Proc. Natl. Acad. Sci. USAVol. 92, Fp. 5214-5218, May 1995Immuno ogy

Transplantation of a 17-amino acid a-helical DNA-binding domaininto an antibody molecule confers sequence-dependentDNA recognition

(DNA-binding antibodies/recombinant antibodies/transcription factors/basic helix-loop-helix protein/TFEB)

KATHRYN E. MCLANE*t, DENNIS R. BURTON*t§, AND PETER GHAZAL*§¶Departments of *Immunology, tMolecular Biology, and 1Neuropharmacology, Division of Virology, The Scripps Research Institute, 10666 North Torrey PinesRoad, R307, La Jolla, CA 92037

Communicated by Frank J. Dixon, The Scripps Research Institute, La Jolla, CA, February 1, 1995

ABSTRACT Recombinant antibodies capable of se-quence-specific interactions with nucleic acids represent aclass of DNA- and RNA-binding proteins with potential forbroad application in basic research and medicine. We describethe rational design of a DNA-binding antibody, Fab-Ebox, byreplacing a variable segment of the immunoglobulin heavychain with a 17-amino acid domain derived from TFEB, a classB basic helix-loop-helix protein. DNA-binding activity wasstudied by electrophoretic mobility-shift assays in whichFab-Ebox was shown to form a specific complex with DNAcontaining the TFEB recognition motif (CACGTG). Similar-ities were found in the abilities of TFEB and Fab-Ebox todiscriminate between oligodeoxyribonucleotides containingaltered recognition sequences. Comparable interference ofbinding by methylation of cytosine residues indicated thatFab-Ebox and TFEB both contact DNA through interactionsalong the major groove of double-stranded DNA. The resultsof this study indicate that DNA-binding antibodies of highspecificity can be developed by using the modular nature ofboth immunoglobulins and transcription factors.

Several classes of DNA-binding proteins are defined on thebasis of common DNA recognition and dimerization motifs,which include the helix-turn-helix, the homeobox, the POUdomain, the zinc finger, the basic leucine zipper, and the basichelix-loop-helix (bHLH) transcription factors (1-4). Structuralstudies of these proteins indicate that the regions involved inDNA binding, dimerization, or other protein-protein interac-tions are discrete domains or modular functional elements (1,3, 5). Antibodies also have domain-like structures-constantregions that maintain general antibody structure and mediateeffector functions and regions of variable amino acid sequencethat are involved in specific antigen recognition. Six shortsequence segments of the light- and heavy-chain variable do-mains form the antigen-binding site. These six segments arereferred to as the complementarity-determining regions (CDRs)(6). The heavy chain CDR3 (HCDR3) is functionally importantin determining antibody-binding specificity and is the most vari-able in length (2-26 residues) and sequence in humans (6, 7).

Antibodies have a number of advantages for displayingprotein motifs. In particular, specific vector systems have beendeveloped for creating antibody libraries that can be eitherexpressed in bacteria or displayed on phage (7-15). Impor-tantly, phage display allows for optimization of affinity ofbinding, including the manipulation, generation, and exami-nation of many variants. Moreover, antibodies are stable,well-characterized proteins which can accommodate variationwithin a constant framework, are easily purified, and could beused in therapy.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Antibodies that are specific for nucleosides, nucleotidesequences, and specific conformations of double-strandedDNA have previously been raised in animals (16-18). Re-cently, we have shown that antibodies that bind to DNA can beselected from semi-synthetic libraries expressed on the surfaceof phage (19). One clone demonstrated a clear preference forpoly(dG-dC) relative to poly(dA-dT). Antibodies capable ofsequence-specific recognition ofDNA could be valuable toolsfor many clinical and experimental purposes (20). A few ex-amples of potential applications include their use in gene diag-nostics, gene mapping, regulation of gene expression, and thera-peutic regimes. In the present study, the HCDR3 region of arecombinant antibody is used as the framework for presenting a17-amino acid DNA-binding domain of an idealized bHLHprotein, TFEB. We show that this engineered antibody is capableof sequence-specific recognition of DNA.

MATERIALS AND METHODSRecombinant Plasmids and Construction ofFab-Ebox. The

heavy chain CDR3 domain of the Fab p313 (12) was replacedby a prototypical Ebox binding domain by using three overlapextension PCR-splicing reactions. The primers were synthe-sized by Operon Technologies (Alameda, CA) and the se-quences were as follows: FTX3, which corresponds to pComb3residues 3990-4011 (21), 5'-GCAATTAACCCTCACTA-AAGGG-3' (22-mer); primer 1A, 5'-GGCAGCTCTACGTCG-CCTCTCAGCGGCCGCATGCGCAGCCTTCTTAGCGGC-TCCTCCTCTCGCACAATAATATATGGCCGT-3' (81-mer);primer 1B, 5'-GCTAAGAAGGCFGCGCATGCGGCCGCT-GAGAGGCGACGTAGAGCTGCCATCAATGGAGGAGC-TGCAGGCGGGATGGACGTCTGGGGCCAAGGG-3' (93-mer); and CGlz (22), 5'-GCATGTACTAGTTTTGTCACAA-GATTTGGG-3' (30-mer). The PCR synthetic reactions wereperformed as described by Horton and coworkers (23, 24) usinga Perkin-Elmer 9600 thermocycler. The conditions used for 40cycles of PCR are indicated in parentheses. After the 40 cycleswere completed, there was an extension reaction of 10 min at72°C. All PCR reaction mixtures contained 1.5 mM MgCl2 and1 unit of TaqDNA polymerase (Promega), and the PCR productswere purified by agarose gel electrophoresis and electroelution.Two synthetic PCR reactions were carried out with 1 ,g of p313DNA (94°C for 30 sec; 50°C for 60 sec; and 72°C for 2 min). Thesereactions produced the following products: (i) double-strandedDNA containing the 5'-end of the heavy chain fragment of Fabp313 and the 3'-end of the Ebox-binding domain [499 bp; the

Abbreviations: bHLH, basic helix-loop-helix; MLP, adenovirus majorlate promoter; CDR, complementarity-determining region; EMSA,electrophoretic mobility-shift assay.tPresent address: University of Minnesota, 10 University Drive,Duluth, MN 55812.§D.R.B. and P.G. made equal contributions and should be consideredjoint senior and corresponding authors.

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product of FTX3 (0.6 ,uM) and primer 1A (3 ,uM)] and (u)double-stranded DNA containing the 5' end of the Ebox-bindingdomain fused to the 3' end of the heavy chain fragment of Fabp313 [432 bp; the product of primer 1B (3 ,M) and CGlz (0.6j,M)]. By virtue of overlap in theDNA sequences, the 499-bp and432-bp PCR products (500 ng each) were spliced by PCR usingprimers FTX3 (0.6 ,uM) and CGlz (0.6 ,uM) (94°C for 30 sec;52°C for 60 sec; and 72°C for 2 min). The modified heavy chainfragment (898 bp) was digested withXho I and Spe I and insertedinto Fab p313 containing only the light chain by using the Xho Iand Spe I restriction sites (12). Escherichia coli XL1-Blue trans-formants were screened by restriction enzyme mapping andautomated DNA sequencing to obtain clones containing thedesired sequence. Cells capable of expressing soluble antibodywere obtained by digesting the DNAwithNhe I and Spe I, ligatingit, and transforming electrocompetent E. coli XL1-Blue cells asdescribed (12). A clone expressing the desired sequence wasdesignated Fab-Ebox.The construction of the recombinant plasmid pTFEBA265

for expressing TFEB in E. coli has been described (25) and wasa kind gift from David Fisher and Philip Sharp.

Electrophoretic Mobility-Shift Assays (EMSAs). EMSAscontained the indicated amount of protein in 20 ,lI of reactionbuffer [25 mM Hepes, pH 7.5/50mM KCl/5 mM MgCl2/0.1%Nonidet P-40/5% (vol/vol) glycerol/10 mM dithiothreitol]containing radiolabeled double-stranded adenovirus majorlate promoter (MLP) probe (200 pg) and a 2000-fold molarexcess of poly(dI-dC)poly(dI-dC) as nonspecific competitor.After 20 min at room temperature, DNA-binding reactionswere run on 5% polyacrylamide gels (29:1 acrylamide to bisacrylamide) containing 0.5 X TBE buffer (lx TBE = 90 mMTris/90mM boric acid/2.5 mM EDTA, pH 8.3) at 8 V/cm. Thegels were dried and autoradiographed at -70°C.

Purification of Recombinant TFEB and Fab-Ebox.TFEBA265 was overexpressed in BL21 bacteria and purifiedby nickel-chelate chromatography (Qiagen, Chatsworth, CA)(26, 27). Fab-Ebox was induced with isopropyl f3-D-thio-galactopyranoside (2 mM) and purified by using goat anti-human F(ab')2 as described (28, 29). Both TFEBA265 andFab-Ebox were dialyzed against 20mM Hepes, pH 7.5/50mMKCl/10 mM MgC92/1 mM dithiothreitol/10% (vol/vol) glyc-erol. Purified proteins were analyzed by SDS/PAGE and silverstaining, which indicated that both TFEBA265 and Fab-Eboxwere -98% pure.

mutagenesis (27). This study showed that, by using a minimalsequence of essential amino acids and replacing nonessentialresidues with alanine to confer a stable a-helical conformationto the peptide segment, the affinity for the TFEB DNA rec-ognition site could be improved compared with the wild-typeTlFEB. Therefore, we hypothesized that a relatively short peptidesequence, 17 amino acid residues in length, containing 10 con-served residues and 7 alanine replacements, might confer DNA-binding specificity on an antibody molecule. We refer to thisminimal sequence as the Ebox-binding domain. Given the x-raycrystal structures of Fab fragments (39, 40), it is likely that theEbox domain will extend out of the antibody forming a relativelylarge protrusion which could interact with the DNA double helix.

Incorporation of the 17-Amino Acid Ebox Binding Domaininto an Antibody (Fab-Ebox) Confers DNA Recognition. DNAencoding the CDR3 of the heavy chain of the recombinant Fab,p313 (22), was replaced with a synthetic insert containing thecodons for the 17-amino acid sequence corresponding to theEbox-binding domain and short flanking Gly-Gly sequences byoverlap-extension PCR (Fig. 1A). The antibody Fab p313 is ananti-tetanus toxoid antibody that was isolated from a humancombinatorial library (12) and does not bind DNA (19). Atransformant was cloned containing the correctly modified Fabheavy chain and was designated Fab-Ebox. The sequence of theEbox binding domain inserted into the CDR3 is given in Fig. 1B.To compare the specificity of DNA binding of Fab-Ebox

with native TFEB, a soluble form of TFEB designatedTFEBA265 was prepared (26, 27). EMSAs were conductedwith double-stranded 32P-labeled 28-mer oligodeoxyribonu-cleotides containing the class B bHLH protein core recogni-tion sequence CACGTG, designated MLP1 and MLP2 (Fig.1C). Both purified Fab-Ebox and TFEBA265 formed com-plexes with 32P-labeled MLP (Fig. 2A). Most likely, MLP wascomplexed with monomeric Fab-Ebox (19) or dimeric TFEB-A265 (27). As previously observed, TFEB also tended to formtetrameric complexes of slower mobility (25, 27). The EMSAresults clearly indicate that transplanting the 17-amino acidEbox-binding domain into the CDR3 region of the heavy chainconfers DNA-binding activity on Fab-Ebox. Note that the parentantibody of Fab-Ebox is unable to bind DNA with any appre-ciable affinity (19). The binding ofFab-Ebox to 32P-labeled MLPprobe in the presence of a 2000-fold molar excess of poly(dI-dC)poly(dI-dC) indicates that the interaction of Fab-Ebox with

RESULTS AND DISCUSSIONRationale for Using the DNA-Binding Motif of TFEB. The

basic domain of the bHLH protein TlEB (30) was selected as amodel sequence for several reasons. First, deletion analysis ofTFEB indicates that, although high-affinity binding requiresdimerization, lower affinity binding can be observed with mutantsthat have deleted dimerization domains (25). These results sug-gested that presentation of a single basic domain derived fromTlFEB might confer DNA binding on an antibody molecule.Second, the specific protein-DNA interactions that occur for thebHLH proteins with DNA have been determined by x-raycrystallography (31-34). Third, the sequence specificity ofDNArecognition by TFEB has been well defined (30, 35). The bHLHproteins bind as dimers to the common core recognition sequenceCANNTG, also known as an "E box" (36), and can be dividedinto two groups: class A, which recognize the palindromic se-quence CAGCTG, and class B, which bind the palindromicsequence CACGTG (37, 38)., TFEB, like other class B bHLHproteins, binds to the core recognition sequence CACGTG,which is found in the MLP. TFEB also recognizes the sequenceCATGTG of the IgM heavy chain enhancer (27, 30) but does notbind the class A sequence CAGCFG.

Finally, the amino acid residues of TFEB that are essentialfor DNA binding have been defined using alanine-scanning

A XhoFTX3

Spe

FR3 CDR3 FR4

1A\ CG1 z

B GM GMZ r AAAGArGM CAcG GM Wr GAG 0G3G G A A K K A A H A A A E R

OG k GCr OC Am AAT GS .Gk GC GCAGOC G3aR R R A A I N G G A A G G

C NLP 5'-C GTGIAGt:CPC C G1G 'G-3'1oP2 5 '-GAC PA OOC Gr CA GIGM MC AM-3'

FIG. 1. Construction of Fab-Ebox by PCR and probes for DNA-binding specificity. (A) Details for the construction of Fab-Ebox areprovided in Materials and Methods. (B) Sequence of an idealized Ebox-binding protein insert. The Ebox-binding domain was designed after amodified form of TFEB (25, 27)- in which amino acids that were notcritical for DNA binding were replaced by alanine residues to stabilize ana-helical conformation of this segment (27). In addition, two glycineresidues were added to both ends of the insert to limit the a-helicalpropensity to the inserted sequence and to provide conformational flex-ibility within the CDR3 (41, 42). (C) Sequences of radiolabeled oligonu-cleotide probes for DNA binding. The sequences of the complementarysingle-stranded synthetic oligodeoxyribonucleotides MLP1 and MLP2that were used to study DNA binding are indicated. The class B bHLHprotein consensus DNA-binding sequence is underlined.

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ATFEBA265 Fab-Ebox

Ig1

pmoles0.5 5.0 20 20 100 200

BTFEBA265 Fab-Ebox

I ~~ ~ ~ - I'

dsMLP poly dl.dC dsMLP poly dl.dCqp CD8 0 0=I

0 co c C°°0 o o °0Z (04= Za c

.?

A ioo - -0 -a

80 4

-o~~~n

m 60I0.la 40 4

20

20

0 40 80 120 160nM

C

FIG. 2. DNAsequence recognition byTFEBA265 and Fab-Ebox. (A)DNA binding at different concentrations of TFEBA265 and Fab-Ebox.The EMSA contained the indicated amounts of TFEBA265 and Fab-Ebox with labeled double-stranded MLP (dsMLP) probe (200 pg; 1.4 x105 cpm). (B) Competition with poly(dI-dC)po1y(dI-dC) and unlabeleddsMLP. DNA-binding assays were performed as descnbed in Materialsand Methods with the exception that TFEBA265 (2 pmol) and Fab-Ebox(100 pmol) were preincubated at roonm temperature for 10 min withunlabeled DNA competitors, prior to addition of 32P-dsMLP probe (200pg; 1.4 x I05 cpm). 411 DNA-binding reactions contained at least a2000-fold excess of poly(dI-dC).poly(dI-dC) and, in addition, containedeither unlabeled dsMLP DNA or additional poly(dI-dC).poly(dI-dC) toa final molar excess over 32P-dsMLP probe as indicated.

MLP DNA is highly specific. The present results suggest that,while dimerization ofbHLH proteins may promote higher affin-ity binding, the dimer does not appear to be required for specificDNA recognition. Indeed, in a recent study by Blackwell et al(43), a related member of the bHLH family of proteins termedSkn-1, which lacks the leucine-zipper dimerization segment, wasshown to bind specific DNA sequences with high affinity as amonomer, demonstrating an underlying submodularity in theseDNA-binding domains.

Sequence Specificity and Groove Geometry ofDNA Bindingby Fab-Ebox. To compare the DNA-binding specificity ofFab-Ebox and TFEBA265, competition EMSAs were per-formed with nonspecific, double-stranded, alternating polymerDNAs poly(dI-dC)-poly(dI-dC), poly(dA-dT)-poly(dA-dT), andpoly(dG-dC)-poly(dG-dC). The results shown in Fig. 2B demon-strate that nonspecific DNA poly(dI-dC)-poly(dI-dC) does notcompete for specific DNA binding of Fab-Ebox and TFEBA265until an excess of 410,000-fold is used relative to the double-stranded MLP probe. Similar results were obtained for compe-tition with poly(dA-dT)-poly(dA-dT) and poly(dG-dC)-poly(dG-dC) (data not shown). Thus, on the basis of these criteria thespecificity of DNA binding to the MLP is comparable forFab-Ebox and TFEBA265.To determine if Fab-Ebox and TFEBA265 differed in their

sequence specificity for DNA binding, several double-stranded28-mer oligodeoxyribonucleotides containing altered recognitionmotifs were used as competitors for binding to the 32P-labeledMLP probe (CACGTG). The altered core recognition sequencestested included CATGTG (E3, the bHLH class B recognitionsequence of the IgM heavy chain enhancer), GAGGTG (mMLP,a double mutant), and CAGCTG (MyoD, the bHLH proteinclass A recognition sequence). In addition, a double-stranded27-mer containing an unrelatedDNA sequence (CRS) was testedto clearly determine the level of nonspecific binding expected forcompetition with DNA comparable in size to the 28-mer 32P-labeled MLP probe. The results of these competition assays areillustrated in Figs. 3 and 4, and data of three IC50 determinationsfor each DNA competitor for Fab-Ebox and TFEBA265 aresummarized in Table 1.The competition studies indicate that TFEBA265 binds the

sequence CACGTG with 10-fold greater apparent affinity

100-

80 -

60

40

20

0 4 802.. ''

01160 40 80 120 160

- E 3 i8v

_ 0

_ 4 _ 0:_0 t _ P

nM

FIG. 3. DNA-binding specificity: Comparison of binding to differ-ent consensus sequences. Competition EMSAs were performed in vastexcess of protein, in which double-stranded oligodeoxyribonucleotidescompeted with 32P-labeled double-stranded MLP for bindingTFEBA265 (20 pmoles) or Fab-Ebox (200 pmol). The unlabeledcompetitor DNA corresponded to double-stranded oligodeoxyribo-nucleotides that contained the following changes in the class B bHLHprotein consensus DNA binding sequence CACGTG found in theMLP sequence (Fig. 1C): mMLP, GAGGTG; E3, CATGTG; andMyoD CAGCTG. CRS is a DNA sequence unrelated to MLP (5'-AGCTTGCAGAGCTCGTTTAGTGAACC-3'). The binding to 32p-labeled dsMLP (200 pg; 2-10 x 105 cpm) was determined, in thepresence of different concentrations of competitors (0-150 nM) andquantitated by using a ,3-scanner (AMBIS) to obtain relative IC5ovalues. The re'sult of this analysis for TFEBA265 is illustrated inA; forFab-Ebox, C. B andD illustrate the autoradiographic results obtainedwhen using a single concentration of competing double-stranded oligo-deoxyribonucleotides: 5 nM for TFEBA265 (B) and 50nM for Fab-Ebox(D). Control indicates the level of binding in the absence of competitor.The results of three IC50 determinations for each of the competingdouble-stranded oligodeoxyribonucleotides are summarized in Table 1.O, MLP; 0, mMLP; z, E3; A, MyoD; and , CRS.

than does Fab-Ebox (IC50 0.5 nM and 5 nM, respectively).The apparent affinities of TFEBA265 for the recognitionsequences CACGTG (MLP) and CATGTG (E3) have previ-ously been shown to differ by 10-fold (25), and our presentresults confirm this apparent relative-affinity ratio. The rela-tive affinities of Fab-Ebox for CACGTG (MLP) and CAT-GTG (E3) also differ by '-10-fold. The ratio of the relativeaffinities of Fab-Ebox and TFEBA265 for MLP (CACGTG)versus MyoD (CAGCTG) are also similar (1:25 and 1:21,respectively). A profound difference in the recognition of thedouble-mutant GAGGTG (mMLP) is found between Fab-Ebox and TFEBA265-i.e., whereas the ratio of apparentaffinities of TFEBA265 for MLP (CACGTG) versus mMLP(GAGGTG) is -1:100, it is only '1:10 for Fab-Ebox. Thisresult indicates that although DNA recognition by Fab-Eboxis sequence dependent, the specificity for DNA sequencerecognition is not as precise as that found for TFEBA265.However, given that.the Ebox sequence binds DNA as amonomer and is out of context of the other structural elementsof the bHLH protein, even this level of sequence-dependentrecognition was not anticipated.The dissociation constant of TFEBA265 for MLP has not

previously been reported. To obtain an approximation of theaffinities of Fab-Ebox and TFEBA265 for MLP, apparent Kdvalues were estimated from the competition assays by using

B 0 0;2^ . ~~~~~~~~,u:_ E

(A

I-> p

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A 100 a

80~~~~~~

80

CD)

g 40 MLP 5'CACGTG 3'

20 MyoD 5 CAGCTG 3'

0

0 10 20 30 40 50 60 70 80 90 100% Displaced/nM Competitor

B

2 1500- 0 TFEB0Fab-Ebox

1000

500- 0

0.0 0.5 1.0 1.5

1/lFree DNA, nM1

FIG. 4. Comparison of class A bHLH versus class B bHLH DNArecognition sequences. (A) Competition EMSAs were performed withdouble-stranded MLP and MyoD as competitors. The data are presentedas the Hofstee plot, where the slope of the line is the ICs0 (nM) and theslopes can be directly compared (44, 45). The IC5o and correlationcoefficients (in parentheses) of the linear regression for these data wereas follows: 0, TFEB/MLP, 0.46 nM (0.98); U, Fab-Ebox, 4.3 nM (0.96);., TFEB/MyoD, 10.9 nM (0.98); and A, Fab-Ebox/MyoD, 91.9 nM(0.94). The results of three IC50 determinations for each of the competingdouble-stranded oligodeoxyribonucleotides are summarized in Table 1.(B) The apparent dissociation constants were determined from compe-tition EMSAs performed with double-stranded MLP as the competitor.The apparentKd valueswere determined by using double-reciprocal plots(1/[bound DNA] versus 1/[free DNA], where the is the slope dividedby the intercept of the axis 1/[bound DNA]). The reciprocal of theintercept is the apparent "Bm." of the EMSA and reflects the concen-tration of protein actively bindingMLP under these conditions (5 pM and15 pM for TFEBA265 and Fab-Ebox, respectively). In the experimentshown, the apparent Kd values for TFEBA265 and Fab-Ebox were 2 nMand'24 nM, respectively. In this analysis, the apparent Kd values aretentative estimates of the true equilibrinum constants.

double-reciprocal plots (1/[bound DNA] versus 1/[free DNA]),as illustrated in Fig. 4B. In this analysis, the values obtained forthe apparent Kds are preliminary estimates of the true equi-librium constant (Kd), since the nonspecific binding of theprotein with poly(dI-dC)-poly(dI-dC) present in each reactionand the potential dissociation'of the protein-DNA complex inthe gel were not taken into account. The average apparent Kdvalues (±SDs; n = 3) obtained for Fab-Ebox and TFEBA265for MLP were 18 8 nM and 1.8 ± -2 nM, respectively. Thesevalues are within the same order of magnitude as the IC5os (5nM and 0.5 nM for Fab-Ebox and TFEBA265, respectively),which reflect a similar 10-fold reduction in the relative affinityof Fab-Ebox for DNA compared with TFEBA265.The DNA base-specific interactions of TFEB with the' core

recognition motif can be inferred from the recent x-ray

cocrystallization of a highly homologous bHLH protein, Max(32, 34), which makes direct contacts along the major grooveof the double helix. Several of these contacts involve interac-

Table 1. Analysis of binding of TFEBA&265 or Fab-Ebox to a setof related DNA target sequences

TFEBA&265 Fab-Ebox

DNA target IC50, nM Target/MLP IC5o, nM Target/MLPMLP 0.52 ± 0.6 1.0 4.5 ± 0.5 1.0mMLP 59 t 5 114 60 ± 2 13E3 5.4 ± 0.9 10 52 t 20 12MyoD 12 ± 2 25 95 ± 14 21CRS 172 ± 18 332 168 ± 35 37MLP-mel 7 ± 1 14 26 ± 6 5MLP-me2 9 ± 2 18 34 ± 7 8MLP-melme2 129 ± 58 250 126 ± 37 28

Competition EMSAs were performed as described in Materials andMethods and the legends to Figs. 3 and 5. The unlabeled competitorDNAs were double-stranded oligodeoxyribonucleotides that con-tained changes in the class B bHLH protein consensus DNA bindingsequence CACGTG, which is found in the MLP sequence (Fig. 1C).mMLP, GAGGTG; E3, CATGTG; and MyoD, CAGCTG. CRS is aDNA sequence unrelated to MLP (see Fig. 3 legend for sequence).MLP-mel is mel [5'-GATCGGTGTAGGC(5-methyl-C)A(5-methyl-C)GTGACCGGGTGT-3'] an-nealed to MLP2 (Fig. 1C);-MLP-me2 isme2 [5'-GATCACACCCGGT(5-methyl-C)A(5-methyl-C)GTGGC-CTACACC-3'] annealed to MLP1 (Fig. 1C); and MLP-melme2 is melannealed to me2. The IC50 values were determined by linear regressionof the Hofstee plot (% displaced versus % displaced/nM competitor)as illustrated in Fig. 4, where the slope is the IC50. The IC50 (mean ±SD) of three experiments are listed. Also shown is the ratio of the IC5ofor'binding to the target DNA to the IC50 for binding to MLP forTFEBA265 or Fab-Ebox as indicated.

tions of the conserved amino acid sequence Glu-Arg-Arg-Argof class B bHLH proteins with a cytosine residue and thephosphate backbone of the sequence CACG of the class Bbinding motif (32). To determine if DNA recognition by Fab-Ebox occurred through interactions with the major groove and tocompare the interactions involved in Fab-Ebox and TFEBA265binding, the effect of methylation of the cytosine residues of theCACGTG sequence was studied. The use of 5-methylcytosine asthe modified base has several advantages: (i) the 5-methyl groupprotrudes into the major groove of the double helix and couldpotentially sterically hinder interactions with cytosine residuesand the phosphate backbone of the sequence of interest, CACG;(Ui) the 5-methyl group of cytosine does not interfere withWatson-Crick base-pair interactions; and (iii) 5-methylcytosinecan be incorporated into synthetic DNA, which allows control ofthe level of methylation of single strands of the MLP sequence.

Competition EMSAs were performed with double-strandedDNA corresponding to the MLP sequence but containing5-methyl-C residues in the core recognition motif in one(MLP-mel and MLP-me2) or both (MLP-melme2) comple-mentary single strands. The results of the competition assaysare illustrated in Fig. 5 and summarized in Table 1. Methyl-ation of the cytosine residues of a single strand of the duplexresulted in a -90% to 95% decrease in the apparent affinityof TFEBA265 for double-stranded MLP and a "80% to 90%decrease in the affinity of Fab-Ebox for the core sequence.DNA containing 5-methyl-C residues in both complementarysingle strands of double-stranded MLP (CACGTG) exhibitedIC5o values for both TFEBA265 and Fab-Ebox that ap-proached those observed for the nonspecific double-strandedoligodeoxyribonucleotide CRS (IC50 >100 nM). These resultsindicate that both TFEBA265 and Fab-Ebox bind double-stranded MLP through interactions with the major groove.

In conclusion, we have used the framework of an antibodyand have constructed a DNA-binding protein by replacingHCDR3 with a 17-amino acid, idealized Ebox-binding domain.The resulting Fab binds DNA specifically and with potentiallyhigh apparent affinity. Remarkably, although less sequencespecific than the parent TFEB protein, Fab-Ebox was found tointeract with the major groove of the DNA helix and discrim-

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20 -

1'*- I'.'I't-I

BE

E E E

v 2 2 "Z ;p-U. jaB N

*§ w.:...I14

40 80 120 160nM

D 0E

1- 4 "

oE EEC) a) a)z

-. I2a , g~, 0

nM

FIG. 5. Comparison of DNA contacts with double-stranded MLP byTFEBA265 and Fab-Ebox: Interference of binding by 5-methylcytosine.The unlabeled competitor DNA corresponded to the following changesfrom MLP: MLP-mel is mel [5'-GATCGGTGTAGGC(5-methyl-C)A(5-methyl-C)GTGACCGGGTGT-3'] annealed to MLP2 (Fig. 1C);MLP-me2 is me2 [5'-GATCACACCCGGT(5-methyl-C)A(5-methyl-C)GTGGCCTACACC-3'1 annealed to MLP1 (Fig. 1C); and MLP-melme2 is mel annealed to me2. A and B are results obtained with

-BA265, and C andD are typical results obtained for Fab-Ebox.B andD represent the results obtained by using a single concentration ofcompetitor (50 nM). Control indicates the level of binding in the absenceof competitor. The results of three IC50 determinations for each of thecompeting double-stranded oligodeoxyribonucleotides are summarizedin Table 1. 0, MLP; *, MLP-mel; , MLP-me2; A, MLP-melme2.

inate in its recognition of the CACGTG motif in a sequence-

dependent manner. The results described demonstrate that themodular nature of immunoglobulins and transcription factorscan be exploited to create unique DNA-binding proteins bymolecular design.

We thank Robin Stanfield and Ian Wilson for molecular modelingof Fab-Ebox and antibodies with related inserts that assisted in thefinal design of the DNA-binding antibody. We thank Roman Rozen-shteyn and Raiza Bastidas for their expert technical assistance.pTFEBA265 was a generous gift from David Fisher and Philip Sharp;pFab313 was a generous gift from Carlos Barbas. This work was

directly supported by funding from institutional and postdoctoralfellowship grants from the American Cancer Society (PF-3667) to

KE.M., and by National Institutes of Health grants to D.R.B. and P.G.P.G. is a Scholar of the Leukemia Society of America. This work is9011-IMM from the Scripps Research Institute.

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