structural prediction and binding analysis of hybridized aptamers

Received: 2 October 2009, Revised: 25 January 2010, Accepted: 4 February 2010, Published online in Wiley Online Library: 29 March 2010

Structural prediction and binding analysis ofhybridized aptamersJing Zhoua, Boonchoy Soontornworajita, Matthew P. Snipesa

and Yong Wanga*

Few studies were performed to investigate the molecular recognition capabilities of hybridized aptamers. This studyis aimed at applying both theoretical algorithms and experimental assays to examine the effects of hybridizationlength and region on the secondary structures and binding functionality of hybridized aptamers. The experimentalresults were significantly different from the structural predictions in many hybridization conditions. To explain thisdifference, we further proposed a novel equilibrium reaction model that can explicitly analyze the molecularinteractions between hybridized aptamers and their targets. We believe that the research findings and the novelmodel can be used to guide numerous hybridized aptamer-based applications. Copyright� 2010 John Wiley & Sons,Ltd.

Supporting information may be found in the online version of this paper

Keywords: nucleic acid aptamer; molecular recognition; intramolecular hybridization; intermolecular hybridization;secondary structure; aptamer discovery; aptamer truncation

INTRODUCTION

Nucleic acid aptamers are single-stranded nucleic acid nanos-tructures that are screened from DNA/RNA libraries with thetechnology named systematic evolution of ligands by exponen-tial enrichment (SELEX) (Ellington and Szostak, 1990; Tuerk andGold, 1990). Nucleic acid aptamers have attracted significantattention in various research areas because they have uniquemerits as affinity molecules. First, in principle, nucleic acidaptamers can be selected against anymolecule with high bindingaffinity and remarkable specificity (Jenison et al., 1994; Liu et al.,2003; Proske et al., 2005; Jaeger and Chworos, 2006; Lee et al.,2006; Bruno et al., 2009). Second, aptamers have tunable stabilityin biological environments and their biodegradability can becontrolled by the degree of nucleotide modification (Lee et al.,2006). For instance, the degradation of amethylated aptamer wasalmost undetectable during 96 h incubation in plasma (Burme-ister et al., 2005). Third, aptamers are small oligonucleotides.Thus, they have little immunogenicity or toxicity and can besynthesized with a standard chemical procedure (Lee et al., 2006).Because nucleic acid aptamers are oligonucleotides, they

can be transformed into hybridization formats in the presenceof a complementary oligonucleotide. The intermolecular hybrid-ization between nucleic acid aptamers and complementaryoligonucleotides has been applied to the development ofbiosensing probes (Wang et al., 2009; Yan et al., 2009), multivalentnanostructures (Di Giusto et al., 2006; Rinker et al., 2008),pretargeting systems (Zhou et al., 2009), intelligent hydrogels(Wei et al., 2008; Yang et al., 2008), and smart nanomachines(Dittmer et al., 2004). Despite the great usefulness of hybridizedaptamers, few studies have been carried out to clearly illustratehow the hybridization affects the molecular recognitioncapabilities of the aptamers.

Therefore, this study was aimed at investigating the effectsof hybridization length and region on the secondary structuresand binding functionality of a model aptamer. The modelaptamer can bind to human T cell lymphoblast-like CCRF-CEMcells with high affinity and specificity by recognizing humanprotein tyrosine kinase 7 (hPTK7) (Shangguan et al., 2006, 2007).Numerous complementary DNA oligonucleotides were usedto hybridize with different regions of this aptamer. Thehybridized aptamers were studied with both theoreticalalgorithms and experimental assays. Through this effort, weacquired important research findings, which not only advancethe understanding of molecular interactions between hybri-dized aptamers and their targets, but also provide valuableinformation for the future development of hybridizedaptamer-based systems.

MATERIALS AND METHODS

Cell culture

CCRF-CEM cells (CCL-119, human T lymphocytic leukemia cellline) were obtained from ATCC (Manassas, VA). All cell culturereagents were purchased from Invitrogen (Carlsbad, CA) unlessotherwise noted. CCRF-CEM cells were cultured and maintained

(wileyonlinelibrary.com) DOI:10.1002/jmr.1034

Research Article

* Correspondence to: Y. Wang, Department of Chemical, Materials & Biomole-cular Engineering, University of Connecticut, 191 Auditorium Road, Storrs, CT06269-3222, USA.E-mail: [email protected]

a J. Zhou, B. Soontornworajit, M. P. Snipes, Y. Wang

Department of Chemical, Materials & Biomolecular Engineering, University of

Connecticut, Storrs, CT 06269-3222, USA

J. Mol. Recognit. 2011; 24: 119–126 Copyright � 2010 John Wiley & Sons, Ltd.

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in RPMI medium 1640 (Hyclone, Logan, UT) supplemented with10% fetal bovine serum (FBS) and 100 units/mL penicillin–streptomycin (Mediatech, Manassas, VA) at 378C.

Secondary structure prediction

The secondary structures of single stranded and hybridizedaptamers were generated with the program RNAstructureversion 4.6 (http://rna.urmc.rochester.edu/rnastructure.html),because RNAstructure uses the most current thermodynamicparameters (Mathews et al., 2004). Of the secondary structuresgenerated, the most stable ones with the lowest free energieswere presented.

Gel electrophoresis analysis

The anti-hPTK7 aptamers and complementary oligonucleotideswere purchased from Integrated DNA Technologies (Coralville,IA). Their sequences are shown in Table 1. The aptamer and thecomplementary oligonucleotide were mixed in washing buffer(Dulbecco’s PBS with CaCl2 and MgCl2 was supplemented with4.5 g/L glucose and 5mM MgCl2). The solution was heated to728C and then slowly cooled down to 48C before being loadedinto a 16% native polyacrylamide gel. After electrophoresis, thegels were stained with ethidium bromide and imaged with theBio-Rad GelDoc XR Imager System.

Flow cytometry analysis

5� 105 CCRF-CEM cells were washed twice with 700mL ofwashing buffer and re-suspended in 200mL of binding solutionon ice. The binding solution was 50 nM of FAM-labeled full-lengthanti-hPTK7 aptamer or a hybridization mixture containing 50 nMof FAM-labeled anti-hPTK7 aptamer and 250 nM of complemen-tary oligonucleotide. The hybridization mixture was heated to728C and then slowly cooled down to 48C before the cell labelinganalysis. After 1 h of incubation, the cells were washed once with1mL of washing buffer and then subjected to flow cytometryanalysis. The flow cytometry was performed using the BDFACSCaliburTM flow cytometer (San Jose, CA). A total of 10 000events were counted. The experiment was performed induplicate. The mock solution was a blank binding buffer(washing buffer supplemented with 1mg/mL BSA and 20% FBS).Flow cytometry was also performed for a competition assay. In

this assay, the binding solutions were prepared with 50 nM ofFAM-labeled full-length aptamer (i.e., apt) and different concen-trations of the unlabeled full-length aptamer, the truncated 40-ntaptamer, or the scrambled 40-nt aptamer.

Confocal microscopy imaging

CCRF-CEM cells were fluorescently labeled with the sameprocedure as described in the flow cytometry experiment. Cellsuspension (40mL) was dropped on a glass chamber slide and

Table 1. Sequences of the original full-length aptamer, the 73-nt aptamer, the 40-nt aptamer, the scrambled 40-nt aptamer, andcomplementary oligonucleotides

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examined by single focal plane imaging. The imaging equipmentwas a Leica SP2 confocal laser scanning microscope equippedwith Ar/ArKr, Gre/Ne, and He/Ne lasers.

RESULTS

Gel electrophoresis analysis of intermolecular hybridization

A series of complementary oligonucleotides were used tohybridize either the 30 or the 50 region of the full-lengthanti-hPTK7 aptamer. We used 15-, 25-, and 35-nt longcomplementary oligonucleotides as examples to illustrate thehybridization efficiency. As shown in the polyacrylamide gelelectrophoresis images (Figure 1), the yield of hybridized aptamerincreased with the molar ratio of complementary oligonucleotideto aptamer. At a molar ratio of 1:5, all the aptamers were virtuallytransformed into their hybridization formats.

Prediction of the secondary structures of hybridizedfull-length aptamers

Previous studies have shown that the full-length anti-hPTK7aptamer has 40 essential nucleotides (Shangguan et al., 2007).The lowest-energy secondary structures of the full-lengthaptamer and the 40 essential nucleotides are shown inFigure 2A and B, respectively. The 40 essential nucleotides formthe same secondary structure in both cases. To confirm thebinding functionality of the truncated 40-nt aptamer andunderstand the role of nonessential nucleotides in affectingthe binding ability of essential nucleotides, a competition assaywas performed (Figure 2C). The binding profiles show that thetruncated 40-nt aptamer without the nonessential nucleotidesexhibits slightly higher binding affinity.When the full-length anti-hPTK7 aptamer is hybridized with

complementary oligonucleotides, the essential nucleotides eitherretain the same structure or change into completely differentstructural formats (Figure 3). It is dependent on the hybridizationregion and length. For hybridization at the 30 region, when theaptamer is hybridized with 3CO15, these essential nucleotidesretain the same lowest-energy structure. When the hybridizationlength is increased over 15-nt, the essential nucleotides exhibit asignificantly different secondary structure. For hybridization atthe 50 region, when the aptamer is hybridized with 5CO15through 5CO33, the essential nucleotides are able to retain thesame secondary structure.

Determination of the binding functionality of hybridizedfull-length aptamers

Because the structure is a dominant factor in determiningthe binding functionality of an aptamer (Jayasena, 1999), the

structural analysis suggests that the hybridized aptamers canretain their binding functionality if the structure of the essentialnucleotides does not change. Otherwise, the binding function-ality should be lost, or at least significantly decreased. However,the results acquired from cell binding assays are only in partialagreement with the structural analysis.For hybridization at the 30 region, when the hybridization

length was 15-nt, the hybridized aptamer could bind to the cellsthe same as the original full-length aptamer (Figure 4A and B).When the hybridization length was increased over 15-nt, the celllabeling efficiency gradually decreased in the range between 15-and 25-nt. After that, there was virtually no labeling. These resultsindicate that hybridized aptamers gradually lost the bindingfunctionality. However, one critical question is whether thedecrease of cell labeling efficiency was due to the competitionbetween complementary oligonucleotides and aptamers to thesame target. To address this question, we performed another twoexperiments. The first one was to use 3CO25 as a model tocompare the binding abilities of apt (i.e., the full-length 88-ntaptamer), 3CO25þapt, apt40 (i.e., the truncated 40-nt aptamerwithout nonessential nucleotides), 3CO25þapt40, and mocksolution. As shown in Supplementary Figure 1, the intermolecularhybridization between 3CO25 and apt40 due to short hybrid-ization length was negligible. If the decrease in cell labeling wasdue to the competition between complementary oligonucleo-tides and aptamers not the hybridization with essentialnucleotides, the cell labeling efficiency would be decreased toa similar level in both 3CO25þapt and 3CO25þapt40 cases.However, the hybridized apt (i.e., 3CO25þapt) showed significantdecrease in cell labeling whereas the apt40 (i.e., 3CO25þapt40)did not (Figure 4C). We also studied the effect of theconcentration of FAM-labeled complementary oligonucleotideon the nonspecific cell labeling. The nonspecific labeling didoccur but was much weaker than the specific cell labeling

Figure 2. Comparison between apt and apt40. (A) Secondary structure

of apt. (B) Secondary structure of apt40. (C): Binding competition; 50 nM

FAM-labeled apt mixed with different concentrations of unlabeled apt,apt40, and scrambled apt40, respectively. The percentages of labeled cells

are presented.

Figure 1. Gel electrophoresis analysis of hybridized aptamers. (A) Effect of molar ratio on hybridization efficiency. (B) Hybridization of the full-lengthaptamer (i.e., apt) with representative complementary oligonucleotides at the 30 region. (C) Hybridization of apt with representative complementary

oligonucleotides at the 50 region. The molar ratio of apt to complementary oligonucleotide: 1 to 5. DL: 10 bp DNA ladder; apt: the full-length aptamer.

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Figure 3. Representative structures of apt hybridized with complementary oligonucleotides. (A) Hybridization at the 30 region. (B) Hybridization at the

50 region.

Figure 4. Determination of binding functionality of hybridized aptamers with flow cytometry. (A–C) Hybridization with complementary oligonucleo-

tides at the 30 region. (D–F) Hybridization with complementary oligonucleotides at the 50 region. Both the histograms and the percentages of labeled cells

are presented for clear legibility.


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(Supplementary Figure 2). Taken together, the results demon-strate that the decrease of binding ability was primarily due to thehybridization of essential nucleotides by complementary oligo-nucleotides.For hybridization at the 50 region, when the hybridization

length was between 15- and 25-nt, the intermolecularhybridization did not affect the binding functionality of theaptamer (Figure 4D and E). Surprisingly, when the hybridizationlength was between 25- and 35-nt, the binding efficiency was awave-shaped function of the hybridization length. This trend wasreproduced in separate experiments (Supplementary Figure 3).To further understand how intermolecular hybridization at the 50

region affects the binding functionality of the aptamer, weincreased the hybridization length over 35-nt. As shown inFigure 4F, the binding ability was significantly decreased with theincrease of hybridization length over 35-nt. These data show thatit is more difficult for the essential nucleotides to form the originalfunctional structure to recognize its target with the increase ofhybridization length in the essential region.We also used confocal microscopy imaging to examine the

cells labeled by the hybridized aptamers. The confocalmicroscopy images were consistent with the flow cytometryresults (Figure 5).

Examination of the properties of hybridized 73-nt aptamers

To better understand the binding function of the aptamer afterthe hybridization at the 50 region, we removed the first 15nucleotides at the 30 region. The essential nucleotides of apt73exhibit a significantly different secondary structure but retain thesame secondary structure when apt73 is hybridized with 5CO15through 5CO33 (Figure 6A). On the other hand, the essentialnucleotides exhibit a completely different secondary structurewhen apt73 is hybridized with 5CO35.In terms of cell labeling, apt73 without hybridization did not

completely lose its binding functionality. Instead, it exhibited aslightly decreased cell labeling efficiency, indicating thatnonessential nucleotides interfere with but did not completelyblock the binding of essential nucleotides. Apt73 could retainthe same binding capability when hybridization length variedbetween 15 and 35 (Figure 6B). These data are significantlydifferent from the results acquired from the hybridized full-length

aptamer (Figure 4D and E). When the hybridization length wasgreater than 35-nt (Figure 6C), the binding capability started tosignificantly decrease.

DISCUSSION

Roles of nucleotides of an aptamer in binding to the target

Nucleic acid aptamers are single-stranded oligonucleotides thattend to form a number of thermodynamic nanostructures,which determine if the aptamers can bind to their targets withhigh affinity or specificity (Jayasena, 1999). However, differentregions of a given aptamer play different roles in binding to itstarget. The nucleotides in contact with the target are usually10–15-nt long and exhibit secondary structures such as hairpinloops, G-quartet loops, bulges, or pseudoknots (Gold et al.,1995). However, it is improper to simply eliminate thenucleotides of an aptamer that do not directly contact thetarget because some of these nucleotides play an importantrole in supporting the interactions between the contactnucleotides and their target (Gold et al., 1995). Thus, thenucleotides that either bind to the target or facilitate thebinding are the essential nucleotides for a functional aptamer.However, a full-length aptamer selected from nucleic acidlibraries is generally 80–100-nt long and the number of essentialnucleotides is approximately 25–40-nt (Jayasena, 1999). Thus, alot of nucleotides in the selected aptamers do not have directcontact with the target or enhance the formation of thefunctional structure. These nucleotides are nonessential andcan be eliminated during aptamer truncation.Interestingly, our experimental results (Figure 4D and E)

showed that the existence of some nonessential nucleotidescould interfere with the aptamer–target interactions when othernonessential nucleotides were hybridized. If this was not the case,the hybridized aptamers should be able to bind to the targetthe same as the full-length aptamer. This analysis was furtherconfirmed by the examination of the binding functionality of the73-nt aptamer. This truncated format does not have the first 15nucleotides of the full-length aptamer at the 30 end. Withoutthese 15 nucleotides, the hybridized 73-nt aptamers could bindto the cells the same as the original aptamer (Figure 6B).

Figure 5. Confocal microscopy imaging of labeled cells. (A) Aptamers were hybridized at the 30 region. (B) Aptamers were hybridized at the 50 region.



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A novel equilibrium reaction model for the analysis ofinteractions between a hybridized aptamer and its target

When hybridized aptamers interact with target molecules, thesystem involves three components: an aptamer, a complemen-tary oligonucleotide, and a target. The aptamer interacts withboth the target and the complementary oligonucleotide. Inaddition, the nucleotides of the aptamer will not only formintramolecular structures, but also form a double-strandedstructure with the complementary oligonucleotide via inter-molecular hybridization. Thus, the intramolecular hybridizationcompetes with the intermolecular hybridization in the system.The complexity of these issues is illustrated by our experimentalresults. However, these issues cannot be completely addressed bythe structural predictions. The difference between the exper-imental results and the structural predictions is presumablybecause current algorithms predict secondary structures in anenvironment without target molecules whereas it is common fornucleic acid aptamers to undergo conformational changes uponbinding to a target (Yang et al., 1996; Williamson, 2000). Here, wepropose a novel equilibrium reaction model for the analysisof interactions between a hybridized aptamer and its target(Figure 7).

This model is used to describe the variation of bindingcapability in different hybridization conditions. If the interactionsbetween nonessential nucleotides and essential nucleotides arequite strong (Figure 7A), the functional structure of the essentialnucleotides is interfered with. Thus, the yield of aptamer–target

Figure 6. Analysis of secondary structures (A) and binding functionality (B and C) of hybridized 73-nt aptamers.

Figure 7. Proposed mechanism of molecular recognition between ahybridized aptamer and its target. (A) The nonessential nucleotides of the

aptamer are hybridized. (B) The essential nucleotides of the aptamer are

hybridized.


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complexes decreases. Otherwise, if the interactions are negli-gible, the essential nucleotides can retain the functional structureand the yield of the aptamer–target complexes is not affected.This model can explicitly explain the results shown in Figure 4Dand E.This equilibrium reaction model (Figure 7B) can also explain

the binding functionality of an aptamer when its essentialnucleotides are hybridized with complementary oligonucleo-tides. The interactions between complementary oligonucleotidesand essential nucleotides become stronger with an increase ofhybridization length because the number of base pairs increases.Thus, it is more and more difficult for the essential nucleotides tore-form the original functional structure. Figures 4 and 6 supportthis analysis. On the other hand, if essential nucleotides havevery strong intramolecular interactions and the intermolecularhybridization between essential nucleotides and complementaryoligonucleotides is short (e.g., the hybridization between 73-ntaptamer and 5CO35 as shown in Figure 6B), the functionalstructures of hybridized aptamers may not be significantlyinterfered with.Our results and model analysis have advanced the under-

standing of the functionality of hybridized aptamers. However,we have to realize that a lot of factors can affect aptamer–targetinteractions, including not only conformational change, but alsoelectrostatic interactions, hydrogen bonding, hydrophobic forces,etc. In addition, though the algorithm-based structural predictionhas been intensively investigated based on experimentallydetermined thermodynamic parameters and free energy mini-mization principles (Zuker, 2003), this method still has limitation.For instance, it is limited for the prediction of tertiary structuresand is still limited for disclosing structural changes at thepresence of target molecules as shown by our experimentalresults. In addition, the model we established is useful forqualitative analysis of dynamic change of secondary structures.However, a quantitative analysis will provide more understanding.

Therefore, one critical issue in our future work will be tosystematically investigate the structures of hybridized aptamerswith other means such as X-ray crystallography, nuclear magneticresonance, and biochemical approaches (e.g., in-line probing)(Patel, 1997; Brunel and Romby, 2000; Wakeman and Winkler,2009).

CONCLUSIONS

In summary, anti-hPTK7 aptamers and numerous comp-lementary oligonucleotides were used to investigate thesecondary structures and binding functionality of hybridizedaptamers. Through this study, we acquired two importantfindings. First, in certain conditions, the existence ofnonessential nucleotides of aptamers can interfere with thebinding of essential nucleotides to their targets. Second,dependent on hybridization length and region, hybridizedessential nucleotides have different capabilities of formingfunctional structures to recognize the targets. Based on thesefindings, we proposed a novel equilibrium reaction model toexplicitly analyze the molecular interactions between hybri-dized aptamers and their targets. The research findings andthe novel model not only advance the understanding ofaptamer–target recognition, but also provide valuableinformation for the future development of hybridizedaptamer-based systems.

Acknowledgements

The authors thank Dr. Carol Norris for technical support. Thiswork is supported in part by grants from the US NationalScience Foundation (DMR-0705716) and the UConn ResearchFoundation.

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structural prediction and binding analysis of hybridized aptamers

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