) rotameric forms of PNA in

CHEMICAL BIOLOGY BIOLOGICAL CHEMISTRY 298CHIMIA 200155 NO4

Fig 5 UV-melting curves (top) and CD-spectra(bottom) of the [321 ]amide-DNA homoaden-osine decamer duplex with the complementaryRNA decamer built from D-ribothymidine (rTO)or the enantiomeric (unnatural) L-ribothymi-dine (rTL)

30

25 a1OrTL10E (Tm = 20degC)c 20g~ 15~c a1OrTD 10e 10~I 5 (Tm = 30degC)~

0

0 20 40 60 80 100Temp rmiddotC)

8

6

4

2gI 0S middot2

-4

220 240 260 280Wavelength (nm)

300 320

complex formation but could also pro-vide fundamental information about theconsequences of replacing a hydrophilicby a hydrophobic functional group withinan isostructural environment

Indeed our preliminary results showthat E-OPA which has its bases structur-ally arranged as in the complexed form ofPNA exhibits subtle differences in pair-ing to DNA compared to PNA Homoba-sic thymine-containing decamers havelost their propensity for triple-helix for-mation and bind with reduced affinity toDNA Furthermore the preferred strandassociation in non self-complementaryadenine- and thymine-containing E-OPA

oligomers has changed from antiparallelto parallel [18] All the information wehave at present indicates that non-trivialelectrostatic properties of the tertiaryamide function rather than geometrydominate complementary DNA recogni-tion In our view this is a prime exampleof our inability to predict and accountfor such subtle effects in the moleculardesign process

The OPAs represent a convenientstructural platform to investigate the roleof electrostatic effects in detail For ex-ample the remaining H-atom of the dou-ble bond in E-OPA can be replaced byappropriate functional groups to study

hydrophobic interactions dipole interac-tions and solvation effects separatelyThis will lead to a better understanding ofthe relative contribution of these effectsto stability and may eventually yieldPNA analogues with tailor-made chemi-cal properties Work in this direction iscurrently in progress

Base-modified OligonucleotideAnalogues for DNA Triple-helixFormation

A related field of interest focuses onrecognition of the major groove of dou-


HN

Baseo

LBaseo

Z-OPA

HN

HN HN~N rBaSe rBase

~OO

HN 0

~rBaSeHN

N rBaSe

~OO

PNA 0

E-OPA

Fig 6 Part of the X-ray structure of a DNAlPNA2 triplex displaying the non-involvement of the carbonyl oxygen of the central amide functionalitybetween the base-linker and the backbone of the PNA-strand in intra- or interstrand hydrogen bonding (left) and molecular structures of the twoanalogues E- and Z-OPA developed in our laboratory (right)

Fig 7 Schematic view of a third strand oligonucleotide (red) aligned in the major groove of a DNAdouble helix (light blue) forming a DNA triple-helix (top left) together with the base-triplesoccurring in the parallel (top right) and the anti parallel (bottom) triple-helical binding motif

ing unnecessary functional groups andheteroatoms in order to increase the in-trinsic basicity of the required aromaticring-nitrogen We thus came up with anaminopyridine-containing C-glycosideas a replacement for cytidine (Fig 8)This heterocycle shows increased basici-

)Pu-Py motifparallel motif

IPu-PymotifanlJparaliel motif

HG

N

N

l H - H N Q---H-N

f J- hN- G~-H---~~ Cgt

N= -NN-H---Q

H

N

oT No N 9

H ~ H~~~-~~~M

N= rNo

Ta N q

H H

~N~~_~~~M~d rN

a

NN

modified oligonucleotide third strandsable to overcome this sequence restric-tion

In order to circumvent the unfavor-able properties associated with the pro-tonation of cytosine by base design wefocused on the simple concept of remov-

ble-stranded DNA by oligonucleotidesforming triple-helical DNA structuresSequence specific blockage of the majorgroove of DNA with oligonucleotidesoffers an alternative means of selectivelyinterfering with the genetic expressionmachinery at the level of transcriptionThis approach is often referred to as theantigene strategy There are two stabletriple helical binding motifs known today(Fig 7) In the pypu-py motif an oligo-nucleotide third strand consisting exclu-sively of pyrimidine bases binds in a par-allel fashion to the purine strand of theunderlying duplex forrningTA-T andC+G-C base-triples In the alternativemotif a purine-rich third strand bindsin an antiparallel fashion to the purinestrand of the duplex to give specific AA-T TA-T or GG-C base-triples The re-quirement for protonation of the cytosinebase in the pypu-py motif an unfavor-able process at neutral pH leads to astrong reduction of affinity under physio-logical conditions This has been identi-fied as one of the major limitations ofthe parallel triple-helical binding motifFurthermore for both recognition motifsit is absolutely required that the targetDNA duplex consists of a homopurinehomopyrimidine sequence tract Thisrequirement severely limits the range ofamenable DNA-targets and has triggeredan intense search for sugar andor base-


Fig 8 DNase I footprint assay of third strand binding to duplex DNA containing four targetcassettes (top) that are identical with the exception of the base-pair (XV) opposite the base ofinterest (2) of the third strand Protected (white) zones on the gel correspond to areas where thirdstrand binding occurred The left part of the gel (third strand with Z = Mep) clearly shows strongbinding to the GC cassette and no binding even at high mM concentrations to all of the othercassettes

Xy

AT

CG

TA

to elucidate the function of newly identi-fied proteins or receptors More sophisti-cated DNA-based analytical tools willcontinue to replace more traditional tech-nologies in applications ranging frommolecular medicine to the forensic sci-ences Biotechnology will see new tools

z = MaC1

05nm - 10 jlM

-AAAAAGA AGAGAGATTTTTCT TCTCTCT-

5-TTTTTCT TCTCTCT-3

ther progress in the future and second orthird generation oligonucleotide ana-logues with improved chemical and phar-macological properties against varioustargets will be developed Furthermoreantisense technology will be of invalu-middotable help in functional genomics in order

Although antisense research hasreached a plateau after a steep initialburst phase there will definitely be fur-

Outlook and Conclusions

ty by ca 2 pKa units We were indeedable to show that replacement of 5-me-thyl cytosine (MeC) by the respective ami-nopyridine unit (Mep) leads to a notableincrease in triplex stability even at a pHas high as 75 and in sequence contextswhere natural DNA third strands notori-ously fail to form triple helices Further-more there was no decrease in selectivityof triplex formation as revealed by DNaseI footprint analysis in a competition ex-periment (Fig 8) [19]

The central problem in DNA triplexchemistry however is its restriction tohomopurinehomopyrimidine target se-quences Despite almost 15 years of in-tense research a solution to this problemis still elusive In one of our projectsaimed at overcoming the sequence recog-nition requirements we proposed theconcept of targeting natural pyrimidinebases by one conventional bond and onenon-classical (hypothetical) CHO hydro-gen bond This concept arose from earlierobservations in experiments with N7-hy-poxanthine as a third strand base [20)and from X-ray data on a RNA hexamerduplex [21)[22] in which the presence ofsuch non-classical CHO hydrogen bondswas either postulated or observed Wewere able to demonstrate that up to threeuracil bases as part of IS-mer DNA targetcassette can be recognized by a thirdstrand oligonucleotide containing N7-hy-poxanthine opposite the uridine residues[23] This corresponds to a DNA targetsite containing 20 pyrimidine basesWe have recently expanded on this themeand have shown that the base 4-deox-othymine (4HT) as part of a third strandrecognizes a cytosine base within the pu-rine target sequence with high selectivitybut moderate affinity [24) (Fig 9)

At this point we have no unambigu-ous confirmation of the existence of suchunconventional hydrogen bonds Nor dowe know yet whether such an arrange-ment of functional groups exhibits asmall positive energetic contribution totriplex stability or whether it is just neu-tral and non-repulsive In any case theintegration of such energetically weakbut potentially selectivity enhancing ele-ments into the design of third strand basesis an interesting addition to standardapproaches involving classical hydrogenbonding schemes


5GCTAAAAAGA AGAGAGATCG3CGATCTCTCT TCTTTTTAGC

5TTTTTCT TCTCTCTTm [OC] Third Strand Dissociation

Z = 4HT

x C G A Ty G C T ApH60 360 298 250 26370 186 89 10780 6(c=16 JlM 10 mM Na-cacodylate 100 mM NaCl025 mM spermine pH 60)

-N~Ht-No

301CHIMIA 200155 No4

Fig 9 Sequence context and four base-triple arrangements with 4-deoxothymine (4HT)as third strand base The table displays Tm data from thirdstrand melting at the conditions indicated The arrangement XYZ = eG 4HT corresponds to the most stable of the four possibilities

arising from the DNA-based expansionof the genetic alphabet enhancing theperformance of expression systems ofproteins containing unnatural amino ac-ids Last but not least a whole new areaof nanotechnology and biocomputingbased on intelligent informational (bio)macromolecules like DNA with distinctlogical properties is being developedTaking all this into account there is plen-ty of room down at the bottom to createnew oligonucleotide analogues with dis-tinct chemical physical and biologicalproperties from simple monomeric unitsyet to be designed

AcknowledgmentsI would like to thank all my past and present

coworkers for their enthusiastic contributions tothis challenging interdisciplinary field of re-search Financial support from the Swiss Na-tional Science Foundation the Roche Founda-tion as well as from Novartis AG Basel andRoche Diagnostics GmbH Penzberg is grate-fully acknowledged

Received February 20 2001

[I] ML Stephenson Pe Zamecnik ProcNatl Acad Sci USA 1978 75 285-288

[2] Pe Zamecnik ML Stephenson ProcNatl Acad Sci USA 1978 75 280-284

[3] M Chee RYang E Hubbell A BernoXC Huang D Stern J Winkler DJ

Lockhart MS Morris SP Fodor Sci-ence 1996 274 610--614

[4] NC Seeman Trends Biotechnol 199917437-443

[5] NC Seeman Curro Opin Struct BioI19966519-526

[6] AP Alivisatos KP Johnsson X PengTE Wilson CJ Loweth MP BruchezJr PG Schultz Nature 1996382609-611

[7] CA Mirkin RL Letsinger RC MucicJJ Storhoff Nature 1996382607-609

[8J MH Caruthers Ace Chern Res 199124 278-284

[9] J Hunziker H-J Roth M Bohringer AGiger U Diederichsen M Gobel RKrishnan B Jaun CJ Leumann AEschenmoser Helv Chirn Acta 1993 76259-352

[IOJ DJ Cram Angew Chern 1988 1001041-1052

[llJ M Meldgaard 1 Wengel J Chern SocPerkin 1 2000 3539-3554

[12J M Bolli e Litten R Schlitz CJ Leu-mann Chern BioI 19963 197-206

[13J R Steffens eJ Leumann J Arn ChernSoc 19991213249-3255

[14] e Epple CJ Leumann Chern BioI19985209-216

[15] BM Keller CJ Leumann AngewChern 2000112 2367-2369

[16J A Egger CJ Leumann Synlett 1999 Sl913-916

[17] E Uhlmann A Peyman G BreipohlDW Will Angew Chern 1998 1102954-2983

[18J R Schiltz M Cantin eD Roberts BGreiner E Uhlmann eJ Leumann An-gew Chern 2000 lJ2 1305-1308

[19J S Hildbrand A Blaser SP Parel CJLeumann J Am Chern Soc 1997 1195499-5511

[20J J Marfurt SP Parel eJ Leumann Nu-cleic Acids Res 199725 1875-1882

[21J MC Wahl M Sundaralingam TlBS19972297-102

[22J Me Wahl ST Rao M SundaralingamNature Struct Bioi 1996 3 24-31

[23J 1 Marfurt CJ Leumann Angew Chern1998110184-187

[24J I Prevot-Halter CJ Leumann BioorgMed Chern Lett 19999 2657-2660


Fig 1 Chemical structures of a selection of oligonucleotide analogues built from conformation-ally constrained nucleoside analogues

Fig 2 The prinCipal differences in the preferred recognition mode of complementary strandsbetween oligopurine sequences of bicyclo-DNA and DNA (top) as a consequence of thedifference in the preferred conformation around torsion angle r (bottom)

H~ n H~~n J minor

~_ ~ --X ~ (N y--- ~N-f ~N W~SOn-CrickY ~N~ WI on-Cn

--0 N=lt mInor --0 N= )Ory

DNA

ning to investigate the [321]amide-DNA

We found the [321]DNA systemto be a competent base-pairing systemforming duplexes with natural DNA andRNA with thermal stability that is infe-rior by ca I-3degC in Tm per base-paircompared to natural DNA [14] Only anti-parallel and no-parallel duplexes wereformed with both natural DNA and itselfas the comp1exing partner which pointstowards a remarkable remote stereocon-trol of the sugar-phosphate backbone indetermining the polarity of strand associ-ation [15] Furthermore the Watson-Crick pairing mode is maintained as in-ferred from CD-spectroscopic analysisBase-mismatch discrimination is similarcompared to natural DNA These factscorroborate our current view that a cyclicunit between base and backbone is not anecessary requirement for obtaining acompetent selective and complementarybase-pairing system provided that thebackbone is structurally preorganized

Io)~JBase

IBicyclo-DNA

0 0 0

O~Base0

o~ O~Base ~BaseII II

-0--0 II

-O-PW 0 Base -O-P-O1

~B-O-P-O

-O~Base ~~0

~Base0

0 0

DNA homo-DNA bicyclo-DNA tricyclo-DNA

In a different design project we havereplaced the cyclic furanose unit of adeoxynucleoside by a linear linker unitwhile maintaining the conformational re-striction of the bonds directly involved inthe repeating unit of the sugar phosphatebackbone Thus we synthesized and eval-uated the two analogues shown in Fig 4While the number of bonds between thebackbone unit and the base is equal tothat of DNA in [32I]DNA it is longerby one bond in [321]amide-DNA Withthis design we tried to determine whethera cyclic sugar unit is a necessary require-ment for obtaining a complementarybase-recognition system and whetherthe three bond rule for the distance be-tween base and backbone must be strictlyobeyed At the outset this was not at allclear since there did not exist a function-al phosphodiester-based DNA analoguewith acyclic connection of the nucleobas-es to the backbone While we have stud-ied a number of oligonucleotides in thecase of [321]DNA we are only begin-

From detailed analysis of a seriesof different bicyc1o-DNA and tricyc1o-DNA oligonucleotide sequences 6-12nucleotide units in length we found thatin contrast to natural DNA homopurinehomopyrimidine sequences of these ana-logues preferentially form duplexes ofthe Hoogsteen and reversed Hoogsteentype with strongly enhanced thermal sta-bility [12][13] This unexpected changein the base-pairing preferences was foundto be mainly the consequence of a geo-metric change of one of the six torsionangles (torsion angle y) defining thebackbone geometry of DNA from thegauche to the antiperiplanar conforma-tion (Fig 2) Tricyclo-DNA behaves sim-ilarly and leads to a very strong Hoogs-teen base-pairing system Denaturationtemperatures (Tm) of homoadenine- andhomothymine- nonamer duplexes of tri-cyclo-DNA can be higher by almost 50degCcompared to the parent natural (Watson-Crick) base-paired duplex

The repertoire of secondary structuralmotifs of RNA is generally much largerthan that of genomic DNA because of thelack of a complement Typically ribo-somal RNA mRNA catalytic RNA andsingle stranded DNA-aptamer sequencestherefore fold intramolecularly to formcomplex three-dimensional structureswith unusual base-base arrangementshairpin-loops and bulges To improve theperformance of catalytic RNA or DNA-aptarners it is sometimes desirable to sta-bilize such secondary structural motifsFig 3 shows a self-complementary DNAdodecamer which occurs as an equilibri-um mixture between a bimolecular duplexand a monomolecular hairpin in solutionDoping the loop-region of the oligonu-cleotide with individual tricyclo-DNAunits leads to distinct differences in theequilibrium mixture and the hairpin sta-bility Exchanging the 5-thymidine resi-due by a tricyc10thymidine unit eg leadsto an increase in Tm of the hairpin by ap-proximately 20degC (red curve) and a com-plete shift of the equilibrium towardsthe hairpin structure as can be inferredqualitatively from the correspondingUV-melting curves Exchanging the 3-de-oxyadenosine residue by a tricyc1o-aden-osine unit (cyan curve) shows the oppo-site effect namely a stabilization andequilibrium shift towards the bimolecularduplex structure Thus besides being astructural probe for local loop conforma-tions such rigid nuc1eosides with alteredbackbone geometry may prove usefulfor enhancing the binding efficiency ofDNA-aptamers or the catalytic efficiencyof ribozymes



10 20 30 40 50 60 70 80 90

Temp [C)

18middot

16

14middot

12

I 10

8

= 6middot

4middot

2middot

Omiddot

-2middotiJ

OHun

a t =


a


il

~Base

Io

1oIo=P-o

3

IoIO=F-O

C 36

I9o=P-o)

H 4NYBase

o










30


0

0 20 40 60 80 100Temp rmiddotC)

8

6

4

2gI 0S middot2

-4


300 320









HN

Baseo

LBaseo

Z-OPA

HN

HN HN~N rBaSe rBase

~OO

HN 0

~rBaSeHN

N rBaSe

~OO

PNA 0

E-OPA






HG

N

N

l H - H N Q---H-N


N= -NN-H---Q

H

N

oT No N 9

H ~ H~~~-~~~M

N= rNo

Ta N q

H H

~N~~_~~~M~d rN

a

NN






Xy

AT

CG

TA


z = MaC1

05nm - 10 jlM


5-TTTTTCT TCTCTCT-3










Z = 4HT


-N~Ht-No


































10 20 30 40 50 60 70 80 90

Temp [C)

18middot

16

14middot

12

I 10

8

= 6middot

4middot

2middot

Omiddot

-2middotiJ

OHun

a t =


a


il

~Base

Io

1oIo=P-o

3

IoIO=F-O

C 36

I9o=P-o)

H 4NYBase

o










30


0

0 20 40 60 80 100Temp rmiddotC)

8

6

4

2gI 0S middot2

-4


300 320









HN

Baseo

LBaseo

Z-OPA

HN

HN HN~N rBaSe rBase

~OO

HN 0

~rBaSeHN

N rBaSe

~OO

PNA 0

E-OPA






HG

N

N

l H - H N Q---H-N


N= -NN-H---Q

H

N

oT No N 9

H ~ H~~~-~~~M

N= rNo

Ta N q

H H

~N~~_~~~M~d rN

a

NN






Xy

AT

CG

TA


z = MaC1

05nm - 10 jlM


5-TTTTTCT TCTCTCT-3










Z = 4HT


-N~Ht-No


































30


0

0 20 40 60 80 100Temp rmiddotC)

8

6

4

2gI 0S middot2

-4


300 320









HN

Baseo

LBaseo

Z-OPA

HN

HN HN~N rBaSe rBase

~OO

HN 0

~rBaSeHN

N rBaSe

~OO

PNA 0

E-OPA






HG

N

N

l H - H N Q---H-N


N= -NN-H---Q

H

N

oT No N 9

H ~ H~~~-~~~M

N= rNo

Ta N q

H H

~N~~_~~~M~d rN

a

NN






Xy

AT

CG

TA


z = MaC1

05nm - 10 jlM


5-TTTTTCT TCTCTCT-3










Z = 4HT


-N~Ht-No

































HN

Baseo

LBaseo

Z-OPA

HN

HN HN~N rBaSe rBase

~OO

HN 0

~rBaSeHN

N rBaSe

~OO

PNA 0

E-OPA






HG

N

N

l H - H N Q---H-N


N= -NN-H---Q

H

N

oT No N 9

H ~ H~~~-~~~M

N= rNo

Ta N q

H H

~N~~_~~~M~d rN

a

NN






Xy

AT

CG

TA


z = MaC1

05nm - 10 jlM


5-TTTTTCT TCTCTCT-3










Z = 4HT


-N~Ht-No


































Xy

AT

CG

TA


z = MaC1

05nm - 10 jlM


5-TTTTTCT TCTCTCT-3










Z = 4HT


-N~Ht-No



































Z = 4HT


-N~Ht-No
































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