Speciﬁc Labeling of Zinc Finger Proteins using ... · in ZFP engineering have enabled the design and construction of “programmable” transcription factors that target and bind

Specific Labeling of Zinc Finger Proteins using Noncanonical AminoAcids and Copper-Free Click ChemistryYounghoon Kim,† Sung Hoon Kim,‡ Dean Ferracane,† John A. Katzenellenbogen,‡

and Charles M. Schroeder*,†,§

†Department of Chemical and Biomolecular Engineering, ‡Department of Chemistry, and §Center for Biophysics and ComputationalBiology, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States

*S Supporting Information

ABSTRACT: Zinc finger proteins (ZFPs) play a key role intranscriptional regulation and serve as invaluable tools for genemodification and genetic engineering. Development of efficientstrategies for labeling metalloproteins such as ZFPs is essential forunderstanding and controlling biological processes. In this work, weengineered ZFPs containing cysteine-histidine (Cys2-His2) motifsby metabolic incorporation of the unnatural amino acidazidohomoalanine (AHA), followed by specific protein labelingvia click chemistry. We show that cyclooctyne promoted [3 + 2]dipolar cycloaddition with azides, known as copper-free clickchemistry, provides rapid and specific labeling of ZFPs at high yields as determined by mass spectrometry analysis. We observethat the DNA-binding activity of ZFPs labeled by conventional copper-mediated click chemistry was completely abolished,whereas ZFPs labeled by copper-free click chemistry retain their sequence-specific DNA-binding activity under native conditions,as determined by electrophoretic mobility shift assays, protein microarrays, and kinetic binding assays based on Forster resonanceenergy transfer (FRET). Our work provides a general framework to label metalloproteins such as ZFPs by metabolicincorporation of unnatural amino acids followed by copper-free click chemistry.

■ INTRODUCTION

Zinc finger proteins (ZFPs) comprise a broad class oftranscription factors in living organisms. ZFPs regulate geneexpression by binding to target sites along chromosomal DNAwith high specificity and moderate-to-high affinity.1 Zinc fingersare classified based on amino acid residues coordinating zincions, with a major class containing a common structuralelement with two cysteine and two histidine residuescomplexed with a zinc ion (Cys2-His2 motif). Recent advancesin ZFP engineering have enabled the design and constructionof “programmable” transcription factors that target and bind tochromosomal DNA in a sequence-specific fashion, therebyenabling robust control of gene expression in live cells.1−3

Synthetic ZFPs consist of tandem arrays of zinc finger modulesassembled via molecular cloning. In addition, zinc fingernucleases (ZFNs) have been constructed by fusing zinc fingermodules with the FokI cleavage domain. ZFNs have been usedfor targeted manipulation of genomes in a variety of cell types,including human cell lines.4−6 Using ZFNs, chromosomal DNAcan be modified by producing double-stranded DNA breaks toinduce site-specific homologous recombination, thereby allow-ing for direct modification of any desired genomic locus. Zincfinger technology has capitalized on the success of rapidconstruction methods for ZFPs, including a modular assemblymethod, combinatorial selection of engineered multifingerproteins (oligomerized pool engineering, OPEN), and context-dependent assembly methods (CoDA).7−9

Engineered ZFPs can be screened for sequence-specific DNAbinding using powerful in vivo methods, including bacterial two-hybrid selection.10 However, measurement of dynamic ZFP-DNA binding and binding kinetics is generally facilitated by invitro methods or molecular-level biochemical tools.11,12

Biochemical analysis can yield valuable mechanistic informationregarding DNA site recognition by zinc finger modules.Fluorescence-based techniques such as Forster resonanceenergy transfer (FRET) allow for measurement of dynamic,nonequilibrium processes that are difficult to access usingtraditional electrophoretic mobility shift assays or immunopre-cipitation methods. Characterization of DNA−protein inter-actions using fluorescence-based methods generally requiressite-specific conjugation of fluorescent probes to minimizeperturbations to protein structure and to retain proteinactivity.13 A common strategy for protein labeling isconjugation of fluorescent tags at existing or engineeredcysteine moieties on the protein backbone. However, thislabeling strategy is generally not feasible if cysteine moieties areessential for protein function. In the context of zinc fingers, anintact and unmodified Cys2-His2 motif is required forcompetency in ZFP-based DNA recognition, and this presentsa major challenge in labeling ZFPs using cysteine-based

Received: May 16, 2012Revised: July 8, 2012Published: August 8, 2012

Article

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© 2012 American Chemical Society 1891 dx.doi.org/10.1021/bc300262h | Bioconjugate Chem. 2012, 23, 1891−1901

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methods.14,15 Targeted cysteine modification or nonspecificconjugation of fluorescent molecules to ZFPs tends to diminishor completely abolish the DNA-binding ability of ZFPs.16 Fromthis perspective, there is a strong need for development of newmethods for specific labeling of ZFPs with fluorescent probes orchemical functional moieties to facilitate the study of DNArecognition, protein function, and dynamic binding of zincfinger modules.12

Development of new strategies for transcription factorlabeling would enable fundamental research in gene networkdynamics, transcriptional regulation, and genetic engineering.Metabolic labeling of ZFPs by direct incorporation ofbioorthogonal moieties (e.g., azide) is a particularly attractivestrategy to label proteins with fluorescent probes using clickchemistry via copper-catalyzed azide−alkyne cycloaddition.17

Click chemistry allows for rapid, robust, and specific chemicalreactions, thereby serving as a practical and widely adoptedlabeling strategy.18,19 Reaction conditions for click chemistryare generally robust, being insensitive to variations in saltconcentration or the presence of additional functional groupssuch as primary amines. Copper (Cu) catalysts are commonlyused to activate click reactions between an azide and a terminalalkyne to generate triazole moieties, and a triazole ligand istypically used to boost reaction yields and to enhance reactionrates in aqueous media.19,20 Although a triazole ligand is not anabsolute requirement for the azide−alkyne cycloadditionreaction, low reaction yields have been observed in the absenceof the triazole ligand in typical aqueous buffers, and such lowyields complicate quantitative studies of dynamic proteininteractions.21 In addition to copper-mediated azide−alkynecycloaddition, copper-free strain-promoted cycloaddition canbe used to avoid potentially toxic copper(I) (Cu1+) ions forclick reactions.22 The conditions for copper-free click reactionsare mild and biologically compatible, which facilitatesbioorthogonal reactions and chemical conjugation in livingcells.23

In this work, we report an efficient method for residue-specific labeling of ZFPs using direct metabolic incorporation ofnoncanonical amino acids followed by copper-free clickchemistry. ZFPs consisting of tandem arrays of zinc fingermodules are expressed by replacing natural methionine withazidohomoalanine (AHA) in cell cultures. In this way,noncanonical AHA amino acids are directly incorporated attargeted locations along the protein backbone to introducereactive sites for click chemistry. Using this strategy, wecompare both copper-mediated and copper-free strain-promoted [3 + 2] cycloaddition as labeling methods, and weassay the DNA binding properties of modified ZFPs using asolution-based FRET method and a microarray-based DNAhybridization imaging technique. Finally, we validate themetabolic labeling strategy for ZFPs by demonstratingcompetent DNA recognition and binding of target sequenceswith high specificity and affinity. Overall, we show thatmetabolic incorporation of unnatural amino acids, coupledwith Cu-free click chemistry, is a powerful approach to studytranscription factor binding using a wide variety offluorescence-based techniques.

■ MATERIALS AND METHODSMolecular Cloning. Standard protocols in molecular

biology were used for molecular cloning, as described in theSupporting Information (SI).24 Plasmid DNA was isolated fromcell cultures using miniprep kits (Qiagen). Plasmid DNA was

digested using restriction endonucleases (New EnglandBiolabs), and gene inserts and linearized vectors were purifiedby agarose gel electrophoresis and gel extraction kits (Qiagen).All molecular subcloning was performed using E. coli strainDH5-alpha, pUC19 plasmid DNA, and a modified version ofpcDNA 3.0 plasmid DNA (Invitrogen).

Design and Modular Assembly of ZFPs. The design andassembly of engineered zinc finger proteins was carried out aspreviously described.25 Detailed procedures describing ZFPassembly are provided in SI.

Protein Expression and Purification. Engineered ZFPswere produced using E. coli protein expression host platforms(M15MA and B834) and purified using fast protein liquidchromatography (FPLC) by affinity-based and size exclusionmethods, as described in SI.

Homology Modeling. Zif268 (pdb 1A1L_A) was used as amodel protein for generating protein structures. Homologymodels were constructed using the Swiss Model server, andprotein structures were rendered using VMD (Visual MolecularDynamics, University of Illinois, Urbana−Champaign).26

Click Chemistry Reactions for Azide-Modified Pro-teins. For copper-mediated click chemistry reactions, purifiedprotein (∼μM concentration) was mixed with copper sulfate(final concentration of 100 μM), tris[(1-benzyl-1H-1,2,3-triazol-4yl)methyl] amine (TBTA) (200 μM), and ascorbicacid (0.5 mM), followed by the addition of Cy5-alkyne (∼5−30× molar excess relative to protein). Ascorbic acid was freshlyprepared for each batch reaction. For copper-free clickchemistry reactions, fluorescein-dibenzylcyclooctyne (DBCO,Click Chemistry Tools, Scottsdale, AZ) or Cy5-DBCO wasdirectly mixed with protein solutions. Cu-free click chemistryreactions were performed in protein elution buffer immediatelyfollowing gel filtration (Superdex) or metal-ion affinityexchange chromatography (Ni-NTA), with the latter reactioncontaining 1 M imidazole. Reaction yields were independent ofthe buffer composition for reactions proceeding at roomtemperature (25 °C) in the presence of up to ∼100× molarexcess dye reactant. To monitor the reaction yield, mixtureswere snap-frozen in ethanol/dry ice solution to stop thereaction. For MALDI-TOF mass spectrometry analysis,unreacted dye was removed by methanol/chloroform extrac-tion. Labeled protein samples were analyzed by MALDI-TOFmass spectrometry, SDS-PAGE and fluorescence imaging usinga Typhoon scanner (GE healthcare) for cyanine dye-labeledproteins, or a Storm scanner (GE healthcare) for fluorescein-labeled proteins.

Fluorescence Anisotropy. A 26-base oligonucleotide(“target1”) encoding for a 9-base ZFP binding site andcontaining a fluorescein dye (6-FAM) at the 5′ terminus wasobtained from Integrated DNA Technologies (Coralville, IA).The oligonucleotide sequence for “target1” was: 5′-6-FAM-TATGGAACTCATCAGCCTCATTTCTA-3′, where the ZFPbinding site is shown as the underlined sequence. The “target1”oligo was converted to duplex DNA by hybridization with acomplementary oligonucleotide, wherein the target andcomplementary strands were annealed by elevating thetemperature to 95 °C for 5 min, followed by gradual coolingto room temperature. Fluorescence measurements wereperformed using a Cary Eclipse Fluorescence Spectrophotom-eter (Varian) equipped with excitation and emission polarizers,automated temperature control, and magnetic solution stirrers.ZFP-DNA binding was performed at 20 °C in the followingreaction buffer: 20 mM Tris/Tris HCl (pH 7.9), 100 mM

Bioconjugate Chemistry Article

dx.doi.org/10.1021/bc300262h | Bioconjugate Chem. 2012, 23, 1891−19011892

NaCl, 0.1 mM ZnCl2, 1 mM TCEP, 20% glycerol, 0.05%Nonidet-40, 2 ng/mL dI-dC, and 50 μg/mL BSA. The optimalbuffer composition for DNA binding was determined over thecourse of several control experiments. Fluorescence anisotropywas measured in solutions containing 20 nM 6-FAM labeledtarget1-dsDNA (duplex DNA) using bandwidths of 20 nm.Fluorescence anisotropy (r) was calculated by

=− ×+ ×

=rI G I

I G IG

II2

withVV VH

VV VH

HV

HH (1)

where the instrument G factor is defined as the ratio ofdetection sensitivities for vertically and horizontally polarizedlight in the system, and H and V refer to the horizontal andvertical positions, respectively, of the excitation (first subscript)and emission (second subscript) polarizers.27 For thesemeasurements, the instrument G factor was carefully measured,and we observed that glycerol did not affect fluorescencemeasurements. For data analysis, a nonlinear regression methodwas used to fit the experimental data. In addition, anisotropydata was analyzed using a Hill equation to characterize theextent of cooperative protein binding.28 Normalized anisotropyvalues were fit to a Hill equation

θ =+K

[protein][protein]

n

nD (2)

where θ is the fraction of occupied DNA bound to protein, KDis the dissociation constant, and n is the Hill coefficientdescribing the extent of cooperative binding.FRET Measurements. A 25-base oligonucleotide (“tar-

get2”) encoding for a 9-base ZFP target DNA binding site andcontaining a Cy5 dye at the 5′ terminus was obtained fromIntegrated DNA Technologies. The oligo sequence for“target2” was 5′-Cy5-TATGGAACTCATCAGCCT-CATTTCT-3′, where the ZFP binding site is shown as theunderlined sequence. The “target2” oligo was converted toduplex DNA by hybridization with a complementaryoligonucleotide. Fluorescence measurements were performedat 20 °C using a Cary Eclipse Fluorescence Spectrophotometer.For FRET measurements, the ZFP-DNA binding buffer was 20mM Tris/Tris HCl (pH 7.9), 100 mM NaCl, 0.1 mM ZnCl2, 1mM TCEP, 20% glycerol, 0.05% Nonidet-40, 2 ng/mL dI-dC,and 50 μg/mL BSA. In addition, Cy3-DBCO-ZF2 protein wasprepared in binding buffer at 600 nM, and “target2” Cy5-DNAwas titrated to the desired final concentration. After incubatingthe reaction for 10 min at room temperature (25 °C),fluorescence spectra were measured. All samples were measured

in triplicate, and disposable cuvettes were used to minimizeresidual fluorescence from previous measurements.

Electrophoretic Mobility Shift Assay (EMSA). Protein−DNA binding was assayed by EMSA using a protocol aspreviously described.29 Briefly, an oligonucleotide (“target3”)encoding for the ZFP binding sequence and containing a Cy3dye at the 5′ terminus was used. The sequence for “target3” was5′-Cy3-AATATGGAACTCATCAGCCTCATTTCTAAA-TATGAAGAGTT-AAGACGTAATG′. The “target3” oligonu-cleotide was converted to duplex DNA by hybridization with acomplementary oligonucleotide. Alternatively, an amplicon(100 bp or 200 bp in length) containing the target bindingsequence was obtained by PCR amplification using phagelambda DNA as a template in a standard PCR reaction (SI).Following PCR, amplicons were purified by PCR cleanup(Qiagen). For EMSA, protein and DNA were mixed in desiredstoichiometric proportions and incubated in binding buffer atroom temperature (25 °C) for 20 min. Following incubation,the mixture was supplemented with DNA loading dye for gelelectrophoresis and run on an 8% polyacrylamide gel. Gels wereimaged using ethidium bromide (EtBr) staining and UVillumination for PCR amplified DNA or a Typhoon scanner forcyanine dye-labeled DNA.

Microarray Measurements. PEGylated glass slides wereprepared using PEG-biotin-NHS ester (MW 3500 g/mol) andmPEG-NHS ester (MW 5000 g/mol) as previouslydescribed.30 PEGylated glass slides were treated withprotein−DNA binding buffer, followed by incubation withneutravidin (0.5 mg/mL). Next, Cy3-biotin-DNA (1 μM inwater), which has the same sequence as “target3” DNA, wasprinted as 1 nL spots on neutravidin treated PEGylated slidesusing a TeleChem Stealth SMP3 pin (Perkin-Elmer) aspreviously reported.31 Briefly, the Cy3-biotin-DNA was dilutedto a final concentration of 1 μM in deionized water, and theDNA solution was printed as spots on the functionalized glassslides in duplicate. After printing, slides were incubated with theDNA solution spots for 5 min. Glass slides were rinsed 4× withbinding buffer to remove excess, unbound Cy3-DNA-biotinfrom the glass surface. Next, Cy5-ZFP (20 nM) in bindingbuffer solution was applied to Cy3-DNA surface array, followedby incubation for 5 min at a relative humidity of 55−60% atambient temperature 25 °C. Glass slide arrays were rinsed withDNA−protein binding buffer to remove unbound Cy5-ZFPlabeled proteins. DNA−protein complexes were imaged using aGenePix Pro 6.5 double laser scanner at wavelengths of 535 and635 nm (Axon Instruments). For imaging, the photomultipliertube voltage was set to 550 V, and the gain was set to 30%. Fordisplay, pseudocolors were applied to Cy3 and Cy5 (green and

Table 1. Summary of Engineered Zinc Finger Proteins Used in This Worka

ZFPprotein molecular weight

(Da)dissociation constant, Kd

(nM)number of zinc finger

modulesfusion protein (post

purification)number ofUAAs location of UAAs

ZFP1 11040 158 3 None 1 N-terminusZFP2 38944 191 3 Cerulean (N-terminal

fusion)4 Cerulean FP

domainZFP3 39505 167 3 mCherry (N-terminal

fusion)11 mCherry FP

domainZFP4 39505 175 3 mCherry (C-terminal

fusion)11 mCherry FP

domainZFP5 8716 ∼2000 2 None 1 N-terminusZFP6 12040 180 3 None 0 N/A

aDissociation constants (Kd) were measured using fluorescence anisotropy (Materials and Methods). The 9-base DNA binding sequence for three-finger ZFPs (ZFP1-4 and ZFP6) was 5′-ATCAGCCTC-3′. Schematics of the protein constructs are shown in SI Scheme S1.



red, respectively), and pseudocolored images were mergedusing Image J (NIH) to show DNA−protein colocalization asoverlapping colors in composite images.

■ RESULTS AND DISCUSSIONEngineering Zinc Finger Proteins for Specific DNA

Binding. Recent advances in ZFP technology have enabled thedesign of chimeric ZFPs with the ability to bind to targetedDNA sequences. Individual zinc finger modules can be linkedin tandem arrays, thereby enabling assembled ZFPs to bind torecognize DNA sequences of variable length. Using thisapproach, we cloned several multifinger ZFPs using a modularassembly method (Table 1).7 A homology model structure forone of the engineered three-finger ZFPs (ZFP1) studied in thiswork is illustrated in Figure 1a, which shows that zinc fingersare stabilized by coordination of a zinc ion in a cysteine-histidine structural binding motif.In an initial set of experiments, we expressed and purified

several of the engineered ZFPs using natural amino acids. TheDNA binding capacity for a three-finger ZFP (ZFP6) wascharacterized using an electrophoretic mobility shift assay(EMSA), as shown in Figure 1b. In this experiment, purifiedZFP was mixed with equimolar amounts of DNA oligonucleo-tides encoding for the 9-base pair recognition sequence andnonspecific DNA without the target sequence. Electrophoreticshift assays clearly show that target DNA bands are up-shifteddue to the formation of a specific protein−DNA complex(dissociation constant Kd = 180 nM), whereas nonspecificDNA did not yield a significant shift. As a control experiment,we determined that a single base pair mismatch in the targetDNA recognition sequence prevents specific binding of ZFPsto target sequences (SI Figure S1). Based on these results, weconcluded that chimeric ZFPs were capable of binding to targetDNA with high affinity and specificity.Residue Specific Noncanonical Amino Acid Incorpo-

ration. Specific labeling of ZFPs with fluorescent tags forquantitative binding assays presents several challenges. Tospecifically label ZFPs with fluorescent tags, we pursued threedistinct approaches: (1) introduction of mutant cysteineresidues as reactive sites, (2) labeling via fluorescent proteinfusion domains, and (3) residue-specific substitution of aminoacids. In the first approach, we engineered cysteine residues atthe N- or C-terminus of a three-finger ZFP, followed by

reaction with a maleimide-conjugated fluorescent dye. Weobserved that the DNA binding capacity for ZFPs labeled usingthis strategy was completely abolished, and we conjecture thatthe natural Cys2-His2 structural motif was perturbed due tocross-reactivity between the labeling probe and natural cysteinemoieties using this strategy.32 In a second approach, wegenerated N- or C-terminal fusions between ZFPs andmCherry or Cerulean fluorescent proteins. We found that thebinding equilibrium of ZFP-fusion proteins is comparable tothe parent ZFPs (SI Figure S2), and ZFPs with N- or C-terminal fusions exhibited similar specificity to target DNAcompared to unlabeled ZFPs. In a series of control experiments,we verified that fluorescent proteins alone did not confer DNA-binding properties to ZFP-fusion proteins. However, incontrast to small molecule organic dyes, fluorescent proteinsgenerally exhibit lower levels of brightness and photostability,which could potentially complicate quantitative fluorescencebinding experiments or single molecule detection assays.In a third approach, we pursued a bioorthogonal labeling

strategy for ZFPs based on metabolic incorporation ofunnatural amino acids (UAAs), followed by conjugation oforganic dyes using azide−alkyne cycloaddition via clickchemistry. For UAA incorporation into recombinant proteins,we adopted the method previously developed by the Tirrellgroup, wherein metabolic incorporation of UAAs is accom-plished by universal replacement of one natural amino acid witha non-natural analogue.17,33 In some cases, universal sub-stitution of methionine can be used for protein labeling becausethe occurrence of methionine is low relative to other aminoacids, yet most proteins have at least one methionine at the N-terminus. In E. coli, wild-type aminoacyl tRNA synthetases cancharge cognate tRNAs with methionine analogues such asazidohomoalanine (AHA), and the ribosomal machineryuniversally incorporates the UAAs into growing protein chainsduring translation.17,34 To ensure universal replacement ofnatural amino acids such as methionine with UAAs, methionineauxotroph strains of E. coli are often employed as proteinexpression platforms. Alternative strategies based on suppres-sion of nonsense mutations at amber codons have also beendeveloped for site-specific incorporation of UAAs intorecombinant proteins.35,36 In contrast to universal residue-specific replacement methods, site-specific incorporationmethods can be used to introduce UAAs into specific target

Figure 1. Engineered ZFPs bind target DNA in a sequence-specific fashion. (a) Homology model structure of a modular ZFP consisting of a tandemarray of three repeating zinc finger modules, each containing a zinc ion in a Cys2-His2 complex (yellow). (b) Electrophoretic gel mobility shift assay(EMSA) shows that a three-finger ZFP (ZFP6) binds specifically to target DNA. The bound fraction was quantified to determine the equilibriumbinding constant Kd. (inset) EMSA image shows that ZFP6 forms a specific complex with target DNA in the presence of nonspecific competitorDNA.



locations within proteins, which minimizes the potential forperturbations to natural protein structure. From thisperspective, site-specific incorporation of UAAs is a versatileapproach for protein modification. However, the residue-specific methionine replacement strategy can be pursued whenthe substituted residue is confined to a well-defined targetsite.37

The vast library of zinc finger modules provides ampleflexibility to engineer ZFPs containing only a single methionineresidue at the protein N-terminus, distal from the DNA bindingregion of the module. The modular assembly method allows forfacile metabolic labeling of ZFPs with UAAs, because the libraryof zinc finger modules is degenerate for any given three-basesequence of DNA.6,25 Therefore, we selected ZFP moduleswithout methionine codons from the Zinc Finger Consortiumlibrary to exclude methionine residues from the DNA bindingregion, thereby preserving the DNA binding activity of thelabeled protein. We used azidohomoalanine (AHA) as amethionine surrogate in E. coli methionine auxotroph strains(Scheme 1a), thereby replacing all methionine residues inrecombinant proteins with AHA. Tandem arrays of zinc fingerswere constructed consisting of two or three tandem modules toallow for specific binding to target DNA sequences (Table 1).Metabolic incorporation of UAAs into ZFPs was carried out

using the E. coli methionine auxotroph strain M15MA or B834as a protein expression host. We observed that the methionine

auxotroph strain M15MA efficiently incorporates AHA intoexpressed proteins in growth medium containing AHA,generally resulting in high levels of incorporation of AHAresidues into recombinant proteins.37 In the absence ofmethionine or AHA, E. coli cell cultures exhibited extremelypoor cell growth over the course of several hours. Prior toinducing protein expression, basal expression was minimal dueto the strong lacIq repressor module in the pQE80L expressionplasmid. Following protein expression, ZFPs harboringmetabolically incorporated UAAs were purified using metal-ion affinity chromatography (Ni-NTA resin), and proteinsamples were carried forward to click chemistry reactions.In the absence of expression tags, three-finger ZFPs generally

exhibited low expression yields in E. coli methionine auxotrophstrains used in this work (B834, M15MA, or T7 Crystal).Without expression tags, the majority of three-finger ZFPsappeared in the insoluble fraction of cell cultures whenexpressed in methionine auxotroph strains. Moreover, wefound that purification of ZFPs from the insoluble fraction ofcell cultures was not feasible due to inefficient refolding ofdenatured ZFPs.38 To enhance the expression yields ofengineered ZFPs in methionine auxotroph strains, we usedfluorescent proteins (FPs) as expression tags (Table 1), similarto previous studies using expression tags that do not interferewith DNA binding.39,40 We observed substantial improvementsin expression yields for ZFPs with N- or C-terminal FP fusions,

Scheme 1. Chemical Structures of (a) Methionine (Met) and the Methionine Analogue Azidohomoalanine (AHA), (b) Cy5-Alkyne, and (c) Cy5-DBCO

Scheme 2. Schematic of Click Chemistry-Mediated Covalent Conjugation of Fluorescent Dyes to ZFPs ContainingMetabolically-Incorporated Azidea

a(a) Copper-mediated click chemistry was used to conjugate dyes containing linear alkynes with ZFPs; however, the ligand TBTA induced proteindenaturation. (b) Copper-free click chemistry was used to conjugate dyes containing DBCO to ZFPs, thereby enabling ZFPs to retain sequence-specific DNA binding activity.



with a significant increase in the amount of expressed ZFP inthe soluble fraction of cell cultures. In some cases, ZFP-FPfusion proteins were used for equilibrium DNA binding assays(SI Figure S2). However, for most three-finger ZFP constructs,we inserted a thrombin cleavage site between genes encodingfor FPs and ZFP, thereby enabling the FP expression tags to becleaved off of the ZFPs during purification after proteinexpression (SI Scheme S1). Using this approach, zinc fingermodules can be cleaved from ZFP-FP constructs while thefusion protein is bound to the Ni-NTA affinity column, therebygenerating high yields of three-finger ZFPs containing UAAs.We used biotinylated thrombin for the cleavage reaction, whichenables facile removal of thrombin from ZFP samples usingmagnetic bead-based separation with streptavidin-coated micro-spheres. Purified ZFP was assayed using electrospray ionizationmass spectrometry, which revealed highly purified protein athigh yields (SI Figure S3).The expression tag method described in this work provides

several advantages for UAA incorporation using N-terminalmethionine replacement. In prokaryotes, it is well-known thatthe N-terminal methionine is typically excised in thepostexpression modification by methionine aminopeptidase(MetAP).41 In a similar vein, MetAP can also remove N-terminal AHA, depending on the residue following the initialAHA.37 Therefore, N-terminal expression tags intrinsicallyprotect AHA residues from cleavage without the need foradditional protein design or cloning steps.Fluorescent Probe Conjugation using Click Chemistry

with Linear Alkynes and Dibenzocyclooctyne. Followingexpression and purification, we labeled ZFPs with fluorescentdyes using two distinct strategies: Cu-mediated click chemistryusing Cy5-conjugated with a propargyl group and Cu-freestrain promoted [3 + 2] cycloaddition using Cy5-conjugateddibenzocycloocyne (DBCO), as denoted in Scheme 2. In thecase of Cu-mediated click chemistry, we observed that additionof copper sulfate or the triazole ligand tris[(1-benzyl-1H-1,2,3-triazol-4yl)methyl] amine (TBTA), which is commonly used toaccelerate Cu-mediated click cyclizations, resulted in proteinprecipitation and loss of protein function (SI Figure S7).Precipitated ZFPs showed a complete loss of DNA-bindingactivity (Figure 2), and precipitation of zinc finger proteins was

irreversible. We attempted to resolubilize ZFPs using an excessamount of zinc ions in solution; however, the DNA bindingactivity of ZFP could not be recovered.Zinc ion binding is a key requirement for efficient folding

and activity of Cys2-His2 ZFPs because zinc finger modulespossess β β α structures, which are stabilized by zinc chelation.Dissociation constants for zinc binding are typically in the rangeof 10−9 to 10−11 M, whereas ligands such as EDTA bindextremely tightly with dissociation constants of approximately

10−16 M.42,43 Only a few minutes of exposure to strongchelating reagents such as EDTA will dissociate zinc ions fromzinc finger proteins,44 and loss of zinc ions induces structuralperturbations. In zinc finger modules, consistent contacts aremade between the DNA backbone and histidine residues.Specific contacts may be especially important in controllingdocking arrangements, because they couple the structural coreof the zinc finger directly to the sugar phosphate backbone ofDNA. Furthermore, metal ion substitution can inducesignificant structural changes in ZFPs.43,45 It is well-knownthat zinc finger modules can associate with Cu2+, Ni2+, or Co2+

ions, and the binding affinity is comparable to that of Zn2+.However, the binding of divalent ions other than zinc candisrupt the β β α structure of zinc finger modules, and the DNAbinding ability of ZFPs cannot be restored upon refolding.46

Therefore, the addition of Cu2+ as a catalyst in Cu-mediatedclick reactions has the potential to induce structuralperturbations in ZFPs.Copper-mediated click chemistry requires a Cu chelating

reagent to increase reaction rates and yields. TBTA has suitablechelating capacity for copper(II) and is widely used for copper-mediated click reactions. Upon addition of TBTA to a solutionof copper(II), the faint blue color immediately turns to a darkerblue, which indicates copper chelation (SI Figure S7).Moreover, we observed that TBTA can strongly chelatezinc(II), as well as copper(II), as evidenced by observing acolor change (green to blue-green) that occurs when zinc ionsare added to solutions containing TBTA and Cu2+ (SI FigureS7c). Under typical copper-mediated click reaction conditionsinvolving ZFPs, zinc ions are present at relatively highconcentrations (∼mM), in vast excess relative to the ZFP insolution. Therefore, it is likely that addition of TBTA as acatalytic ligand for copper-mediated click reactions will bebound by free zinc ions, rather than copper, which will reducethe efficiency of TBTA on the copper-mediated click reactions.In addition, we observed that addition of copper sulfate tosolutions containing ZFPs resulted in immediate proteinprecipitation (SI Figure S7a), which indicates that Cu2+ ionscan interact with ZFPs in undesired ways. We further foundthat TBTA or copper ions, alone or together, can induce theprecipitation of ZFPs (SI Figure S7b). We found that theprecipitation of ZFPs was irreversible, because it could not bereversed by extensive dialysis against a zinc-rich solution.Although ZFP recovery after denaturation has rarely been

successful,47 a limited number of wild-type ZFPs have beendemonstrated to refold after denaturation and extensivepurification. However, protein refolding is time-consuming,labor-intensive, and generally a low-yield process.48 For“synthetic” or engineered proteins such as ZFPs constructedby modular assembly methods, the overall protein fold andcorresponding energy minimization may be suboptimal due tothe “cut and paste” assembly method of the individual zincfinger modules, thereby making it very challenging to efficientlyrefold zinc finger proteins into the native β β α proteinarchitecture stabilized by coordination of a zinc ion. Toovercome the problems associated with ZFP denaturation dueto loss of zinc ions, we used Cu-free strain promoted [3 + 2]cycloaddition, which does not require catalysts or ligands forprotein labeling.49,50

To demonstrate ZFP labeling using UAAs and Cu-free clickchemistry, ZFPs were expressed and purified, and Cu-free clickchemistry was used to label azide-modified ZFPs withfluorescein, Cy3, and Cy5-conjugated dibenzocyclooctyne

Figure 2. Gel electrophoretic mobility shift assay (EMSA) for a three-finger ZFP. (a) Under native conditions, ZFPs clearly form a specificcomplex with target DNA. (b) Upon addition of the triazole ligandTBTA and copper ions, ZFPs no longer form a specific complex withDNA, and the DNA binding activity is ablated.



(DBCO).51 Click reactions were monitored using MALDI-TOF and ESI (Q-TOF) mass spectrometry analysis andfluorescence-based gel imaging (Figure 3). More than 70% of

the monofunctionalized three-finger ZFP was labeled with Cy5using Cu-free click chemistry, as revealed by MALDI-TOFanalysis (Figure 3c). The labeling reaction appeared to saturatein excess of 20:1 molar ratio of DBCO to ZFP followingovernight reaction at 4 °C, as shown in Figure 3d. In addition,we observed no cross reactivity between natural amino acidsand fluorescein−DBCO, according to MALDI-TOF analysis,even in the presence of ∼100× molar excess dye to protein inthe reaction mixture, which is a clear advantage ofbioorthogonal labeling chemistries. Bioorthogonal labelingstrategies generally ensure highly specific labeling with minimalperturbations to protein structure, provided that the target

labeling site is carefully selected based on protein structure/function considerations. In addition, Cu-free click chemistryalso provided flexibility in reaction conditions for proteinlabeling. ZFPs labeled with fluorescein−DBCO at 4 °Covernight compared favorably with protein samples labeled25 °C for two hours in terms of reaction yields, though highertemperatures generally showed slightly improved reactionyields, as shown in SI Figure S5. Over the course of twohours, reactions carried out at 55 °C resulted in ∼85% yield forZFP5, whereas the reactions carried out at 25 °C resulted in∼63% yield. Moreover, we observed Cu-free click labeling ofZFPs to proceed with high yields at 4 °C with overnightincubation, giving yields comparable to labeling reactions at twohours at 25 °C. In order to preserve the stability of purifiedtranscription factor proteins such as ZFPs, it may be preferableto perform labeling reactions at 4 °C.

Binding Assays for ZFPs Labeled by Cu-Free StrainPromoted [3 + 2] Cycloaddition. We used fluorescenceanisotropy to measure the binding isotherms of wild-type ZFP1(Met-ZFP1), azide-labeled ZFP1 (AHA-ZFP1), and Cy5-DBCO-clicked ZFP1 (Cy5-ZFP1), as shown in Figure 4a.Metabolically labeled ZFP1 (AHA-ZFP1) and Cy5-DBCO-clicked ZFP (Cy5-ZFP1) exhibit nearly identical DNA bindingproperties compared to wild-type Met-ZFP1, as revealed byfluorescence anisotropy. The dissociation constants Kdcalculated from fluorescence anisotropy experiments were asfollows: 158 nM, 184 nM, and 123 nM for Met-ZFP1, AHA-ZFP1, and Cy5-ZFP1, respectively. Therefore, the dissociationconstants were nearly the same for these three ZFP1 proteinspecies, within experimental error, based on a Hill model fittingequation (Materials and Methods). The measured values ofdissociation constants for engineered ZFPs are in agreementwith previously published data for ZFPs.40

We also investigated the DNA binding properties of labeledZFPs using gel electrophoretic mobility shift assays (EMSA). Inthese experiments, Cy5-clicked zinc finger proteins (Cy5-ZFP2) were bound to Cy3-labeled duplex DNA containing the9-base “target2” DNA site for the three-finger tandem array ofassembled ZFPs (Materials and Methods). EMSA revealedsequence-specific DNA binding of Cy5-ZFP2 to target Cy3-DNA in the presence of nonspecific competitor DNA (Figure4b), which agrees with fluorescence anisotropy data for thesame proteins (SI Figure S4). In these experiments, we used afixed concentration of DNA (1 nM) and varied theconcentration of labeled ZFP, which revealed the specificbinding of ZFPs protein to target DNA. Unlike fluorescenceanisotropy, EMSA is a quasi-steady-state method for assayingDNA binding; however, binding isotherms calculated fromEMSA data were comparable to fluorescence anisotropyexperiments, as shown in Figure S4. At a DNA:AHA-ZFP2ratio of ∼1:60, nearly all DNA is bound to the zinc fingerprotein, as shown in Figure 4b. In addition, EMSA shows adistinct single band corresponding to the bound ZFP-DNAcomplex, which is suggestive of a well-defined protein−DNAcomplex in the bound state. Overall, these experimentsdemonstrate the utility of specific labeling of three-fingerZFPs with conserved DNA binding capacity of transcriptionfactor proteins to target DNA sites.

Solution-Based FRET Binding Assays and MicroarrayAssays for Labeled ZFPs. To demonstrate the utility oflabeling ZFPs using UAAs and Cu-free click chemistry for invitro DNA binding assays or single molecule fluorescenceexperiments, we studied the binding between Cy3-clicked ZFP2

Figure 3. Copper-free click chemistry for specific labeling of ZFPswith fluorescent tags. (a) Fluorescent gel image and (b) SDS-PAGEanalysis for a gel showing dye conjugation to three-finger ZFPsfollowing copper-free click reactions. Lane 1, Met-ZFP1; Lane 2,AHA-ZFP1; Lane 3, Cy5-clicked-ZFP1; Lane 4, Cy3-clicked-ZFP1;Lane 5, protein MW ladder. (c) Protein mass spectrophotometricanalysis (MALDI-TOF) of labeled ZFPs. AHA-ZFP1 (MW 11 035Da) was reacted with Cy5-DBCO (MW 914 Da) using copper-freeclick chemistry, and the shift in mass confirms conjugation of a singleCy5 dye to ZFP1. Reaction yield calculated from the peak height is72%. (d) Fluorescent gel showing that dye loading on ZFP5 increasesupon increasing the stoichiometric ratio between fluorescein-DBCOand ZFP5 in the click reaction (1 h at 25 °C). An equal mass ofprotein was loaded in each lane.



(Cy3-ZFP2) and Cy5-labeled target DNA (Cy5-DNA,“target2” DNA) using Forster resonance energy transfer(FRET). We designed a FRET dye pair using a Cy3-donor/Cy5-acceptor located on the labeled ZFP and target DNA,respectively. The binding site for DNA was designed to belocated at a distance of 5 nm from DNA terminus containingthe Cy5 dye molecule, which is within the Forster radius of 56Å for a Cy3/Cy5 dye pair.27 In these experiments, we used afixed concentration of Cy3-ZFP2 in solution, and we varied theamount of Cy5-DNA while monitoring the FRET signal(Figure 5a). Samples were illuminated using an excitationwavelength of 520 nm to excite the Cy3 donor dye. Uponaddition of Cy5-acceptor DNA to solution, we observed adecrease in Cy3 emission and a concomitant increase in Cy5emission arising from FRET between the Cy3/Cy5 dye pairwithin the ZFP-DNA complex. FRET signals reached amaximum for solutions containing a 7.5:1 ratio of ZFP/DNA(600 nM Cy3-ZFP2 with 80 nM Cy5-DNA). As a controlexperiment, 80 nM Cy5-DNA alone did not show anyconsiderable emission at 670 nm in the absence of Cy3-ZFP2. Overall, the FRET experiments reported here serve as

proof-of-principle demonstration for studying dye-labeledtranscription factors using fluorescence-based methods, whichwill enable kinetic binding experiments or transcription factorlocalization studies via bulk-phase or single moleculefluorescence imaging.Inspired by equilibrium FRET measurements, we monitored

the dynamic dissociation of ZFPs to target DNA, as shown inFigure 5b. Initially, an excess amount of unlabeled target DNAwas added to a solution of Cy3-ZFP2 and Cy5-DNA, and thedecrease in FRET was monitored over time. The fluorescencesignal was fit to a single exponential decay, and we determineda characteristic time for dissociation as t1/2 = 57 s. Kineticbinding/unbinding measurements typically require specializedequipment such as surface plasmon resonance (Biacore) orrelated techniques.52 However, in our work, direct labeling oftranscription factors allows for these measurements to beobtained using standard fluorescence spectrophotometers.Several surface-based methods are available to study

protein−DNA or protein−protein binding interactions, includ-ing label-free surface plasmon resonance, surface-basedfluorescence detection using DNA microarrays and single

Figure 4. DNA binding assays for labeled ZFPs. (a) Fluorescence anisotropy shows that fluorophore-conjugated ZFPs are competent for sequence-specific DNA binding. Three-finger ZFPs were studied before and after click reactions, including the wild-type protein (Met-ZFP1, square),metabolically labeled azide containing protein (AHA-ZFP1, circle), and Cy5-clicked protein (Cy5-ZFP1, triangle). (b) Gel electrophoretic mobilityshift assays (EMSA) show that labeled ZFPs effectively bind to target DNA in sequence-specific manner. Three-finger ZFPs (ZFP2) labeled withCy5 (red) bind to Cy3-labeled DNA (green), and the protein−DNA complex (yellow) migrates through the gel with a clear mobility shift.

Figure 5. Solution-based FRET and surface-based DNA binding assays for labeled ZFPs. (a) Solution-based FRET reveals specific bindinginteractions between Cy3 dye labeled ZFPs and target Cy5-DNA. The concentration of Cy3-ZFP2 was maintained at 600 nM, and the Cy5-DNAconcentration was varied. (b) Unbinding kinetics for a bound complex of Cy5-ZFP2 and Cy3-DNA measured by FRET. (c) Microarraysdemonstrate colocalization between Cy5-labeled ZFP2 and surface-immobilized Cy3-DNA encoding for the target DNA binding sequence.



molecule fluorescence methods. Fluorescence-based singlemolecule techniques typically require surface passivation withPEG or polyelectrolyte multilayers, followed by immobilizationof the target biomolecule to a glass surface. Here, we studiedZFP-DNA binding interactions on a glass surface passivatedwith poly(ethylene glycol) (PEG) using specific chemistry forbiomolecule attachment (Figure 5c). In this way, we adaptedDNA binding studies involving labeled ZFPs to assays involvingsolid supports, such as general microarray-based screening toolsfor proteomics or genomics. We used a microarray system fordetection of ZFP-DNA binding that utilizes similar surfacechemistry for surface-immobilized single molecule fluorescenceexperiments.In this experiment, glass slides were first pretreated with PEG

to minimize nonspecific protein binding. Next, Cy3-DNA(green) was printed on the PEGylated glass surface within aconfined area, and Cy5-ZFP2 (red) was applied to the DNA-printed glass slide, incubated for 5 min, and copiously rinsed toremove unbound protein. Overlaid fluorescence images of Cy3-DNA-biotin and Cy5-ZFP2 show specific binding interactionsbetween DNA and ZFP, as revealed by a distinctive yellowcolor. In particular, the ZFP binding pattern (red) preciselycolocalizes with the DNA printing area (green), with the lowbackground being the result of the hydrophilic PEG-treatedsurface.

■ CONCLUSIONSZFPs comprise a broad class of transcription factor (TF)proteins. TFs are fundamental elements of genetic control andplay a key role in transcriptional regulation in living organisms.In this work, we developed an efficient method for specificlabeling of ZFPs using metabolic incorporation of UAAs,followed by copper-free click chemistry. Modular ZFPscontaining tandem arrays of three zinc fingers were successfullyexpressed using a universal amino acid replacement strategy bysubstituting azide-containing AHA for natural methionine inauxotroph strains. We demonstrate residue-specific labelingwith dye-conjugated DBCO using copper-free click chemistry,generally achieving reaction yields in excess of ∼70%.Moreover, the specific DNA binding activity was retained forZFPs labeled via copper-free click chemistry. To our knowl-edge, this work represents an important advance in residue-specific labeling of ZFPs, which can be useful conducting invitro screens for assembled ZFPs via solution-based FRETassays or microarray-based hybridization assays. Furthermore,this work will open new avenues for the molecular-levelexploration of dynamic processes involving ZFP binding insolution-based assays and in surface-based fluorescence experi-ments using surface-immobilized protein strategies.Bioorthogonal chemistries such as copper-catalyzed azide−

alkyne cycloaddition broaden the toolset for investigation ofprotein−protein or protein−DNA interactions using solution-phase or surface-based assays. For microarrays or surface-basedbiosensing assays, biomolecules such as DNA or proteins aretypically linked to surfaces using specific chemical attachment,and the binding partners are introduced in solution. Manysurface-based assays rely on biotin−avidin linkage chemistry toimmobilize biomolecules, which strongly motivates the need foradditional orthogonal chemistries for fluorescent labeling ofbiomolecules. In the case of ZFPs, copper-mediated clickchemistry completely abolishes the binding activity of zincfingers to target DNA by disruption of the Cys2-His2 zinc ioncomplex caused by additives in the reaction, such as copper and

chelating reagent. On the other hand, copper-free clickchemistry provides an efficient strategy for residue-specificlabeling of ZFP by incorporation of unnatural amino acids andcopper-free click chemistry, which circumvents cross-reactivitywith natural amino acid residues and retains protein function.Overall, we anticipate that this labeling strategy will be useful

in quantifying transcription factor dynamics using fluorescence-based methods, which will motivate new studies in genenetwork dynamics or regulation.11 Until now, the study oftranscription factor localization and dynamics has mainly reliedon using fluorescent proteins as labeling probes. Using UAAsand copper-free click chemistry, ZFPs can be labeled with smallmolecule organic dyes for improved fluorescence brightness,which will enable quantitative studies of transcription factorbinding kinetics.53 Finally, the labeling method described in thiswork provides a general framework for labeling metalloproteinscontaining iron, zinc, or copper cofactors for mechanisticstudies of protein function.

■ ASSOCIATED CONTENT*S Supporting InformationExperimental data. This information is available free of chargevia the Internet at http://pubs.acs.org/

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected] authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by a Packard Fellowship from theDavid and Lucile Packard Foundation and an NIH Pathway toIndependence Award (4R00HG004183-03 to C.M.S.) and anNIH MERIT Award (5R37DK-15556 to J.A.K.). We thankProfessor A. J. Link (Princeton University) for generouslyproviding E. coli auxotroph strain M15MA.

■ REFERENCES(1) Urnov, F., Miller, J., Lee, Y., Beausejour, C., Rock, J., Augustus, S.,Jamieson, A., Porteus, M., Gregory, P., and Holmes, M. (2005) Highlyefficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646−651.(2) Hockemeyer, D., Soldner, F., Beard, C., Gao, Q., Mitalipova, M.,DeKelver, R., Katibah, G., Amora, R., Boydston, E., and Zeitler, B.(2009) Efficient targeting of expressed and silent genes in humanESCs and iPSCs using zinc-finger nucleases. Nat. Biotechnol. 27, 851−857.(3) Rebar, E., and Pabo, C. (1994) Zinc finger phage: affinityselection of fingers with new DNA-binding specificities. Science 263,671.(4) Bibikova, M., Beumer, K., Trautman, J., and Carroll, D. (2003)Enhancing gene targeting with designed zinc finger nucleases. Science300, 764.(5) Hockemeyer, D., Soldner, F., Beard, C., Gao, Q., Mitalipova, M.,DeKelver, R., Katibah, G., Amora, R., Boydston, E., and Zeitler, B.(2009) Efficient targeting of expressed and silent genes in humanESCs and iPSCs using zinc-finger nucleases. Nat. Biotechnol. 27, 851−857.(6) Kim, H., Lee, H., Kim, H., Cho, S., and Kim, J. (2009) Targetedgenome editing in human cells with zinc finger nucleases constructedvia modular assembly. Genome Res. 19, 1279.(7) Wright, D., Thibodeau-Beganny, S., Sander, J., Winfrey, R., Hirsh,A., Eichtinger, M., Fu, F., Porteus, M., Dobbs, D., and Voytas, D.



http://pubs.acs.org/

mailto:[email protected]

(2006) Standardized reagents and protocols for engineering zinc fingernucleases by modular assembly. Nat. Protocols 1, 1637−1652.(8) Maeder, M. L., Thibodeau-Beganny, S., Sander, J. D., Voytas, D.F., and Joung, J. K. (2009) Oligomerized pool engineering (OPEN):an’open-source’protocol for making customized zinc-finger arrays. Nat.Protocols 4, 1471−1501.(9) Sander, J. D., Dahlborg, E. J., Goodwin, M. J., Cade, L., Zhang, F.,Cifuentes, D., Curtin, S. J., Blackburn, J. S., Thibodeau-Beganny, S.,and Qi, Y. (2010) Selection-free zinc-finger-nuclease engineering bycontext-dependent assembly (CoDA). Nat. Methods 8, 67−69.(10) Joung, J., Ramm, E., and Pabo, C. (2000) A bacterial two-hybridselection system for studying protein DNA and protein-proteininteractions. Proc. Natl. Acad. Sci. U.S.A. 97, 7382.(11) Wang, Y., Guo, L., Golding, I., Cox, E. C., and Ong, N. (2009)Quantitative transcription factor binding kinetics at the single-molecule level. Biophys. J. 96, 609−620.(12) Carroll, D., Morton, J., Beumer, K., and Segal, D. (2006)Design, construction and in vitro testing of zinc finger nucleases. Nat.Protocols 1, 1329−1341.(13) Chen, I., and Ting, A. Y. (2005) Site-specific labeling of proteinswith small molecules in live cells. Curr. Opin. Biotechnol. 16, 35−40.(14) Lee, Y. M., and Lim, C. (2011) Factors controlling the reactivityof zinc finger cores. J. Am. Chem. Soc.,.(15) Ahel, I., Ahel, D., Matsusaka, T., Clark, A. J., Pines, J., Boulton,S. J., and West, S. C. (2008) Poly (ADP-ribose)-binding zinc fingermotifs in DNA repair/checkpoint proteins. Nature 451, 81−85.(16) Aldovini, A., and Young, R. A. (1990) Mutations of RNA andprotein sequences involved in human immunodeficiency virus type 1packaging result in production of noninfectious virus. J. Virol. 64,1920−1926.(17) Link, A., and Tirrell, D. (2003) Cell surface labeling ofEscherichia coli via copper (I)-catalyzed [3+ 2] cycloaddition. J. Am.Chem. Soc. 125, 11164−11165.(18) Kolb, H., Finn, M., and Sharpless, K. (2001) Click chemistry:diverse chemical function from a few good reactions. Angew. Chem.,Int. Ed. 40, 2004−2021.(19) Meldal, M., and Tornoee, C. (2008) Cu-Catalyzed azide- alkynecycloaddition. Chem. Rev. 108, 2952−3015.(20) Kolb, H., Finn, M., and Sharpless, K. (2001) Click chemistry:diverse chemical function from a few good reactions. Angew. Chem.,Int. Ed. 40, 2004−2021.(21) Natarajan, A., Du, W., Xiong, C., DeNardo, G., DeNardo, S., andGervay-Hague, J. (2007) Construction of di-scFv through a trivalentalkyne azide 1, 3-dipolar cycloaddition. Chem. Commun. 2007, 695−697.(22) Baskin, J. M., Prescher, J. A., Laughlin, S. T., Agard, N. J., Chang,P. V., Miller, I. A., Lo, A., Codelli, J. A., and Bertozzi, C. R. (2007)Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl.Acad. Sci. U.S.A. 104, 16793.(23) Chang, P. V., Prescher, J. A., Sletten, E. M., Baskin, J. M., Miller,I. A., Agard, N. J., Lo, A., and Bertozzi, C. R. (2010) Copper-free clickchemistry in living animals. Proc. Natl. Acad. Sci. U.S.A. 107, 1821−1826.(24) Ausubel, F. M., Brent, R., Kingston, R., Moore, D. D., Seidman,J. G., Smith, J. A., Struhl, K. (2011) Current Protocols in MolecularBiology, Wiley.(25) Wright, D., Thibodeau-Beganny, S., Sander, J., Winfrey, R.,Hirsh, A., Eichtinger, M., Fu, F., Porteus, M., Dobbs, D., and Voytas,D. (2006) Standardized reagents and protocols for engineering zincfinger nucleases by modular assembly. Nat. Protocols 1, 1637−1652.(26) Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD: visualmolecular dynamics. J. Mol. Graphics 14, 33−38.(27) Lakowicz, J. R. (2006) Principles of Fluorescence Spectroscopy, 3rded., Springer, New York.(28) Barbara, A. L., and Owen, C. T. M. (2009) Rapid method formeasuring DNA binding to protein using fluorescence anisotropy. Nat.Protocols,.

(29) Hellman, L. M., and Fried, M. G. (2007) Electrophoreticmobility shift assay (EMSA) for detecting protein-nucleic acidinteractions. Nat. Protocols 2, 1849−1861.(30) Selvin, P., and Ha, T. (2008) Single-molecule techniques: alaboratory manual, Cold Spring Harbor Laboratory Press.(31) Kim, S. H., Tamrazi, A., Carlson, K. E., Daniels, J. R., Lee, I. Y.,and Katzenellenbogen, J. A. (2004) Estrogen receptor microarrays:subtype-selective ligand binding. J. Am. Chem. Soc. 126, 4754−5.(32) Lee, Y. M., and Lim, C. (2011) Factors controlling the reactivityof zinc finger cores. J. Am. Chem. Soc.,.(33) Kiick, K., Saxon, E., Tirrell, D., and Bertozzi, C. (2002)Incorporation of azides into recombinant proteins for chemoselectivemodification by the Staudinger ligation. Proc. Natl. Acad. Sci. U.S.A. 99,19.(34) Kiick, K., Saxon, E., Tirrell, D., and Bertozzi, C. (2002)Incorporation of azides into recombinant proteins for chemoselectivemodification by the Staudinger ligation. Proc. Natl. Acad. Sci. U.S.A. 99,19.(35) Chin, J. W., Cropp, T. A., Anderson, J. C., Mukherji, M., Zhang,Z., and Schultz, P. G. (2003) An expanded eukaryotic genetic code.Science 301, 964−967.(36) Wang, L., Brock, A., Herberich, B., and Schultz, P. G. (2001)Expanding the genetic code of Escherichia coli. Science 292, 498−500.(37) Wang, A., Winblade Nairn, N., Johnson, R. S., Tirrell, D. A., andGrabstein, K. (2008) Processing of N-Terminal Unnatural AminoAcids in Recombinant Human Interferon-β in Escherichia coli.ChemBioChem 9, 324−330.(38) Rauscher, F. J., Morris, J. F., Tournay, O. E., Cook, D. M., andCurran, T. (1990) Binding of the Wilms’ tumor locus zinc fingerprotein to the EGR-1 consensus sequence. Science 250, 1259.(39) Kim, M. S., Stybayeva, G., Lee, J. Y., Revzin, A., and Segal, D. J.(2011) A zinc finger protein array for the visual detection of specificDNA sequences for diagnostic applications. Nucleic Acids Res. 39, e29−e29.(40) Sander, J., Zaback, P., Joung, J., Voytas, D., and Dobbs, D.(2009) An affinity-based scoring scheme for predicting DNA-bindingactivities of modularly assembled zinc-finger proteins. Nucleic AcidsRes. 37, 506.(41) Ben-Bassat, A., Bauer, K., Chang, S. Y., Myambo, K., Boosman,A., and Chang, S. (1987) Processing of the initiation methionine fromproteins: properties of the Escherichia coli methionine aminopeptidaseand its gene structure. J. Bacteriol. 169, 751.(42) Pabo, C. O., Peisach, E., and Grant, R. A. (2001) Design andselection of novel Cys2His2 zinc finger proteins. Annu. Rev. Biochem.70, 313−340.(43) Michael, S. F., Kilfoil, V. J., Schmidt, M. H., Amann, B. T., andBerg, J. M. (1992) Metal binding and folding properties of aminimalist Cys2His2 zinc finger peptide. Proc. Natl. Acad. Sci. U.S.A.89, 4796.(44) Nyborg, J. K., and Peersen, O. B. (2004) That zincing feeling:the effects of EDTA on the behaviour of zinc-binding transcriptionalregulators. Biochem. J. 381.(45) Hutchens, T. W., Allen, M. H., Li, C. M., and Yip, T. T. (1992)Occupancy of a C2-C2 type ’Zinc-finger’protein domain by copperDirect observation by electrospray ionization mass spectrometry. FEBSLett. 309, 170−174.(46) Predki, P. F., and Sarkar, B. (1994) Metal replacement in″ zincfinger″ and its effect on DNA binding. Environ. Health Perspect. 102,195.(47) Carroll, D., Morton, J., Beumer, K., and Segal, D. (2006)Design, construction and in vitro testing of zinc finger nucleases. Nat.Protocols 1, 1329−1341.(48) Nurmemmedov, E., and Thunnissen, M. (2006) Expression,purification, and characterization of the 4 zinc finger region of humantumor suppressor WT1. Protein Express. Purif. 46, 379−389.(49) Baskin, J. M., Prescher, J. A., Laughlin, S. T., Agard, N. J., Chang,P. V., Miller, I. A., Lo, A., Codelli, J. A., and Bertozzi, C. R. (2007)Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl.Acad. Sci. U.S.A. 104, 16793.



(50) Lutz, J. F. (2008) Copper free azide-alkyne cycloadditions: newinsights and perspectives. Angew. Chem., Int. Ed. 47, 2182−2184.(51) Ning, X., Guo, J., Wolfert, M., and Boons, G. (2008) Visualizingmetabolically labeled glycoconjugates of living cells by copper free andfast huisgen cycloadditions. Angew. Chem. 120, 2285−2287.(52) Szabo, A., Stolz, L., and Granzow, R. (1995) Surface plasmonresonance and its use in biomolecular interaction analysis (BIA). Curr.Opin. Struct. Biol. 5, 699−705.(53) Elf, J., Li, G. W., and Xie, X. S. (2007) Probing transcriptionfactor dynamics at the single-molecule level in a living cell. Science 316,1191.



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Speciﬁc Labeling of Zinc Finger Proteins using ... · in ZFP engineering have enabled the design and construction of “programmable” transcription factors that target and bind