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Single-stranded DNA (ssDNA) production in DNA aptamer generation

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Citartan Marimuthu,a Thean-Hock Tang,*a Junji Tominaga,b Soo-Choon Tanc and Subash C. B. Gopinath*b
Received 29th September 2011, Accepted 2nd January 2012 DOI: 10.1039/c2an15905h The discovery that synthetic short chain nucleic acids are capable of selective binding to biological targets has made them to be widely used as molecular recognition elements. These nucleic acids, called aptamers, are comprised of two types, DNA and RNA aptamers, where the DNA aptamer is preferred over the latter due to its stability, making it widely used in a number of applications. However, the success of the DNA selection process through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments is very much dependent on its most critical step, which is the conversion of the dsDNA to ssDNA. There is a plethora of methods available in generating ssDNA from the corresponding dsDNA. These include asymmetric PCR, biotinstreptavidin separation, lambda exonuclease digestion and size separation on denaturing-urea PAGE. Herein, different methods of ssDNA generation following the PCR amplication step in SELEX are reviewed.

1. Introduction
The inherent capabilities of the nucleic acids to fold into apparent three-dimensional structures accord them the ability to interact with various target molecules. In principle, nucleic acids with the highest binding afnity and specicity against the target molecules can be selectively generated.1 These nucleic acids are termed aptamers, which are single stranded oligonucleotides DNA/RNA. The term aptamer derives from the word aptus which is dened as tting, while meros denotes particle.2 Aptamers are generated by the process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The

a Infectious Disease Cluster, Advanced Medical & Dental Institute (AMDI), Universiti Sains Malaysia, 13200 Kepala Batas, Penang, Malaysia. E-mail: tangth.amdi@usm.edu.my; Fax: +6045792960/5742099; Tel: +604-579 1990 b Nanoelectronics Research Institute, National Institute of Advanced Industrial Science & Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, 3058562, Japan. E-mail: gopi-subashchandrabose@aist.go.jp; Fax: +81-29861 2930; Tel: +81-298-60 5620 c Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Penang, Malaysia

Citartan Marimuthu is currently a PhD candidate in the Advanced Medical and Dental Institute, Universiti Sains Malaysia since 2009. He has been working in the eld of SELEX (Systematic Evolutions of Ligands by Exponential Enrichment) for the past 2 years. Throughout his period of candidature, he has published 2 papers.
Citartan Marimuthu

Assoc. Prof. Thean-Hock Tang obtained his doctorate degree in the eld of RNomic from the University of Muenster, Germany. Currently, he is Head of Infectious Disease Cluster, Advanced Medical & Dental Institute, Unversiti Sains Malaysia, Malaysia. His research group focuses on Experimental RNomic identication and characterization of non-protein-coding RNA Thean-Hock Tang (npcRNA) from infectious agents. Besides, he is also interested in the development of molecular diagnostic methods based on new npcRNA gene markers and RNA technology, i.e. SELEX technology for the generation of aptamers.
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process commences with the incubation of the large library of randomized nucleotide sequences (forming different folding structures) with the target of interest. Following this, the unbound sequences are separated from the bound molecules. The separation method is one of the most signicant steps in any SELEX procedure and includes separation using nitrocellulose lters,3 sepharose,4 magnetic beads,5 Capillary Electrophoresis (CE) separation, Flow Cytometry (FC),6 Electrophoretic Mobility Shift Assay (EMSA)7 and Surface Plasmon Resonance.8 The target-bound sequences are then eluted and amplied. This process of selection and amplication is repeated till the ligands with the highest binding afnity to the target are obtained. These molecules are cloned and sequences obtained are analyzed (Fig. 1). To date, aptamers have been selected against a myriad of target molecules such as proteins,914 amino acids,1518 drugs,19,20 cofactors2125 and metal ions.26,27 The utilization of the aptamer in any application requires the aptamer to be stable, and resistant to the enzymatic activities of nucleases. DNA aptamers are more stable than RNA aptamers due to the absence of the hydroxyl group at the 20 -end of the

DNA molecule. On the other hand, due to their reactivity the RNA aptamers may lead to the generation of cyclic-20 ,30 -phosphate, resulting in degradation.28 The afnity of DNA aptamer and RNA aptamer does not differ much.29 However, compared to RNA in vitro selection, DNA in vitro selection requires only a single enzymatic step in comparison to the three enzymatic steps of the former (transcription, reverse transcription, and PCR amplication).30 To ensure the successful production of DNA aptamer, efcient generation of ssDNA from dsDNA following the amplication step of SELEX is important as only ssDNA can form diverse structural conformations to enable binding to target molecules. Three-dimensional conformations of ssDNA are comprised of unpaired sites and sites that form stable secondary structures such as hairpin structures, pseudoknots,31,32 and quadruplex structures formed by planes of two neighboring G-quartets.33 These conformations are necessary to facilitate spatial arrangement of recognition elements for interaction with target molecules.34,35 However, the same secondary

Principal Research Scientist of the National Institute of Advanced Industrial Science and Technology (AIST), Japan, and visiting Professor of Craneld University (UK) and Tohoku University (Japan). He was the former director of Center for Applied Near-Field Optics Research in AIST by March 2010. He received his PhD (Materials) from Craneld Institute of Technology (CranJunji Tominaga eld University), UK, in 1991. After the R&D of rewritable phase-change optical discs in TDK Corporation, he moved to AIST in 1997. His specialities are thin lm coating and inorganic materials.

Fig. 1 Schematic representation of SELEX cycles, which involve the following steps to generate the DNA aptamer. Incubation of random oligonucleotide pool with the target molecule; Separation of bound from unbound nucleic acids; Elution of the bound nucleic acids; Amplication of the bound nucleic acids, ssDNA generation; and Cloning and sequence analysis.

Soo-Choon Tan

Dr S.C. Tan obtained his PhD in Pharmaceutical Analysis from Kings College London in 1996. He then joined the Doping Control Centre in University Science Malaysia, Penang, as a laboratory manager until 2011. He is currently an associate professor in the Institute for Research in Molecular Medicine in University Science Malaysia, Penang. His research interests include bioanalysis using chromatography and mass spectrometry, immunoassay, chiral drug metabolism and pharmacogenetics.

Subash C: B: Gopinath

Dr Subash C. B. Gopinath earned his doctorate degree from the University of Madras in 1999. The same year, he moved to Taiwan to work in the Institute of Academia Sinica. In 2003, he was awarded as JSPS Fellow and at present, he is holding a position in the National Institute of Advanced Industrial Science and Technology, Japan. His interest is in aptamer technology, biosensors, RNAligand interactions and vault non-coding RNAs in the multidrug resistance.

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structures cannot be displayed by double-stranded DNA which can only assume double helix conguration and contains no unpaired regions. In this article, different methods of ssDNA generation following dsDNA production are reviewed.

2. PCR amplication in SELEX

In the amplication step of any SELEX experiment, nucleic acids bound to the target molecules are usually recovered by heating with chaotropic reagents such as urea.36 Urea disrupts the hydrogen bonds and hydrophobic interactions within proteins, rupturing the secondary structures of the protein,37 and thereby releasing the bound DNA molecules from the target. These DNA molecules present in very minute amounts are concentrated by ethanol precipitation and amplied by PCR amplication. Optimization of the PCR cycles in SELEX experiment is very important to avoid overamplication. Overamplication increases background binders, which entails more SELEX cycles to achieve eventual good binding afnity and specicity towards the target molecule. This overamplication also leads to opportunistic mispairing, as the ssDNA template having a myriad of different sequences can actually pair with each other, leading to spurious and aberrant PCR products at a higher number of PCR cycles.38 It is imperative to ensure that the PCR cycles are added in the increments of 1 or 2 PCR cycles starting from the lowest PCR cycles possible (for example 4 cycles) in every SELEX round till the band of the expected size appears (Fig. 2). This is followed subsequently by large-scale PCR amplication to provide a large amount of template for the generation of ssDNA using the same number of PCR cycles obtained from the optimization. Bibby et al.39 have performed quantitative PCR (qPCR) on the supernatant from before and after incubation with the target molecule, wash fractions and on the eluate containing the target-bound sequences, in order to roughly estimate the number of PCR cycles required for the subsequent round of SELEX cycles to avoid overamplication. However, there are also several cases of DNA aptamer generation whereby the same number of PCR cycles are maintained throughout the course of SELEX, without determining the optimum number of PCR cycles required for amplication in each round of SELEX.16
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Bruno and Kiel40 empirically determined the optimum annealing temperature during the amplication step of SELEX though the annealing temperature is not critical for short PCR products such as DNA aptamers. This parameter is more signicant for PCR reactions involving long products or total genomic DNA as the template.40,41 Optimization of MgCl2 or primer concentrations, though not critical for dsDNA production, can also be carried out during the amplication of the starting pool of ssDNA library for SELEX. The optimum MgCl2 or primer concentrations are maintained throughout the SELEX cycles.

3. Asymmetric PCR
One of the most common forms of generating ssDNA in DNA aptamer production is by employing asymmetric PCR amplication which produces ssDNA due to the unequal concentration of sense and antisense primers used in the reaction mixture. There are two phases of amplication in the asymmetric PCR. The rst phase involves dsDNA production which proceeds exponentially followed by the second phase of linear amplication that produces ssDNA (Fig. 3a and b). This ssDNA generation continues until the amplication reaches a stage whereupon the increase in the number of DNA copies is restricted by the amount of enzyme present in the reaction mixture.42 In asymmetric PCR amplication, the most critical aspect that must be heeded is the ratio of primer amounts. Most of the asymmetric PCR amplication is unidirectional, relying on either the forward or reverse primer or dependent on the presence of both primers with different amounts, using dsDNA as the template. Boiziau et al.43 have generated DNA aptamer against TAR RNA element by performing asymmetric PCR amplication using dsDNA PCR product as the template with 100 pmol of forward primer. Following 30 PCR cycles, ssDNA is puried by phenol chloroform extraction and ethanol precipitation. Likewise, in the isolation of DNA aptamer against uorophore sulforhodamine B, double-stranded DNA prepared by large-scale PCR reaction was diluted ten-fold, to be used as the template for amplication using only the reverse primer with 10 cycles of amplication. This is followed by selective gel excision from polyacrylamide (PAGE) gel electrophoresis.44 Similarly, using the puried dsDNA PCR product as the template, DNA aptamer generation against the target chitin was also accomplished. By utilizing asymmetric PCR amplication of 45 cycles using the forward primer alone, the resulting ssDNA was puried by gel extraction from agarose.45 A similar procedure was widely exploited in the generation of DNA aptamers against tubulin,46 thrombin47 and kinetoplastid membrane protein-11 (KMP-11) of Leishmania infantum.48 Ding et al.49 have performed large-scale asymmetric PCR using the dsDNA as the template, with 100 pmol and 1 pmol of forward primer and reverse primer, respectively. Also, in several DNA aptamer studies, antisense and sense primers of different amounts and ratios were also utilized, as summarized in Table 1.5053 Asymmetric PCR has become the most prevalent procedure of generating ssDNA in DNA aptamer production, as it is economic and less tedious, involves the same parameters of the PCR amplication step in SELEX (except for primer ratios). Moreover, the PCR cycles can be increased to obtain higher amounts of ssDNA due to the non-exponential nature of amplication in asymmetric PCR.54 In addition, in a modied
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Fig. 2 Optimization of PCR cycles during amplication step of SELEX cycle 1, performed on the target protein erythropoietin. PCR amplication carried out in increments of 2 PCR cycles till the band of the expected size appears and aliquots analyzed on 3% agarose gel electrophoresis. Lane 1: 25 bp DNA ladder; Lane 2: 4; Lane 3: 6; Lane 4: 8; Lane 5: 10; Lane 6: 12; Lane 7: 14 PCR cycles. The optimum number of PCR cycles for large-scale amplication is 12.

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gel. However, there are also cases where the nal asymmetric PCR product was incubated directly with the target without further purication. In the DNA aptamer generation against 1,3-b-D-glucans and Asp f1 allergen of the pathogenic fungus Aspergillus, the asymmetric PCR product (following 8 PCR cycles with only 15 pmol forward primer) was used directly on these targets without any prior purication step.57,58 Equivalently, Ogawa and colleagues have applied the asymmetric PCR product directly for incubation onto the Arg-Gly-Asp region of the bronectin immobilized agarose gel column.59 Asymmetric PCR is the most economical method of generating ssDNA from very low starting material (dsDNA template). However, since it generates both ssDNA and dsDNA, the need for the subsequent purication of ssDNA can be circumvented by the alternative method of generating ssDNA, which is the biotinstreptavidin separation method.
Fig. 3 (a) Illustration of asymmetric PCR product analysis on agarose gel electrophoresis. Lane 1: symmetric PCR product (PCR with same amount of sense and antisense primer); Lane 2: asymmetric PCR product (PCR with a skewed amount of either sense or antisense primer). (b) Asymmetric PCR product analysis on 3% agarose gel electrophoresis. Lane 1: 25 bp DNA ladder; Lane 2: 50 pmol of sense primer only; Lane 3: 150 pmol of sense primer only; Lane 4: negative control (amplication without template). Asymmetric PCR product produces both ssDNA and dsDNA, which requires subsequent purication.

4. Biotinstreptavidin separations
The binding between streptavidin and biotin is the strongest noncovalent and biological interaction. Having the dissociation constant, Kd, in the order of 4 1014 M, biotinstreptavidin separation is the core of the derivation of ssDNA from the biotinylated PCR product.60 In the method of biotinstreptavidin separation, one of the primers is biotinylated and the resulting biotinylated PCR product is immobilized onto the streptavidincoated magnetic beads. Due to the high afnity of the streptavidin towards the biotin, the desired non-biotinylated strand is separated from the biotinylated strand by alkaline treatment (NaOH), and subsequently concentrated by ethanol precipitation (Fig. 4).61 Stevenson et al.62 relied on biotinstreptavidin separation to generate ssDNA from dsDNA in the SELEX of 14-3-3 proteins. In a different investigation using the intact viable B-cell Burkittss lymphoma cell line as the target, the ssDNA production was made possible by biotinstreptavidin separation, using the poly(ethylene glycol) unit to introduce biotin to one of the primers. The

asymmetric PCR known as Linear-After-The Exponential (LATE)-PCR, the efciency of the conventional asymmetric PCR is enhanced by adjusting the length and sequence of the primers, so that the melting temperature of the limiting primer is always equal or greater than that of the excess primer.55,56 However, the real problem in asymmetric PCR amplication is the production of ssDNA together with the dsDNA that can also bind non-specically to the target molecules. This necessitates selective ssDNA purication from polyacrylamide gel or agarose
Table 1 DNA aptamers generated by asymmetric PCR amplication Target Trans-activation response (TAR) RNA element Fluorophore sulforhodamine B Cellobiose Chitin Tubulin Kinetoplastid membrane protein-11 (KMP-11) of L. infantum Arg-Gly-Asp region of the bronectin MUC1 tumor marker Lipopolysaccharide of Escherichia coli Thermostable DNA mismatch binding protein (MutS) from Thermus aquaticus 1,3-b-D-Glucans Asp f1 allergen Thrombin Fibrils of Saccharomyces cerevisiae Sup35p PCR cycles 30 10 30 45 45 10 30 99 15 8 8 90 30 Primer

Dissociation Constant/nM 20 9 190 20 100 19 400 47.3 10 0.303 0.067 93.62 21.84 0.2 240

References 43 44 53 45 46 48 59 52 49 50 57 58 47 51

Forward (100 pmol) Reverse Forward (2667 pmol) Reverse (78 pmol) Forward Forward Digoxigenin labelled forward/reverse Forward (670 pmol) Reverse (20 pmol) Forward (100 nmol) Reverse (4 nmol) Forward (100 pmol) Reverse (1 pmol) Fluorescently labelled forward (50 pmol) Biotinylated reverse (2.5 pmol) Forward (15 pmol) Forward(15 pmol) Biotinylated forward Forward (50 pmol) Reverse (5 pmol)

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incorporation of poly(ethylene glycol) avoids the interference of biotin with the spatial formation of the aptamer. The streptavidin beads were packed into a mini column to capture the dsDNA PCR product containing biotin in one of the strands and was heated with Tris(2-carboxyethyl)phosphine to release the ssDNA.63 Similarly, biotinstreptavidin separation was widely used in the DNA aptamer generation of abrin toxin,64 tenascin C,65 Campylobacter jejuni strain A9a,66 Pseudomonas aeruginosa,67 Carcinoembryonic antigen,68 Phospholamban,69 Thermus aquaticus DNA polymerase,70 Colorectal cancer cell71 and Acetamiprid.72 Pagratis73 has demonstrated the streptavidin-induced electrophoretic mobility shift assay in preparing ssDNA. In this investigation, the resulting biotinylated PCR product following the interaction with streptavidin was resolved on denaturing urea-PAGE, generating two different bands migrating at different ratios. The much faster free ssDNA constituting the non-biotinylated strand can be puried from the PAGE.73 The SMART system is a combination of asymmetric PCR and biotinstreptavidin separation. The asymmetric PCR product containing biotin at one end was loaded onto the streptavidin immobilized column. Following washing to remove excess dNTPs, primers and other PCR components, streptavidin-bound ssDNA was eluted upon treatment with NaOH.74 The ease of removing the non-biotinylated strand from biotinylated strands with NaOH (which weakens the hydrogen bond between these strands) is responsible for the wide use of biotinstreptavidin separation to generate ssDNA in a number of DNA aptamer studies (Table 2). Though separation by biotin streptavidin interaction is very much favored, there are also possibilities for the undesired biotinylated strand to re-anneal with the complementary strands, resulting in the loss of tertiary structure required for target binding as well as increasing the number of non-specic binders. This is due to NaOH treatment that also fractures the hydrophobic, van der Waals forces, hydrogen bonds between biotin and streptavidin, as well as rupturing the surface loop of streptavidin that holds the biotin inside the b-barrel of the streptavidin molecules. As a result, the biotinylated strand enters the solution and re-anneals with the complementary strand.75 Moreover, streptavidin can also be the additional target for the binding of the nucleic acid along the course of the SELEX process. For example, due to its RGD motif-mimicking sequence, streptavidin presents an additional SELEX target in a cell-SELEX

experiment which leads to the aggregation of the cells.76 However, these problems associated with streptavidin can be alleviated by using lambda exonuclease digestion or size separation of the PCR product derived from the modied primers (on denaturing urea polyacrylamide gel electrophoresis).

5. Lambda exonuclease digestions

Originating from bacteriophage l, lambda exonuclease is an enzyme that involves in the repair of double-stranded breaks of the viral DNA.77 As an exodeoxyribonuclease, it selectively digests the phosphorylated strand(s) of double stranded DNA (dsDNA) from the 50 to the 30 end. Having 20 times more afnity for a phosphorylated 50 -end than a hydroxylated 50 -end, only non-phosphorylated single-stranded DNA remains after digestion (Fig. 5a).78 However, this enzyme has greatly reduced activity on single-stranded DNA, non-phosphorylated DNA and no activity detectable against nicked DNA and gapped DNA.79 A fast and efcient method of generating ssDNA with high quality and yield relying on lambda exonuclease digestion is possible. In our recent study, lambda exonuclease digestion of the PCR product from SELEX and subsequent purication of ssDNA by phenol/chloroform extraction were completed within 6068 min. This greatly reduces the time spent on one SELEX cycle. Though phenol/chloroform extraction results in some loss of ssDNA, this step eliminates the lambda exonuclease enzyme, which can be an additional target for the SELEX experiment.80 With the purpose of determining the progress of lambda exonuclease digestion on the dsPCR product, time course analysis of lambda exonuclease digestion can be carried out once before the initial step of the SELEX experiment. This can determine the optimum incubation period of the enzyme and the substrate (Fig. 5b). Bibby et al.39 have generated DNA aptamer against prion protein, PrPc, using lambda exonuclease digestion of the phosphorylated PCR product, whereby the reverse primer is phosphorylated. Lambda exonuclease digestion was carried out in a SELEX methodology called Volume Dilution Challenge with Microuidic Separation (VDCSELEX), where the volume dilution challenge was integrated with the microuidic selection process. The resulting ssDNA was puried with phenol/chloroform extraction followed by ethanol precipitation.81 There are several studies of DNA aptamer generation associated with lambda exonuclease digestion (Table 3).8284 Although lambda exonuclease digestion is efcient, this method is not in vogue due to the cost associated in purchasing the enzyme. In addition, incomplete digestion of the phosphorylated dsPCR product leads to the accumulation of the dsDNA in the reaction mixture. Phenol chloroform extraction to selectively purify ssDNA from the lambda exonuclease digestion mixture of incomplete digestion is challenging as this procedure can co-purify both ssDNA and dsDNA. Selective purication with denaturing-urea PAGE is impossible as both ssDNA and dsDNA migrate at almost the same rate. On the other hand, native PAGE purication results in a very low nal yield of ssDNA from the lambda exonuclease digestion product. The smeary appearance of ssDNA on the native PAGE due to different conformations of three-dimensional structures makes the selective excision of ssDNA tedious.8587 However, the problem of incomplete digestion can
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Fig. 4 Diagram of the ssDNA generation from biotinylated PCR product using streptavidin. Following the attachment of the biotinylated PCR product onto streptavidin, treatment with NaOH breaks the hydrogen bonds between the strands, releasing the unmodied ssDNA strand.

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Table 2 Examples of DNA aptamers generated by biotinstreptavidin separation Biotinylated primer Reverse Forward Reverse Forward Reverse Reverse Reverse Forward Reverse Reverse Reverse Reverse

Target T. aquaticus DNA polymerase Tenascin C B-cell Burkittss lymphoma cell line Carcinoembryonic antigen (CEA)

Dissociation constant/nM 40 1 43 560 20 28 9 292.8 53.1 0.7 0.2 4980

References 70 65 63 68 62 69 64 66 71 67 72

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14-3-3 Protein Phospholamban Abrin toxin C. jejuni strain A9a Colorectal cancer cell P. aeruginosa Acetamiprid

Fig. 5 (a) Illustration of the lambda exonuclease digestion on the phosphorylated PCR product. (b) Time course analysis of 10 units of lambda exonuclease digestion performed at 37  C using 1 mg of dsDNA. Lane 1: 25 bp DNA ladder; Lane 2: 15 min; Lane 3: 20 min; Lane 4: 25 min; Lane 5: 30 min; Lane 6: 35 min. The optimum time of lambda exonuclease digestion on 1 mg of the substrate is 1520 min.

be tackled during manufacturing by ensuring that the primer is phosphorylated to the highest degree possible. Upon the completion of synthesis, the blocking group which temporarily protects the phosphate group throughout the oligonucleotide synthesis must be completely removed.88,89

6. Size separations on denaturing urea polyacrylamide gel electrophoresis

A detectable differential migration of two strands of an amplicon on urea-denaturing PAGE can greatly facilitate selective excision
Table 3 DNA aptamer studies based on lambda exonuclease digestion

of any of the strands. Walder et al.90 have carried out PCR with one of the primers synthesized to contain any of the ribose residues (cleavable by ribonuclease) at the 50 and 30 regions. Following amplication, the ribose residue which is at the junction between the primer binding region and the randomized region of the SELEX amplicon can result in truncation of the primer binding region upon treatment with ribonuclease. Following the treatment, the resulting amplicon will separate into two strands of differing lengths, migrating at contrasting rates on denaturing urea-PAGE, enabling ssDNA purication (Fig. 6a). Similar principles apply in the study conducted by Williams and Bartel,91 in which one of the primers contains hexaethylene glycol and an extension of 20-nucleotides length of a string of adenosine. These modications impede positive strand elongation and create a size difference of the amplicon strands, respectively (Fig. 6b). Equivalently, Keefe et al.92 also demonstrated that introduction of the pH-labile base at the 30 -end of the reverse primer enables strand breakage of the amplicon upon alkaline treatment. The resulting strands of different sizes are subsequently separated on denaturing urea-PAGE. Urea breaks the hydrogen bonds between the strands of the amplicon as the DNA melting temperature is lowered, reducing by as much as 1 kcal mol1 in 3 M urea.9395 This creates differential migration that enables selective excision of the strand of interest with the aid of UV shadowing96 and by uorescence in the case of uorophore-modied primer.97 Moreover, the resolving ability of this amplicon can be further augmented by using silver-stained PAGE.98 In the generation of DNA aptamer against Diclofenac, the resulting amplicon of uorescently labeled forward primer was size-separated on urea-denaturing PAGE. The ssDNA was then puried with the aid of UV shadowing.99 Kim and coworkers have isolated DNA aptamer against ibuprofen, a widely used anti-inammation drug using size separation of amplicons

Target Hepatitis C virus (HCV) subtype 3a polymerase Prion protein Ovine follicle-stimulating hormone a subunit (oFSHa) Human epidermal growth factor receptor 2 (HER2) Streptavidin

Modied primer Phosphorylated reverse primer Phosphorylated reverse primer Phosphorylated reverse primer Phosphorylated reverse primer Phosphorylated reverse primer

Dissociation constant/nM 1.3 0.3 18 36.2 3.6

References 82 39 84 83 81

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requires different techniques (for example different subsequent purication), different optimizations, as well as various costs. Before starting any SELEX experiment, investigators must thoroughly revise each and every procedure accessible, to guarantee that pure ssDNA can be attained, with minimum background amplication to achieve maximum binding afnity towards the target molecule.
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Acknowledgements
Fig. 6 Size separation of the amplicons-derived-modied primers on urea-denaturing PAGE. The inclusion of the riboU (a) and HEGL (b) in the primers creates a size difference of the two strands of the PCR products, which migrate at different rates upon resolving on the urea denaturing-PAGE.

This work was funded by USM RU Grant 1001/CIPPT/813043. We would like to thank Universiti Sains Malaysia for awarding Academic Staff Training Scheme to C.M. for this study and the support rendered by the Advanced Medical and Dental Institute, USM, for his travelling and subsistence to Japan.

References
on urea-denaturing PAGE. In this study, one of the primers is modied with hexaethylene glycol (C13H287, HEGL), spacer that blocks the amplication of the poly-adenine region, creating a size difference upon resolving on urea-denaturing PAGE.100 Although the method of size separation on urea-denaturing PAGE is very efcient as the resulting ssDNA is clearly distinguishable, this method is relatively expensive. The cost of this procedure is associated with the extra functional groups that have to be conjugated to the primers compared to the unmodied primers. Furthermore, this method is very lengthy as much time is spent on completing the denaturing PAGE electrophoresis followed by selective purication of ssDNA, as is evident with very few reported DNA aptamers selected via this procedure (Table 3).101103
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7. Conclusions
The success of any DNA in vitro selection experiment (represented by the dissociation constants in Table 14) is not inuenced by the type of methods employed in generating ssDNA. Studies have proved that different schemes of selection will lead to the acquirement of aptamers that have similar structures, binding to the same epitope of the target. During the course of SELEX, only one epitope on the surface of the target becomes dominant in the presence of many other potential binding sites.104 Hence, the most important point is to ensure that the resulting ssDNA is selectively separated and puried as much as possible from the dsDNA. This is vital as dsDNA also stands the similar probability of being co-amplied alongside ssDNA, compromising the binding afnity of the true binders towards the target molecules. However, each of these methods available

Table 4 DNA aptamer studies based on size separation on denaturing urea-PAGE Dissociation Constant (nM) 172 160 22 42.7 1500 15

Target Multiple myeloma monoclonal protein Zinc nger protein Diclofenac Ibuprofen CD16a on natural killer cells

Modied primer Hexaethylene glycol-modied forward primer Hexaethylene glycol-modied reverse primer Fluorescently labelled primer Hexaethylene glycol-modied primer 30 -Ribo-modied reverse primer

References 101 102 99 100 103

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