Published: February 07, 2011

r 2011 American Chemical Society 388 dx.doi.org/10.1021/bc100376x | Bioconjugate Chem. 2011, 22, 388–396

ARTICLE

pubs.acs.org/bc

Chemical Functionalization of Oligodeoxynucleotides with MultipleBoronic Acids for the Polyvalent Binding of SaccharidesAmanda E. Hargrove,† Andrew D. Ellington,*,† Eric V. Anslyn,*,† and Jonathan L. Sessler*,†,‡

†Department of Chemistry and Biochemistry, University of Texas at Austin, 1 University Station A5300, Austin, Texas 78712-0165,United States‡Department of Chemistry, Yonsei University, Seoul, 120-749 Korea

bS Supporting Information

ABSTRACT: A novel saccharide host containing four boronicacid recognition units on a single DNA duplex terminus wasconstructed. This construct allowed boronic acid sugar recognitionin the context of double-stranded DNA to be established whilehighlighting the benefits ofmultivalency. Following the solid-phasesynthesis of a bis-boronic acid tag, two end-functionalized oligo-nucleotides with complementary sequences were functionalizedthrough amide ligation. By annealing the boronic acid-DNAconjugates, a tetra-boronic acid DNA duplex was assembled. Thesaccharide binding ability of this tetra-boronic acid host wasrevealed through cellulose paper chromatography in the absenceand presence of various saccharides. While no appreciable saccharide binding was seen in the case of a bis-boronic DNAconjugate, the increased migration of the tetra-boronic acid host relative to the control sequences in the presence of selectedmonosaccharides underscored the importance of multivalent effects. We thus identified a requirement for multiple recognitionsites in these conjugate systems and expect the results to facilitate future efforts toward applying synthetic recognition systems tothe realm of macromolecules.

’ INTRODUCTION

The selective binding of saccharides, particularly oligosaccha-rides, in a physiological environment remains an unsolved prob-lem, despite the biological importance andmedicinal relevance ofthese compounds.1 In small molecule systems, the binding ofsaccharides has been most commonly achieved through the useof boronic acids, which form stable cyclic boronate esters withsaccharide diols.2,3 However, the efficacy of boronic acid recog-nition units has rarely been investigated in the context of themacromolecules that would be present in a biological system.4,5

One specific concern is whether multivalent interactions (i.e., theuse of multiple boronic acid subunits) will be required to achieveefficient binding in such an environment. In order to investi-gate the viability of boronic acid-based saccharide recognition inthe context of DNA, we designed a system that allows for theevaluation of the effect of 0, 2, or 4 boronic acid units located atone terminus of a short (20 base pair) DNA duplex. The oligo-nucleotide derivatization scheme was based on a standard peptidecoupling reaction between a bis-boronic acid-functionalized di-peptide and two amine-functionalized single strand (ss) oligonu-cleotides with complementary base sequences (Scheme 1). Threedifferent constructs were prepared by mixing the boronic acid-ssDNA conjugates with the complementary unfunctionalized andfunctionalized sequences. Evaluation of the saccharide binding

ability of these constructs was performed through cellulose paperchromatography, a technique that requires minute amounts ofmaterial (<1 pmol) and boasts a long history in DNA char-acterization.6-10 The mobility of these constructs in the presenceand absence of various small saccharides was investigated. In theseexperiments, the tetra-boronic acid DNA duplex displayed sig-nificant interactions with saccharides, while a bis-boronic acidDNA duplex does not appear to bind saccharides appreciably. Wethus conclude that boronic acid-sugar recognition is viable in thecontext of DNA provided that a sufficient number of bindingsubunits is used, in this case, four boronic acid moieties. Theobserved importance of multivalency may prove critical in futureembodiments of hybrid small molecule-biomacromolecule recog-nition systems.

’EXPERIMENTAL PROCEDURES

Materials and Instrumentation. All chemicals were pur-chased from Aldrich Chemical Co., Acros Organics, or EM andused without further purification unless otherwise noted. Allamino acid derivatives and coupling reagents were purchased

Received: August 30, 2010Revised: December 12, 2010

389 dx.doi.org/10.1021/bc100376x |Bioconjugate Chem. 2011, 22, 388–396

Bioconjugate Chemistry ARTICLE

from Novabiochem. All solvents were of reagent-grade qualityand purchased commercially. Dry CH2Cl2 (DCM) refers tosolvent freshly distilled over CaH2. Dry DMF andmethanol wereobtained by passing high-grade commercial material through twocolumns of molecular sieves as part of an SGWater USA solventdelivery system. All water used was filtered through a Millipurefiltration system. TBE buffer refers to a solution of 89 mM Tris,89 mM boric acid, and 2 mM EDTA at pH 8.3. Oligonucleotideswere purchased from Integrated DNA Technologies (IDT) andgel-purified prior to use. Other biological reagents were pur-chased or prepared as indicated. NMR spectra were recorded onVarian INOVA 600 or Varian Mercury 400 instruments. Smallmolecule UV-vis spectra were recorded on a Beckman DU 640spectrophotometer. Oligonucleotide UV-vis spectra were re-corded on a ThermoScientific NanoDrop 1000 spectrophot-ometer. HR electrospray ionization (ESI) mass spectra wererecorded on a Varian 9.4T QFT-ESI ICR system. Gel and paperimaging was performed using a GE Storm 840 Imager andquantified using the ImageQuant software program. HPLCpurification was performed on Beckman Gold system equippedwith a Waters Xterra MS C18 column (2.5 μm) kept at 60 �C.Compounds were eluted using a 0.1 M triethylammoniumacetate (TEAA)-acetonitrile solvent gradient.N6,N-Bis((2-dihydroxyborono)benzyl)-lysinoglycine (BBA).

Fluorenylmethyloxycarbonyl (Fmoc)-Gly-Wang resin (4.00 g,2.44 mmol) was swollen in dry DCM (40 mL) by shaking overthe course of 1 h. Fmoc Deprotection: After filtration to removethe solvent, a solution of 20% piperidine in DMF was added tothe resin, and themixture was shaken for 20min. The solvent wasremoved, and the piperidine treatment repeated one more time.Wash: The resin was washed three times each with 40 mL of thefollowing solvents in succession: DMF, DCM, methanol, DMF,andDCM.Coupling:To the washed resin was added a solution ofFmoc-Lys(Fmoc)-OH (8.65 g, 14.64 mmol), 2-(1H-benzo-triazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU, 5.55 g, 14.64 mmol), 1-hydroxybenzotriazole (HOBt,2.24 g, 14.64 mmol), and H€unig’s base (4.25 mL, 24.4 mmol) in

1:1 DMF/DCM (40 mL), and the mixture was allowed to shakeat room temperature overnight. The resin was then takenthrough the steps of washing, Fmoc deprotection, and a secondwashing as described above. The resin was then dried in vacuo for3 h.Reductive Amination: A solution of formylphenyl boronic acid(3.66 g, 24.4 mmol) and anhydrous methanol (4 mL) in freshlydistilled DCM (40 mL) was added to the dry resin. After shakingovernight at room temperature, 10.3 g of sodium triacetoxyboro-hydride was added slowly. After addition of acetic acid(0.8 mL), the mixture was shaken for 10 h. The resin was washedas above, and the reductive amination procedure was repeatedone additional time. The resin was washed and dried in vacuo for4 h. A solution of 2.5% water and 2.5% triethylsilane in TFA wasadded to the resin, and the mixture was shaken for 3 h. Thesupernatant was drained and collected, and the resin rinsed twotimes with TFA (40 mL). The TFA fractions were combined andthe solvent removed in vacuo. The residue was further purifiedvia chromatography on a 10 g C18 SepPak (eluting 0-95%methanol in water) to give a white solid (186.4 mg, 40% yieldover four steps). Mp > 230 �C (decomposition). 1H NMR (400MHz, DCl (2.3 mol %) in D2O) δ (ppm) = 0.087-1.01 (m, 2H,C-CH2-C), 1.12-1.27 (m, 2H, C-CH2-C), 1.32-1.53 (m,2H, C-CH2-C), 2.57 (t, 2H, N-CH2-C), 3.30-3.53 (m, 3H,N-CH2-C), 3.78-3.98 (m, 4H, N-CH2-C), 6.90-7.06 (m,6H, C(Ph)-H), 7.27 (d, 2H, C(Ph)-H). LRMS (ESIþ): 471(Mþ). 13CNMR (150MHz, DCl (2.3 mol %) in D2O) δ (ppm)= 20.9, 24.6, 29.4, 40.8, 46.2, 51.1, 51.6, 59.2, 128.8, 129.0, 130.9,131.1, 131.6, 134.0, 135.0, 135.2 (note: the similarity of the twophenyl rings leads to significant overlap in the aromatic region ofthe spectrum; as a result, not all peaks could be cleary identified),168.6, 171.9. HRMS (ESIþ): calcd. m/z for C22H28B2N3O5

þ

[MþH-2(H2O)]þ 436.22096; found m/z 436.22122.

General Precipitation Procedure. To a solution of oligo-deoxynucleotide in water was added 0.1� vol 3 M aqueoussodium acetate (pH 5.2) and 2.3� vol absolute ethanol.After addition of glycogen (2 μL, 40 μg), the sample was cooledat -80 �C for 30 min and then centrifuged for 45 min at 4 �C.

Scheme 1. Construction of Host

390 dx.doi.org/10.1021/bc100376x |Bioconjugate Chem. 2011, 22, 388–396

Bioconjugate Chemistry ARTICLE

The supernatant was removed and the pellet washed carefullywith 70% ethanol in water. The pellet was dried under vacuumand then suspended in 20 μL of water.General Denaturing Polyacrylamide Gel Electrophoresis

(PAGE) Procedure. Polymerization was accomplished via theaddition of 120 μL 10% v/v ammonium persulfate (APS) and 30μL of N,N,N0,N0-tetramethylethylenediamine (TEMED) to asolution of 10-20% acrylamide and urea (7 M) in TBE buffer(25 mL). The solution was immediately poured into a gel frameconstructed with 0.75 mm spacers and allowed to polymerize for1 h. Samples were denatured via the addition of 1� vol of 2�stop dye (98% formamide, 20 mM EDTA, 0.05% (w/v) bromo-phenol blue), followed by heating at 65 �C for 3 min. Electro-phoresis was then performed in TBE buffer until the dye reachedthe bottom of the gel. The gel was then stained by shaking in asolution of SybrGold (Molecular Probes) in TBE buffer andimaged. Radiolabeled products were visualized by exposing thegel to a phosphorscreen overnight.General Gel Purification Procedure. Purchased oligonu-

cleotides were purified through denaturing PAGE using 20%acrylamide. The product band was visualized by irradiation of thegel on a TLC plate. The band was then removed using a razorblade, and the gel chunks incubated with water overnight at37 �C. The supernatant was removed and subjected to standardprecipitation conditions.Oligonucleotide Coupling Procedure. The carboxylic acid

tag (BBA, 2.61 mg, 5.54 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 1.59 mg, 8.31 μmol) were dissolvedin DMF (0.55 mL). H€unig’s base (1.37 μL, 8.31 μmol) was

added and the solution allowed to react at room temperature for2 h with rotational mixing. To an aliquot of the activated solution(100 μL) was added 35 μL DMF followed by 10 nmol of theamine-functionalized oligonucleotide in water (NH2-DNA1 orNH2-DNA1c) and enough additional water to produce a 9:1DMF/water solvent ratio (150 μL final volume). The finalreaction mixture was allowed to react at room temperature withrotational mixing. After 24 h, the mixture was diluted with 60 μLof 0.1 M TEAA and heated to 85 �C for 3 min. The solution wasimmediately filtered using an Ultrafree-MC microcentrifugefilter tube (0.45 μm, Millipore). The resulting solution wasdivided into two equal aliquots, and each aliquot was subjectedto HPLC purification. Purification of BBA-DNA1 and BBA-DNA1c was performed using a 5-25% 0.1 M TEAA-aceto-nitrile gradient. Fractions corresponding to product or startingmaterial peaks were collected in 15 mL conical centrifuge tubes(VWR). After freezing at-80 �C, the samples were submitted tolyophilization. The dry products were then resuspended in 300μL of water and precipitated according to standard procedures.The isolated yield of each reaction was estimated to be ca. 10%(1 nmol) as inferred from absorbance spectroscopy. The purityof the samples was analyzed through PAGE (10% acrylamide,7 M urea).General Procedure for the 50-32P-End Labeling of Oligo-

nucleotides. Oligonucleotide substrate (20 pmol) was broughtto 1 μM in forward reaction buffer (New England Biolabs). Tothis solution was added γ-32P-ATP (1 μL, 23.8 μmol, 0.143 mCi,Perkin-Elmer) and T4 polynucleotide kinase (20 units, NewEngland Biotechnologies). The reaction mixture was incubatedfor 1 h at 37 �C and immediately subjected to denaturing gelpurification. Band visualization was achieved by first obtaininga phosphorimage of the gel. The printed image was thenaligned with the gel beneath a glass plate and the band excised.The remainder of the gel purification proceeded as describedabove.Duplex Formation Procedure. An equimolar mixture of

functionalized or unfunctionalized DNA-1 and DNA-1c washeated to 80 �C in a standard PCR buffer (1.5 mM MgCl2, 50mM KCl, 10 mM Tris, pH 8.3). The solution was then slowlycooled at a rate of 0.1 �C per second.Native PAGE Procedure. Polymerization was accomplished

by addition of 300 μL 10% v/v APS and 30 μL TEMED to asolution of 20% acrylamide and 5% v/v glycerol in 0.25� TBEbuffer (50 mL). The solution was immediately poured into a gelframe constructed with 1.5 mm spacers and allowed to poly-merize for 1 h. The gel was preheated by running at 120 V for 90min in 0.25� TBE buffer. Samples were combined with 6�Orange G (disodium (8Z)-7-oxo-8-(phenylhydrazinylidene)naphthalene-1,3-disulfonate) dye (Molecular Probes). Electro-phoresis was then performed in 0.25� TBE buffer at 200 V withfan cooling until the dye had progressed 2/3 of the distance to thebottom of the gel. The gel was then stained by shaking in asolution of SybrGold (Molecular Probes) in TBE buffer andvisualized through fluorescence imaging.Cellulose Paper Chromatography Procedure. Duplexes

were first synthesized using radiolabeled samples of BBA-DNA1c or DNA1c. Approximately 0.5 pmol of the duplexes orradiolabeled CELAPT 14.11 (a cellulose aptamer described inref 11) was spotted separately using a pipetter ca. 2 cm above thebottom of 10 cm�15 cm cellulose blotting paper (VWR, grade703). The paper was then suspended in a large glass chroma-tography chamber containing PBS (150 mM NaCl, 10 mM

Scheme 2. Oligonucleotide Substrates

391 dx.doi.org/10.1021/bc100376x |Bioconjugate Chem. 2011, 22, 388–396

Bioconjugate Chemistry ARTICLE

Na2HPO4, pH 7.4) or other buffers as indicated. Displacementassays were performed in the PBS buffer with added 5 mMMgCl2 and 0.1 M saccharide, with the final solution adjusted, ifnecessary, to pH 7.4. After the buffer migrated approximately 8cm, the paper was removed, dried, and exposed to a phosphor-screen. The retention factor (r.f.) of the duplexes was determinedby plotting the intensity of pixels along a line from the origin tothe solvent front in ImageQuant software. The pixel positionswere then normalized to the total distance from the origin tosolvent front. The data was plotted in the Origin softwareprogram and the peaks fit to Gaussian curves. The peak maximawere taken to be the r.f. values.

’RESULTS

Design of Boronic Acid-DNA Conjugates. In order toexplore the efficacy and valency of boronic acid recognition unitsin the context of DNA, three constructs were designed based onthe general procedure outlined in Scheme 1. The constructshown, BBA-DNA1:BBA-DNA1c, contains four boronic acids,while a construct consisting ofDNA1 andBBA-DNA1c (DNA1:BBA-DNA1c) only contains two such binding units. Thesefunctionalized duplexes could then be compared to the nativeduplex (DNA1:DNA1c). To synthesize the BBA-DNA1 andBBA-DNA1c conjugates, we focused our functionalization ef-forts on amide bond formation reactions due to the robustness ofsuch techniques in bioconjugation reactions and the readyavailability of primary amine-functionalized oligonucleotides.12

The oligonucleotide sequences (Scheme 2) were selected toachieve duplex stability at room temperature and minimize theprobability of sequence mismatch. The solid-phase synthesis of abis-boronic acid tag containing a free carboxylic acid moiety wasdesigned based on previous examples of solid-phase functiona-lization published by the Anslyn group.13

Synthesis of Bis-Boronic Acid Recognition Unit. Thesynthesis of BBA was performed as shown in Scheme 3. Generalsolid-phase synthesis procedures were performed according tomanufacturer recommendations (Novabiochem). First, Wangresin functionalized with an Fmoc-protected glycine residue (1)was submitted to standard Fmoc cleavage conditions using apiperidine/DMF solution. The coupling of a bis-Fmoc-protected

lysine residue was performed using HBTU as the coupling agentwithHOBt andH€unig’s base as additives. The Fmoc groups werethen removed to expose two primary amine reaction sites (4).Boronic acid functionalization was achieved through the reduc-tive amination of 2 equiv of 2-formylphenylboronic acid usingtriacetoxyborohydride. The resin was then submitted to TFAcleavage conditions, which yielded the desired bis-boronic aciddipeptide (BBA) after purification.Conjugation of Bis-Boronic Acid Tag (BBA) with Oligonu-

cleotides. The carboxylic acid moiety of BBAwas first activatedwith EDC and H€unig’s base in anhydrous DMF. This solutionwas then added to an aqueous solution of DNA1 or DNA1ccontaining 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES) buffer at pH 7.4, and the reaction was monitored byPAGE (Figure 1). Staining with SybrGold revealed a new bandwith reduced mobility relative to the starting oligonucleotide.Quantification of the band intensity was used to estimate thedegree of reaction completion. The maximum observed conver-sion was estimated to be 35% in these reactions. The new mate-rial could also be distinguished as a unique peak via reverse-phaseHPLC analysis (Figure 2). The product was isolated throughpreparative reverse-phaseHPLC and precipitated with ethanol toremove any remaining organic material. PAGE gel shift analysiswas used to confirm the purity of the isolated material. Theconcentration of the product was determined from the absor-bance at 260 nm (note: Themolar absorptivity ofBBA at 260 nmwas found to be 719M-1 cm-1 in 10mMphosphate buffer at pH7.4. This value was less than 0.5% of the oligonucleotide molarabsorptivities reported by the manufacturer at this wavelength.Concentration estimates of the BBA-DNA conjugate solutionswere thus based on the molar absorptivities of the oligonucleo-tide sequences). These reactions routinely resulted in an isolatedyield of 10%.Formation of Functionalized Duplexes. The ability of the

synthesized DNA conjugates to form double-stranded duplexstructures was analyzed through gel shift analysis on a nondena-turing (native) polyacrylamide gel (Figure 3). In order to annealthe appropriate conjugates, a 1:1 mixture of BBA-DNA1 andBBA-DNA1c was heated to 80 �C for 10 min in a standard PCRbuffer solution. The solution was then slowly cooled to 20 �C.After the addition of Orange G dye, the reaction solution was

Scheme 3. Synthesis of BBA

392 dx.doi.org/10.1021/bc100376x |Bioconjugate Chem. 2011, 22, 388–396

Bioconjugate Chemistry ARTICLE

loaded onto a native polyacrylamide gel. Control solutions ofDNA1 alone and BBA-DNA1 alone were submitted to identicalconditions. A significant reduction in electrophoretic mobilitywas observed for the solution of BBA-DNA1/BBA-DNA1c over

the single BBA-DNA1, as expected for the formation of a DNAduplex. This procedure was repeated with a 1:1 mixture ofDNA1and BBA-DNA1c to form the bis-boronic acid duplex.Cellulose Paper Chromatography of Functionalized Du-

plexes. The retention of the native duplex (DNA1:DNA1c),the bis-boronic acid duplex (DNA1:BBA-DNA1c), and thetetra-boronic acid duplex (BBA-DNA1:BBA-DNA1c) on cellu-lose paper chromatography was explored as an indicator ofboronic acid-diol interactions. It was expected that the boronicacid functionalized duplexes would migrate a shorter distancethan the native duplex due to increased binding to the hydroxylgroups available on the cellulose paper. The migration of thesynthesized duplexes was compared to that of a cellulose aptamer(CELAPT14.11) developed by the Breaker laboratory andshown to display greatly reduced mobility on cellulose paper ascompared to random DNA sequences.11 Both BBA-DNA1c andDNA1c sequences were labeled with phosphorus-32 at the 50-terminus to allow for the use of phosphorimaging methods.In the initial assay, the duplex and aptamer solutions, respec-

tively, were spotted on a sheet of cellulose paper using a singlechannel pipetter, and the paper was then developed in a PBSbuffer. As seen in Figure 4, CELAPT14.11 (the positive control)was largely retained at the origin (A) while the native duplexDNA1:DNA1c (the negative control) traveled with the solventfront (C). The bis-boronic acid duplexDNA1:BBA-DNA1c alsotraveled with the solvent front, while the tetra-boronic acidduplex BBA-DNA1:BBA-DNA1c was slightly retained (B).The above result provided initial support for the notion that

the tetra-boronic acid species interacts with cellulose (a saccha-ride-rich substrate). This prompted the study of a number of

Figure 1. Gel mobility assays for the product of the BBA-DNA1conjugation reaction. Lanes (5 ng oligonucleotide): 1, BBA-DNA1/NH2-DNA1 reaction mixture; 2, NH2-DNA1. Conditions: 10% acryla-mide, 7M urea PAGE gel at 0.75mm thickness visualized with SybrGoldstain.

Figure 2. HPLC assay for BBA-DNA1 conjugation reaction. (a) Red,NH2-DNA1; green: reaction mixture; conditions: reverse-phase (C18)column; 0.1 M triethylammonium acetate (TEAA)/acetonitrile eluant.

Figure 3. Gel mobility analysis of the product of the BBA-DNA1:BBA-DNA1c duplex forming reaction. Lanes (5 ng): 1, BBA-DNA1:BBA-DNA1c; 2, BBA-DNA1; 3, NH2-DNA1. All samples were submitted tothe duplex formation procedure. Conditions: 20% acrylamide PAGE gelcontaining 5% glycerol (v/v) at 1.5 mm thickness visualized withSybrGold stain.

393 dx.doi.org/10.1021/bc100376x |Bioconjugate Chem. 2011, 22, 388–396

Bioconjugate Chemistry ARTICLE

buffer conditions in the hopes of optimizing the response. Theconditions included 2� PBS, 0.5� PBS, PBS in the absence ofNaCl, and PBS in the presence of 5 mM MgCl2. The mostsignificant differentiation between BBA-DNA1:BBA-DNA1cand DNA1:DNA1c was observed in the PBS þ MgCl2 buffersystem (Figure 5, images for other conditions shown in theSupporting Information). The migration of these duplexes wasalso tested in a Tris buffer (20 mM Tris, 100 mM NaCl, 5 mMMgCl2) at pH values of 7.4, 8.2, and 9.0. No significant depen-dence on the pH was seen under these conditions (see Support-ing Information). The PBS þ MgCl2 buffer system was thuschosen for further analysis.SaccharideDisplacement Studies. Wenext investigated the

ability of various saccharides to displace the tetra-boronic acidduplex BBA-DNA1:BBA-DNA1c from the cellulose paper.Solutions of DNA1:DNA1c, DNA1:BBA-DNA1c, and CE-LAPT14.11 were again included as controls. The BBA-DNA1:BBA-DNA1c signal was observed to migrate differently whendeveloped in various 0.1 M saccharide solutions (Figure 6 andSupporting Information). For each saccharide, the position of thehost signal relative to the origin and solvent front (i.e., theretention factor (r.f.)) was then determined. First, the Image-Quant program was used to draw a single-pixel line from thesample origin to the solvent front. The intensity of each pixel onthe line and its relative position were then extracted and replottedas a chromatogram in the Origin software program.14 Using thisprogram, the major peak of each chromatogram was fit to aGaussian curve. The intensity maximum of the calculated Gaussiancurve was taken as the r.f. value (Table 1). Due to the difficultyassociated with resolving peaks close to the origin, only peaks with r.f. values greater than 0.15were subject to analysis.Measurements formaltose, galactose, and trehalose were conducted in triplicate toallow the generation of error bars (Table 1). These errors werefound to be approximately 15%.

’DISCUSSION

DNA duplexes containing zero, two, or four boronic acidunits, respectively, were designed in order to investigate the sugarrecognition ability of boronic acid groups in the presence ofnucleic acid structures. The synthesis of these hosts relied on theend-functionalization of complementary single-stranded oligo-deoxynucleotides through amide bond ligation. Hybridization ofthese strands was expected to allow for the determination ofwhether the proximity of boronic acid moieties influences thevalency of binding and the concomitant recognition of saccha-rides under these conditions. After radiolabeling and hybridiza-tion, the sugar recognition ability of these hosts was testedthrough cellulose paper chromatography in the absence andpresence of various saccharides.Construction of Boronic Acid-DNA Conjugate. The

synthesis of the boronic acid tag focused on the solid-phasederivatization of a dipeptide containing a terminal lysine residue.With the goal of optimizing previously reported conditions forthe solid-phase reductive amination of 2-formylphenylboronicacid,13 we developed a procedure based on reports by Hindsgauland co-workers for the solid-phase synthesis of carbohydrates.15Thesuccessful final reaction used dichloromethane to swell the resin andalso featured sodium triacetoxyborohydride as the reducing agent, asafer alternative to the sodium cyanoborohydride agent previouslyreported. These conditions allowed the final bis-boronic aciddipeptide tag (BBA) to be isolated as a white powder.Conditions for the DNA conjugation reaction were optimized

for this system from reports by Cravatt and co-workers that usedN-hydroxysuccinimide to facilitate activation of the carboxylicacid group;16 however, this additive did not facilitate the couplingof the boronic acid tag in our hands. In the present system,successful conjugation of the tag was achieved through activation

Figure 4. Paper chromatography in PBS buffer. Lanes (0.25 pmol): 1,BBA-DNA1:BBA-DNA1c; 2, DNA1:BBA-DNA1c; 3, DNA1:DNA1c;4, CELAPT14.11. Figure 5. Paper chromatography in PBS buffer with 5 mM MgCl2.

Lanes (0.25 pmol): 1, BBA-DNA1:BBA-DNA1c; 2, DNA1:BBA-DNA1c; 3, DNA1:DNA1c; 4, CELAPT14.11.

394 dx.doi.org/10.1021/bc100376x |Bioconjugate Chem. 2011, 22, 388–396

Bioconjugate Chemistry ARTICLE

with EDC and a non-nucleophilic base in dry DMF. A largeexcess of this solution was then added to a concentrated aliquotof the oligonucleotide in aqueous solution, thus minimizing thewater content while retaining solubility of the nucleotide. HPLCpurification followed by ethanol precipitation allowed for theisolation of the desired conjugate in ∼1 nmol quantities. Nearlycomplete duplex formation was observed through native PAGEexperiments, a finding that provided support for the conclusionthat the final receptor construct had been formed. However,extensive efforts to further characterize these systems throughmass spectrometry were unsuccessful. This is ascribed to theinterference caused by the boronic acid functionality.Boronic Acid Valency Requirement. Simple phenylboronic

acids generally display dissociation constants with saccharides inthe millimolar range in aqueous media, with higher bindingobserved at lower salt concentrations.17 However, few reportshave analyzed boronic acid-saccharide interactions in the con-text of biopolymers. These factors raise concerns about the utilityof such receptors in a physiological environment and led us toinvestigate the possible impact of avidity in DNA-structuredhosts. While the functionalized duplexes were not able to beretained on traditional saccharide resins at low concentrations,paper chromatography experiments revealed significant reten-tion of the tetra-boronic acid duplex BBA-DNA1:BBA-DNA1con cellulose paper even at high salt concentrations. In contrast, the

native (unfunctionalized) duplex DNA1:DNA1c traveled with thesolvent front. Perhaps even more intriguing is the fact that the bis-boronic acid duplex was not retained by the cellulose paper.From the above results, we infer that BBA-DNA1:BBA-

DNA1c interacts with cellulose more strongly than the DNA1:DNA1c, presumably due to the paired boronic acid recognitionunits. The different behavior of the differently functionalizedduplexes may be attributable to an avidity effect in which fourboronic acid recognition units promote significant saccharideinteractions under the conditions of our experiment while twoboronic acid units do not. The need for multiple convergentboronic acid units to achieve saccharide binding will be critical tothe design of physiologically relevant boronic acid receptors.BBA-DNA1:BBA-DNA1c Displacement from Cellulose by

Saccharides. In order to probe further the nature of the tetra-boronic acid duplex BBA-DNA1:BBA-DNA1c with saccharides,cellulose chromatography was performed in concentrated sugarsolutions. It was expected that competition of the small mole-cules for theBBA sites would cause this duplex to adhere less wellto the cellulose support. In accord with such expectations, cleardisplacement was observed in the presence of galactose, maltose,trehalose, and both R- and β-lactose. However, little to nodisplacement was observed with the other saccharides tested(Table 1). To the extent that the degree of displacement can betaken as an indicator of relative binding affinity, the strength of

Figure 6. Paper chromatography in PBS buffer with 5 mM MgCl2 and 0.1 M of the indicated saccharide. Lanes (0.25 pmol): 1, BBA-DNA1:BBA-DNA1c; 2, DNA1:BBA-DNA1c; 3, DNA1:DNA1c; 4, CELAPT14.11.

Table 1. Cellulose Paper Chromatography Retention Factors for BBA-DNA1:BBA-DNA1c in the Presence of Various Saccharidesin PBS Buffer at pH 7.4

r.f. description r.f. description

none <0.15 - cellobiose <0.15 β-Glc-(1f4)-Glc

ribose <0.15 Rib sucrose <0.15 R-Glc-(1f2)-β-Fru

fructose <0.15 Fru R-lactose 0.19 β-Gal-(1f4)-R-Glcglucose <0.15 Glc β-lactose 0.22 β-Gal-(1f4)-β-Glc

galactosea 0.21 (0.03) Gal lactulose <0.15 β-Gal-(1f4)-Fru

maltosea 0.33 (0.01) R-Glc-(1f4)-Glc melibiose <0.15 R-Gal-(1f6)-Glc

trehalosea 0.77 (0.12) R-Glc-(1f1)-R-Glc raffinose <0.15 R-Gal-(1f6)-R-Glc-(1f2)-β-FruaAverage of three trials (standard deviation).

395 dx.doi.org/10.1021/bc100376x |Bioconjugate Chem. 2011, 22, 388–396

Bioconjugate Chemistry ARTICLE

the saccharide-BBA-DNA1:BBA-DNA1c interactions does notfollow the order generally observed for simple monoboronic acidreceptors.17 We ascribe these differences to the effects of multi-valency and the interactions between two or more boronic acidunits of the functionalized duplex and certain saccharides.18 Forinstance, a lack of migration is seen in the presence of fructose,the monosaccharide that is generally bound the most effectivelyby monoboronic acid receptors. The monosaccharide galactose,on the other hand, boasts two 1,2-cis diol units when present aseither the R-pyranoside or R-furanoside form. The increasedmigration observed in the presence of galactose supports bindingwith two boronic acid units, and subsequent studies will furtherexplore this possibility.19,20 A large r.f. was also observed forBBA-DNA1:BBA-DNA1c in the presence of trehalose, an effectthat mimics what was seen in the case of CELAPT14.11, theanticellulose aptamer used as a positive control. Similarly, bothour host and the aptamer were unresponsive to cellobiose,despite the similarity of this disaccharide and the cellulose matrix.The similar binding patterns for CELAPT14.11 and our systemprovides support for our interpretation that the supramolecularhost is interacting with sugar moieties, although nonspecificeffects or some other fortuitous basis for this concordance cannotbe fully ruled out at the present time.

’CONCLUSION

While boronic acids show great promise as recognition unitsfor biologically important saccharide targets, the efficacy of thesegroups in the context of biological macromolecules has not beenextensively studied. To address this issue, we attached sugarbinding boronic acids at the terminus of a DNA duplex. It wasfound that boronic acid recognition units are compatiblewith traditional amide-based bioconjugation reactions and thatboronic acid-DNA conjugates readily undergo duplex formation.The ability of the tetra-boronic acid duplexes to interact with sugar-rich substrates was confirmed through cellulose paper chromato-graphy. The lack of binding displayed by the bis-boronic acid duplexand the native duplex provides support for the intuitively appealingnotion that multivalent interactions are required to achieve anappreciable degree of conjugate-cellulose interaction. Furtherevidence for saccharide binding came from displacement studies,which revealed that some mono- and disaccharides could competeeffectively with cellulose. The bioconjugate host was able to selectspecific saccharides among the various analytes tested and did sowith a pattern that deviated from the previously reported prefer-ences of monoboronic acid receptors. Trehalose was found to effectthe migration of the tetra-boronic acid duplex, just as it did withCELAPT14.11, an aptamer selected to bind cellulose. However, inthe case of other smaller saccharides notable differences were seenfor the present synthetic conjugates and this latter aptamer. Thisspeaks to the selectivity of our conjugates.

In conclusion, we have demonstrated that the recognition ofsaccharides can be achieved via the use of boronic acid-function-alized DNA duplexes, although multiple recognition sites arerequired for success. The present results thus provide a blue printfor the effective translation of synthetic recognition systems intothe context of macromolecules.

’ASSOCIATED CONTENT

bS Supporting Information. Additional cellulose paperchromatography images and example of curve fitting line. This

material is available free of charge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*Towhomcorrespondence should be addressed. Phone:þ1 512 4714781. E-mail: anslyn@austin.utexas.edu; sessler@mail.utexas.edu.

’ACKNOWLEDGMENT

The authors gratefully acknowledge the contributions of MattLevy and Gwen Stovall. Funding was provided by the NationalInstitutes of Health (CA-68682 to J.L.S.), the Robert A. Welch(F-1018 to J.L.S., F-1654 to A.D.E., F-1151 to E.V.A.), theNational Science Foundation (CHE-0716049 to E.V.A.), andthe Office of Naval Research (N0014-09-1-1087 to A.D.E.).

’REFERENCES

(1) Varki, A., and Sharon, N. (2009) Historical background andoverview, Essentials of Glycobiology (Varki, A., Cummings, R. D., Esko,J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., and Etzler,M. E., Eds.) Chapter 1, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY.

(2) Mader, H. S., and Wolfbeis, O. S. (2008) Boronic acid basedprobes for microdetermination of saccharides and glycosylated bio-molecules. Microchim. Acta 162, 1–34.

(3) Jin, S., Cheng, Y., Reid, S., Li, M., and Wang, B. (2010)Carbohydrate recognition by boronolectins, small molecules, and lec-tins. Med. Res. Rev. 30, 171–257.

(4) Li, M., Lin, N., Huang, Z., Du, L., Altier, C., Fang, H., and Wang,B. (2008) Selecting Aptamers for a Glycoprotein through the Incorpora-tion of the Boronic Acid Moiety. J. Am. Chem. Soc. 130, 12636–12638.

(5) Yang, X., Dai, C., Molina, A. D., and Wang, B. (2010) Boronicacid-modified DNA that changes fluorescent properties upon carbohy-drate binding. Chem. Commun. 46, 1073–1075.

(6) Vischer, E., and Chargaff, E. (1947) The separation and char-acterization of purines in minute amounts of nucleic acid hydrolysates.J. Biol. Chem. 168, 781–782.

(7) Markham, R., and Smith, J. D. (1949) Chromatography ofnucleic acid derivatives. Nature 163, 250–251.

(8) Lawley, P. D. (1957) The relative reactivities of deoxyribonu-cleotides and of the bases of DNA towards alkylating agents. Biochim.Biophys. Acta 26, 450–451.

(9) Schnitzler, B. E., Thebo, P. L., Tomley, F. M., Uggla, A., andShirley, M. W. (1999) PCR identification of chicken Eimeria: asimplified read-out. Avian Pathol. 28, 89–93.

(10) Zhang, S., Li, B. W., and Weil, G. J. (2000) Paper chromatog-raphy hybridization: a rapid method for detection of Onchocercavolvulus DNA amplified by PCR. Am. J. Trop. Med. Hyg. 63, 85–9.

(11) Boese, B. J., and Breaker, R. R. (2007) In vitro selection andcharacterization of cellulose-binding DNA aptamers. Nucleic Acids Res.35, 6378–6388.

(12) http://www.idtdna.com/catalog/Modifications/.(13) Edwards, N. Y., Sager, T. W., McDevitt, J. T., and Anslyn, E. V.

(2007) Boronic acid based peptidic receptors for pattern-based saccha-ride sensing in neutral aqueous media, an application in real-life samples.J. Am. Chem. Soc. 129, 13575–13583.

(14) http://www.originlab.com/.(15) Hummel, G., Jobron, L., and Hindsgaul, O. (2003) Solid-

phase synthesis of a 1-thio-β-D-GlcNAc carbohydrate mimetic library.J. Carbohydr. Chem. 22, 781–800.

(16) Adam, G. C., Sorensen, E. J., and Cravatt, B. F. (2002)Trifunctional chemical probes for the consolidated detection andidentification of enzyme activities from complex proteomes. Mol. CellProteomics 1, 828–835.

396 dx.doi.org/10.1021/bc100376x |Bioconjugate Chem. 2011, 22, 388–396

Bioconjugate Chemistry ARTICLE

(17) Springsteen, G., andWang, B. (2002) A detailed examination ofboronic acid-diol complexation. Tetrahedron 58, 5291–5300.(18) Sandanayake, K. R. A. S., Nakashima, K., and Shinkai, S. (1994)

Specific recognition of disaccharides by trans-3,30-stilbenediboronicacid - rigidification and fluorescence enhancement of the stilbeneskeleton upon formation of a sugar-stilbene macrocycle. Chem. Commun.1621–1622.(19) Morin, G. T., Paugam, M. F., Hughes, M. P., and Smith, B. D.

(1994) Boronic acids mediate glycoside transport through a liquidorganic membrane via reversible formation of trigonal boronate esters.J. Org. Chem. 59, 2724–2728.(20) Gray, C. W., Johnson, L. L., Walker, B. T., Sleevi, M. C.,

Campbell, A. S., Plourde, R., and Houston, T. A. (2005) Specific sensingbetween inositol epimers by a bis(boronate). Bioorg. Med. Chem. Lett. 15,5416–5418.