WO2011060557A1

WO2011060557A1 - Method for selection of aptamers

Info

Publication number: WO2011060557A1
Application number: PCT/CA2010/001887
Authority: WO
Inventors: Xing-fang LI; Jing Zhang; Yanming Liu; X. Chris Le
Original assignee: Governors Of The University Of Alberta
Priority date: 2009-11-20
Filing date: 2010-11-22
Publication date: 2011-05-26

Abstract

A method of selecting aptamers which specifically bind to target molecules is provided in which the target molecules are separated by gel electrophoresis and transferred onto a membrane. The membrane is incubated with a randomized mixture of aptamers, particularly single stranded DNA, under conditions to form aptamer-target molecule complexes on the membrane. The membrane is washed or contacted with DNAse I to remove unbound aptamers and weakly bound aptamers. The bound aptamers are eluted from the aptamer-target molecule complexes, amplified and sequenced.

Description

METHOD FOR SELECTION OF APTAMERS

FIELD OF INVENTION

The present invention relates to a method for efficient and simultaneous selection of aptamers which specifically bind to target molecules.

BACKGROUND OF THE INVENTION

Aptamers are short oligonucleotides (single stranded DNA or RNA) which can form three-dimensional structures that specifically bind to a wide range of targets including, for example, proteins, organic molecules, and inorganic molecules with high affinity and specificity (Ellington and Szostak, 1990; Tuerk and Gold, 1990; Jayasena, 1999; Patel and Suri, 2000; Clark and Remcho, 2002; Luzi et al, 2003; You et al, 2003). The binding affinities of aptamers to proteins are similar or even higher than those of antibodies with typical dissociation constants (Kd values) of micromolar to low picomolar range (Jenison et al, 1994).

Compared to antibodies, aptamers are easier to produce and inexpensive since the generation process occurs in vitro without the need for animals. In theory, aptamers can be generated against any target protein, and the binding target site of the protein can be determined. Once sequenced, aptamers can be synthesized at lower cost than antibodies, and can be easily modified with different chemical groups to enhance chemical properties such as stability or resolvability, and to achieve various functions (Nimjee et al, 2005). Aptamers may be used in a variety of analytical, bioanalytical, therapeutic and diagnostic applications including, for example, protein identification and purification; inhibition of receptors or enzyme activities (Mann et al, 2005); and detection of proteins from bacteria in environmental or clinical samples.

The conventional approach for generating aptamers is through systematic evolution of ligands by exponential enrichment (SELEX) by which target-specific aptamers are selected and synthesized in vitro from a random aptamer library (Ellington and Szostak, 1990; Tuerk and Gold, 1990). SELEX typically involves incubation of ligand sequences with a target; partitioning of ligand-target complexes from unbound sequences via affinity methods; and amplification of bound sequences (FIG. 1). In the incubation step, nucleic acid libraries are incubated with target molecules in an appropriate buffer at a desired temperature. After binding, the R A/ssDNA aptamer-target complexes are separated from nonspecific molecules. Bound sequences are regenerated by enzymatic amplification processes. The amplified molecules are then used in the next round of selection. Selecting sequences which have the highest specificity and affinity against the target typically requires eight to twelve cycles. The selected

oligonucleotides are analyzed for their sequences and structures after cloning and sequencing. After the sequence of an aptamer is determined, the aptamer can be easily generated through nucleic acid synthesis, and its binding affinity and specificity to a specific target can be validated.

However, SELEX is time-consuming, taking weeks to months to achieve high efficiency and specificity. The number of aptamers with high specificity is limited due to low throughput since one specific aptamer is typically generated for one target protein at one time. Aptamers with high specificity for detection of bacteria are rare, with development of such aptamers requiring tedious separation and purification of multiple target proteins from culture. A capillary electrophoresis based SELEX method can be used to generate aptamers against multiple proteins; however, capillary electrophoresis separation of proteins in bacterial lysates tends to be incomplete and identification of the target proteins is difficult.

SUMMARY OF THE INVENTION

In one aspect, the invention comprises a method of selecting aptamers which specifically bind to target molecules comprising the steps of: (a) separating the target molecules by gel electrophoresis;

(b) transferring the separated target molecules out of the gel and onto a membrane; (c) incubating the membrane with a randomized mixture of aptamers under conditions to form aptamer-target molecule complexes on the membrane;

(d) washing the membrane to remove unbound aptamers and weakly bound aptamers;

(e) optionally, contacting the membrane with DNAse I to digest unbound aptamers and weakly bound aptamers; and

(f) eluting the bound aptamers from the aptamer-target molecule complexes.

In one embodiment, the eluted aptamers may be amplified and the sequences of the eluted aptamers determined. In one embodiment, the aptamers may be selected with a single cycle of binding, washing, elution and characterization of the eluted aptamers.

In one embodiment, the aptamer comprises single-stranded DNA (ss-DNA) or single stranded RNA (ss-RNA). In one embodiment, the method further comprises amplifying uneluted ss-DNA remaining on the membrane, and determining the sequences of the uneluted ss-DNA. In one embodiment, the gel electrophoresis is sodium dodecyl sulfate-polyacrylamide gel

electrophoresis. In one embodiment, the membrane is a polyvinyldifluoride membrane. In one embodiment, the target molecules are bacterial proteins. In one embodiment, the bacterial protein is E. coli protein formamidopyrimidine DNA glycosylase. In one embodiment, the bacterial proteins are extracted from cell lysate and separated by 2D gel electrophoresis followed by western blotting. In one embodiment, the target molecules are viral proteins. In one embodiment, the viral protein is the core protein of hepatitis B virus.

In one embodiment, the target molecules are mammalian cell proteins. In one embodiment, the mammalian cell proteins are extracted from cell lysate and separated by 2D gel electrophoresis followed by western blotting. In one embodiment, the washing of step (d) is conducted at least twenty-five times. In one embodiment, the elution of step (f) is conducted at least fifteen times. In one embodiment, the elution of step (f) comprises gradient elution with stepwise increases of urea concentration. In one embodiment, the amplification is conducted for 19 to 20 cycles. In one embodiment, the amplification includes adding dimethyl sulfoxide to a PC mixture. In one embodiment, the amplification is conducted at an annealing temperature of 56°C.

In another aspect, the invention may comprise a method of selecting and identifying an aptamer which specifically binds to a target molecule, comprising a single cycle of the steps of:

(a) transferring the target molecules onto a physical support;

(b) incubating the physical support with a randomized mixture of aptamers under conditions to form aptamer-target molecule complexes on the membrane;

(c) washing the membrane to remove unbound aptamers and weakly bound aptamers;

(d) optionally, contacting the membrane with DNAse I to digest unbound aptamers and weakly bound aptamers;

(e) eluting the bound aptamers from the aptamer-target molecule complexes; and

(f) characterizing the eluted aptamers.

The physical support may comprise a nitrocellulose or polyvinylidene difluoride membrane. The eluted aptamers may be characterized by amplification and sequencing.

Additional aspects and advantages of the present invention will be apparent in view of the description, which follows. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of an exemplary embodiment with reference to the accompanying simplified, diagrammatic, not-to-scale drawings:

FIG. 1 is a schematic diagram showing a prior art SELEX method.

FIG. 2 is a schematic diagram showing one embodiment of the method of the invention. FIG. 3 is a photograph of a PVDF membrane stained with Coomassie Brilliant Blue showing low range SDS-PAGE molecular weight standards (lanes 1 and 2), and BSA standards (mw -67 kDa) with two different loadings (5 μg for lanes 3 to 5; 15 μg for lanes 6 and 7).

FIG. 4 is a photograph of a PVDF membrane stained with Coomassie Brilliant Blue after five washes with binding buffer and three washes with elution buffer showing low range SDS- PAGE molecular weight standards (lane 1), and BSA standards (mw -67 kDa) with two different loadings (5 μg for lanes 2 to 4; 15 μg for lanes 5 and 6).

FIG. 5 is a photograph of a PVDF membrane with or without washing and elution showing low range SDS-PAGE molecular weight standards (ovalbumin included) (lanes 3 and 6); BSA and thrombin (lanes 1 and 4); and Fpg protein and lysozyme (lanes 2 and 5). The PVDF membrane was stained with Coomassie Brilliant Blue after thirty washes with binding buffer and twenty washes with elution buffer (left panel). The PVDF membrane was stained with

Coomassie Brilliant Blue after transfer (right panel).

FIG. 6 is a photograph of a 12% polyacrylamide gel of PCR products showing 100 bp DNA ladder (lane 1) and PCR products applied with PCR cycle numbers 19-24 (lanes 2-7).

FIG. 7 is a photograph of a 12% polyacrylamide gel of PCR products with and without DMSO (5% PCR mixture volume) showing 100 bp DNA ladder (lanes 1 and 6); nineteen cycles (lanes 2-5); twenty cycles (lanes 7-10); and PCR products under different conditions: without DMSO (lanes 2 and 7); with DMSO (lanes 3 and 8); without template and DMSO (lanes 4 and 9); with DMSO without template (lanes 5 and 10).

FIG. 8 is a graph of the actual concentration of DNA samples and the calculated concentration for calibration of UV absorbance detection.

FIG. 9 is a photograph of PCR results of washing solutions 11-15 showing 100 bp DNA ladder (lane 1); washing solutions 11-15 after washing step (lanes 2-6); and negative control (lane 7).

FIG. 10 is a photograph of PCR results of washing solutions 16-20 showing 100 bp DNA ladder (lanes 1 and 5); washing solutions 16-18 (lanes 2-4); washing solutions 19 and 20 (lanes 6 and 7); and negative control (lane 8).

FIG. 11 is a photograph of PCR results of elution solutions 5-10 with the target protein being BSA and showing 100 bp DNA ladder (lanes 1 and 7); elution solutions 6-10 (lanes 2-6); and negative control (lane 8).

FIG. 12 is a photograph of PCR results of elution solutions 5-10 with the target protein being thrombin and showing elution solutions 6-10 (lanes 1-5); 100 bp DNA ladder (lane 6); and negative control (lane 7).

FIG. 13 is a photograph of PCR results of elution solutions 5-10 with the target protein being lysozyme and showing 100 bp DNA ladder (lanes 1 and 8); elution solutions 6-10 (lanes 2-6); and negative control (lane 7).

FIG. 14 is a photograph of PCR results of elution solutions 5-10 with the target protein being Fpg protein and showing 100 bp DNA ladder (lane 1); elution solutions 6-10 (lanes 2-6); and negative control (lane 7). FIG. 15 is a photograph of PCR results of elution solutions 5-10 with the target protein being ovalbumin and showing 100 bp DNA ladder (lane 1); elution solutions 6-10 (lanes 2-6); and negative control (lane 7). FIG. 16 is a photograph of PCR results of washing solutions 16-23 showing 100 bp DNA ladder (lane 1); washing solutions 16-23 (lanes 2-9); and negative control (lane 10).

FIG. 17 is a photograph of PCR results of washing solutions 24-30 showing 100 bp DNA ladder (lane 1); washing solutions 24-30 (lanes 2-8); and negative control (lane 9).

FIG. 18 is a photograph of PCR results of elution solutions 6-10 showing 100 bp DNA ladder (lane 1); elution solutions 6-10 (lanes 2-6); and negative control (lane 7).

FIG. 19 is a photograph of PCR results of elution solutions 11-15 showing 100 bp DNA ladder (lane 1); elution solutions 11-15 (lanes 2-6); and negative control (lane 7).

FIG. 20 is a photograph of PCR results of elution solutions 6-10 showing 100 bp DNA ladder (lane 1); elution solutions 6-10 (lanes 2-6); and negative control (lane 7). FIG. 21 is a photograph of PCR results of elution solutions 11-15 showing 100 bp DNA ladder (lane 1); elution solutions 11-15 (lanes 2-6); and negative control (lane 7).

FIG. 22 is a photograph of PCR results of elution solutions 6-10 showing 100 bp DNA ladder (lane 1); elution solutions 6-10 (lanes 2-6); and negative control (lane 7).

FIG. 23 is a photograph of PCR results of elution solutions 11-15 showing 100 bp DNA ladder (lane 1); elution solutions 11-15 (lanes 2-6); and negative control (lane 7).

FIG. 24 is a photograph of PCR results of elution solutions 6-10 showing 100 bp DNA ladder (lane 1); elution solutions 6-10 (lanes 2-6); and negative control (lane 7). FIG. 25 is a photograph of PCR results of elution solutions 1 1-15 showing 100 bp DNA ladder (lane 1); elution solutions 11-15 (lanes 2-6); and negative control (lane 7).

FIG. 26 is a photograph of PCR results of elution solutions 6-10 showing 100 bp DNA ladder (lane 1); elution solutions 6-10 (lanes 2-6); and negative control (lane 7).

FIG. 27 is a photograph of PCR results of elution solutions 1 1-15 showing 100 bp DNA ladder (lane 1); elution solutions 1 1-15 (lanes 2-6); and negative control (lane 7). FIGS. 28 and 29 show comparisons of ssDNA sequences which may bind to thrombin.

FIGS. 30 and 31 show comparisons of ssDNA sequences which may bind to lysozyme.

FIGS. 32 and 33 show comparisons of ssDNA sequences which may bind to Fpg protein.

FIG. 34 is a schematic diagram showing the sequences of the pCR™ 4-TOPO™ plasmid. DESCRIPTION OF VARIOUS EMBODIMENTS The present invention relates to a method for efficient and simultaneous selection of aptamers which specifically bind to target molecules. When describing the present invention, all terms not defined herein have their common art-recognized meanings. To the extent that the following description is of a specific embodiment or a particular use of the invention, it is intended to be illustrative only, and not limiting of the claimed invention. The following description is intended to cover all alternatives, modifications and equivalents that are included in the spirit and scope of the invention, as defined in the appended claims.

To facilitate understanding of the invention, the following definitions are provided. The terms "aptamer" or "aptamer sequence" mean single stranded nucleic acids (RNA or DNA) whose distinct nucleotide sequence determines the folding of the molecule into a unique three dimensional structure.

The term "binding" means an interaction or complexation between a target and an aptamer (ss-DNA or ss-RNA), resulting in a sufficiently stable complex.

The term "target" or "target molecule" may include, but is not limited to, a polypeptide, peptide, enzyme, protein, lipid, glycoprotein, carbohydrate, or cell surface molecule such as a receptor, ion channel or extracellular matrix molecule.

The present invention relates to a method for efficient and simultaneous selection of aptamers which specifically bind to target molecules. In one embodiment, the method is shown generally in FIG. 2 as comprising a single round of selection including the steps of incubation or binding, partitioning which comprises washing and elution, and PCR amplification.

In one embodiment, the invention is directed to a method of selecting aptamers which specifically bind to target molecules comprising the steps of:

(a) transferring the target molecules onto a membrane;

(b) incubating the membrane with a randomized mixture of aptamers under

conditions to form aptamer-target molecule complexes on the membrane;

(c) washing the membrane to remove unbound aptamers and weakly bound aptamers;

(d) optionally, contacting the membrane with DNAse I to digest unbound aptamers and weakly bound aptamers; and

(e) eluting the bound aptamers from the aptamer-target molecule complexes.

The eluted aptamers may then be amplified and sequenced using techniques well known to those skilled in the art. Following a single cycle of a method described herein, different groups of sequences which bind to their specific target molecules may thus be generated efficiently and

simultaneously. The target molecules may be initially separated by gel electrophoresis, such as by SDS-

PAGE or native gel electrophoresis, and then transferred onto a physical support which permits their binding with aptamers and partitioning steps. Suitable physical supports include nitrocellulose or polyvinylidene difluoride (PVDF) membranes. The following is a specific example of one embodiment of the present invention. This example demonstrates how the method of the present invention can be used in selecting and identifying aptamers which specifically bind to target molecules efficiently and simultaneously, with a single cycle of binding, partitioning and amplification. This example is offered by way of illustration and is not intended to limit the claimed invention in any manner.

Five test proteins (thrombin, lysozyme, formamidopyrimidine DNA glycosylase (Fpg), bovine serum albumin (BSA), and ovalbumin) were separated according to their molecular weights and in their denatured states using one-dimensional (ID) gel electrophoresis (Examples 1 and 2). In one embodiment, the ID gel electrophoresis is SDS-PAGE. 2-mercaptoethanol and SDS were included in the sample loading buffer. 2-mercaptoethanol breaks the disulphide bridges in the tertiary structure of proteins. SDS is an anionic detergent which breaks hydrogen bonds between proteins, and affects the hydrophobic interaction in a protein and the beta-sheet in the secondary structure. SDS molecules bind to denatured proteins or peptides generally in a mass ratio of 1.4: 1 , and confer a negative charge to the polypeptides. The denatured

polypeptides become "rods" of negative charge cloud with equal charge densities per unit length.

Target proteins originating from bacterial cells may require initial extraction and separation according to their isoelectric points and molecular weights by two-dimensional (2D) gel electrophoresis. Proteomics research has led to the establishment of a data library of 2D gel separation results, including 2D separation of bacterial proteins from various bacterial species. 2D gel separation may thus be used to obtain the desired bacterial proteins without necessitating further purification steps.

In one embodiment, the target molecules are viral proteins. In one embodiment, the viral protein is the core protein of hepatitis B virus.

Aptamers generated against proteins in their native conformations may be more useful for practical applications. The proteins separated on SDS-PAGE were thus restored to their native conformations using Western blotting (Example 2). Transfer of proteins to nitrocellulose or polyvinylidene difluoride (PVDF) membranes removes SDS and 2-mercaptoethanol, resulting in the restoration of proteins to their native conformations. In one embodiment, the membrane is PVDF. It is important to retain the aptamer-protein complexes on the PVDF membrane, and to remove non-specific DNA molecules during washing at the partitioning stage. The retention of proteins on the PVDF membrane was thus evaluated. The protein amounts on the PVDF membrane with and without washes were visualized using Coomassie Brilliant Blue staining. BSA was used as the target protein and transferred from an electrophoretic gel to PVDF membrane. The intensities of the BSA bands were similar without washing (FIG. 3) and with washing (five times with binding buffer followed by three times with elution buffer; FIG. 4), indicating that washing does not appear to affect the retention of proteins on the PVDF membrane. Protein ladders and five standard proteins were used to examine the retention of different proteins (FIG. 5). Washing was increased to thirty times with binding buffer and twenty times with elution buffer, with no apparent loss of proteins on the PVDF membrane. PVDF membrane thus exhibits protein retention properties and mechanical strength which ensures that target protein-aptamer complexes are able to sustain repeated wash conditions. To reduce non-specifically bound aptamers in the blank region of the membrane, the membrane may be blocked with BSA.

In one embodiment, multiple target proteins on the PVDF membrane may be incubated simultaneously to bind ssDNA/R A to the target proteins to form ssDNA/R A-target protein complexes. After transfer, the five test proteins immobilized to PVDF membrane were incubated with a random ssDNA library. In one embodiment, the ssDNA library is an 80-nt-long ssDNA library (SEQ ID NO: 61). The production or acquisition of randomized ssDNA or ssRNA libraries is within the skill of one skilled in the art. Randomized libraries may also be commercially available.

The partitioning stage comprises washing and elution steps (Example 4). In the washing steps, unbound and weakly bound ssDNA/RNA is removed by washing from the ssDNA/RNA- target protein complexes which are retained on the PVDF membrane. In the elution step, the ssDNA/RNA bound to the target proteins are eluted from the complexes. In one embodiment, the number of washing and elution steps may be optimized. To determine optimal numbers of washing and elution steps, all washing and elution solutions were collected individually.

Different fractions of elution solutions contain ssDNA with various affinities to the target proteins. The ssDNA with highly specific affinities to their target proteins are selected from among these fractions. After ethanol precipitation, ssDNA from fractions of washing and elution solutions were dissolved in autoclaved deionized water. The twenty washing solutions were designated as washing solutions 1-20. For each target protein, there were ten elution fractions which were designated as elution solutions 1-10. The five target proteins resulted in five groups of ten elution fractions.

To obtain high quality ssDNA from PCR amplification, the PCR conditions (i.e., the cycle number, PCR mixture, and annealing temperature) were initially optimized to eliminate or minimize non-specific amplification and to enhance the amplification of the specific ssDNA as set out below (Example 4). a) The number of PCR cycles

The number of PCR cycles affects the specificity of the amplification products. Nonspecific amplifications increase when the PCR cycles are increased over twenty. dsDNA products are generated when PCR is performed for nineteen cycles (Musheev and Krylov, 2006). When PCR cycles are increased to above twenty, non-specific amplification of other unwanted products, such as ss-ds DNA byproducts, occurs and reduces desired dsDNA products. To obtain specific amplification of desired PCR products, the number of PCR cycles ranging from 19 to 24 for the same 80-bp length DNA samples was examined, while keeping all other parameters constant (FIG. 6). The amplified products were separated by 12% DNA PAGE. Clean bands of dsDNA products from cycle numbers 19 and 20 were observed (lanes 2 and 3). When the PCR cycles were increased from 21 to 24, non-specific amplifications increased (lanes 4-7). PCR amplification with 19 and 20 cycles was thus applied in subsequent experiments. b) The PCR mixture

DNA with high GC contents may form stem-loop secondary structures and induce the generation of shorter sequences of products. The addition of dimethyl sulfoxide (DMSO) in PCR can reduce the production of shorter DNA products (Kang et al. , 2005). PCR was performed with and without DMSO in the mixtures under the same conditions (FIG. 7).

Addition of DMSO to the PCR mixtures generated much cleaner bands of the desired products. DMSO was included in all PCR amplifications to improve the specificity of full-length products. The PCR results from 20 cycles showed non-specific amplifications after the addition of DMSO (lanes 7-8) compared to PCR amplification with nineteen cycles. When the DNA concentration is low, the number of PCR cycles may be adjusted to achieve the best amplification results. c) Annealing temperature

Two annealing temperatures (55°C and 56°C) were tested, with the temperature of 56°C increasing specific amplifications (data not shown).

UV absorbance detection of OD₂6o is commonly used to estimate DNA concentration. To determine the best dynamic range of this detection (Smartspec™ 3000; BioRad), DNA samples with known concentrations were tested. Based on the OD¾o values (1 unit of OD₂6o equals 33 pg/mL ssDNA), detected ssDNA concentrations were calculated for comparison. The calculated concentrations match the actual concentrations when OD₂₆o is in the range of 0.05 to 0.2 (Au), corresponding to concentrations of DNA from 1.65 to 6.6 μg mL (FIG. 8). The lowest concentration of ssDNA for detection is thus 1.65 x 10^"3 g/L, which equals 62.5 nM of 80-nt- long ssDNA. This calculation is based on 1 M of 80-nt ssDNA being equivalent to 26400 g/L. The OD₂6o measurement cannot detect such low DNA concentration in the twenty washing solutions and ten elution solutions from each target protein. When the DNA samples were amplified, the gel bands could be clearly observed in the washing solutions and elution solutions. Instead of UV absorbance detection, PCR amplification was thus used to determine the number of washing steps in the partitioning stage. The washing steps reduce interference from nonspecific or unbound ssDNA in order to generate the most specific aptamer against the target proteins.

PCR results of twenty washing solutions and ten elution solutions are shown in FIGS. 9 to 10. Negative controls were included in each set of PCR experiments. There was no contamination in the PCR amplification, as confirmed by the absence of an amplification band in the negative control. The PCR products from the washing solutions 11-20 were separated on a electrophoretic gel (FIGS. 9 and 10). Non-specific amplification products were observed along with the expected products (80 bp) in washing solutions 13 and 15 (FIG. 9, lanes 4 and 6). High concentrations of DNA were present in these washing solutions to generate non-specific amplification under the optimized PCR conditions, indicating that twenty washes were insufficient to remove non-specific binding DNA.

After twenty washes, PVDF membrane pieces to which the five target proteins were immobilized were separated into five tubes. Each PVDF membrane piece was eluted ten times with the elution buffer (1 mL). The individual elution solutions were collected separately and designated as elution solutions 1-10. The elution solutions 5-10 in each group were precipitated separately with ethanol and amplified by PCR. FIGS. 1 1 to 15 show the PCR products from the elution solutions 5-10 for the five proteins.

There is one band in the negative control (lane 7, FIG. 14), but the product was not the 80-bp product. Based on the position of the band in the gel, this band was most likely amplified from the primer, not the template DNA, and was not due to contamination.

Specific amplified PCR products were obtained from the elution solutions 5-10 from the five target proteins. Although the bands in FIGS. 11, 14 and 15 are weak, they are observable. DNA molecules which specifically bind to the target proteins were still obtained after ten elutions, when the PVDF membrane had already been washed twenty times. These results indicate that twenty washes and ten elutions were insufficient. To further optimize the number of washes and elutions at the partitioning stage, thirty washes and fifteen elutions were performed. ssDNA and proteins immobilized to PVDF membrane were washed thirty times with binding buffer and fifteen times with elution buffer. All the solutions were collected individually, and ssDNA were precipitated and dissolved in the same volume of autoclaved deionized water for PCR amplification.

Washing solutions 1-30 were collected and the fractions 16-30 were PCR amplified. When the number of washes was increased, more DNA was collected in washing solutions 24- 30 (FIG. 17) compared to the washing solutions 16-23 (FIG. 16), suggesting that DNA was removed from the membrane with the target proteins with increasing numbers of washes.

The band of washing solution 25 (FIG. 17, lane 3) was very weak compared to the band of washing solution 26 (lane 4). The bands of washing solutions 27 and 28 (lanes 5 and 6) are weaker. However, the bands of washing solutions 29 and 30 (lanes 7 and 8) are stronger than those of washing solutions 27 and 28. These results suggest that when increasing the number of washing steps, DNA sequences which specifically bind to their target proteins may also be released from the binding complexes, resulting in the loss of specific DNA sequences for the target proteins. Based on these results, twenty-five washes were chosen for the washing step to remove free or unbound DNA from the binding complexes.

Optionally, the number of washes may be reduced by using DNase I enzyme which digests unbound and weakly bound DNA from the PVDF membrane. Strongly bound DNA, which is protected within the complex of DNA-target molecules, is not digested by DNase I enzyme. Briefly, the membrane was placed in 1 xDNase I reaction buffer containing 5U DNase I enzyme. The mixture was left at room temperature for one hour to allow for digestion of DNA. After one hour, 5 uL of 25 mM EDTA was added to the mixture which was then heated to 65°C for 10 minutes to terminate enzymatic digestion. The membrane was then washed with once with 40 mM Tris-HCl (pH 8.0) before the subsequent elution of bound DNA. DNase I enzyme digestion enhances the removal of unbound and weakly bound DNA from the PVDF membrane, thus minimizing the washing steps. After thirty washes, fifteen elution solutions for each target protein were collected and designated as elution solutions 1-15. The elution solutions 6-15 from each group were amplified under the same PCR conditions (FIGS. 18 to 27).

BSA and ovalbumin have very low binding affinities to ssDNA sequences, and no clear band was observed on 12% DNA PAGE even after PCR amplification (FIGS. 18-21), indicating that use of BSA as the blocking buffer may not affect the incubation binding of ssDNA and their target proteins.

In a comparison of all PCR results of elution solutions 11-15 from the five groups (FIGS. 19, 21, 23, 25, and 27), no clear product band was observed. This confirms that after thirty washes in the washing step, ten elution fractions are sufficient, and there is no need to wash the membrane fifteen times with elution buffer to elute DNA from the binding complexes.

However, if twenty-five washes in the washing step are used, it is preferable to wash the membrane fifteen times with elution buffer in the eluting step to ensure that as much ssDNA as possible can be eluted from the binding complexes.

For elution solutions 6-10, it is difficult to observe amplification products in the groups of DNA which may specifically bind to BSA (FIG. 21) or ovalbumin (FIG. 23). BSA and ovalbumin may have very low binding affinity to ssDNA. The remaining three groups of ssDNA (which may specifically bind to thrombin, Fpg protein, or lysozyme) were selected for DNA cloning and sequencing (Example 5). Following a single cycle of the method described herein, different groups of sequences which may bind to their specific target proteins may thus be generated efficiently and simultaneously.

Optionally, a gradient elution method may be used rather than the above elution steps. The method comprises a stepwise increase of elution strength by increasing the concentration of urea in the elution buffer solution. As an example, three elution buffer solutions were used:

(A) 7 M urea, 40 mM Tris-HCl (pH 8.0), 3M NaCl, and 0.02% Tween™20;

(B) 8 M urea, 40 mM Tris-HCl (pH 8.0), 3M NaCl, and 0.02% Tween™ 20; and

(C) 9 M urea, 40 mM Tris-HCl (pH 8.0), 3M NaCl, and 0.02% Tween™ 20. The membrane was first placed in solution "A" three times at two minutes each (3 x 2 min) before the membrane was taken out and placed in solution "B" for 3 x 2 min, and subsequently placed in solution "C" for 3 x 2 min. Each of the elution solutions were saved for amplification of DNA. The membrane was also saved for amplification of remaining DNA on the membrane. The DNA in the solutions and the DNA on the membrane represent sequences that have increasing binding affinity to the target molecules on the membrane support.

Several ssDNA sequences from three groups of elution solutions were amplified by PCR, and the amplified dsDNA products were inserted into plasmid DNA. Bacteria with inserted recombinant plasmids were cultured on selective LB medium. Several single colonies from each selective medium were picked, and eighty colonies were selected for DNA sequencing.

Approximately sixty sequences of ssDNA which may specifically bind to their target proteins (thrombin, Fpg protein, or lysozyme) were obtained after DNA sequencing. Based on the primer sequences, sequences of the selected aptamers were determined from raw data and grouped into two groups. Common bases of sequences in the same group were compared, with the assumption that the sequences having strong binding affinities to their target proteins share many common bases.

Sequences of ssDNA which may specifically bind to thrombin are set out in Table 1. the sequences were grouped by their primer sequences. Common bases were compared for groups 1 (FIG. 28) and 2 (FIG. 29). Table 1. Sequences of ssDNA which may specifically bind to thrombin

5'-TTC ACG GTA GCA CGC ATA GG TAG ACT GCC GGA GCT ATT 13 ATA ACC AAG GAC ACT GAT AG GT CA TCT GAC CTC TGT GCT GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG AGC ATA TCA TAA ACC ACA 14 AAT CTT GAC AAT TCA TGT TC GA CA TCT GAC CTC TGT GCT GCT-3'

Sequences of ssDNA which may specifically bind to lysozyme are set out in Table 2. All the sequences were grouped by their primer sequences. Common bases were compared for groups 1 (FIG. 30) and 2 (FIG. 31).

Table 2. Sequences of ssDNA which may specifically bind to lysozyme

GAA-3'

5'-AGC AGC ACA GAG GTC AGA TG TAG CAT TTA AGA CTG CGT 25 TGG GCG GGG GCT GGC CGG GT CA CC TAT GCG TGC TAC CGT GAA-3'

5'-AGC AGC ACA GAG GTC AGA TG ATT TCA CTC AAG AAC ACT 26 TAT GGA GCG CAA TGA ACT CT GG CC TAT GCG TGC TAC CGT

GAA-3'

5'-AGC AGC ACA GAG GTC AGA TG CTG AAT GAC TGG CGC GGG 27 CTC CGT CTG GAC CTG CTC TT GG CC TAT GCG TGC TAC CGT

GAA-3'

5'-AGC AGC ACA GAG GTC AGA TG CCG ATA GTC ACC ACT ATC 28 CGC GGC CCT TGC GGT GGA TG TC CC TAT GCG TGC TAC CGT

GAA-3'

5'-AGC AGC ACA GAG GTC AGA TG TAC GTA ACT TCA CGC TCC 29 TTA TCT TGA CGC ACT TGG CC CG CC TAT GCG TGC TAC CGT

GAA-3'

5'-AGC AGC ACA GAG GTC AGA TG GTG GCT CAA CTC TAT GCA 30 CCG ATG TCG GTC TAT CTG AT CG CC TAT GCG TGC TAC CGT

GAA-3'

5'-AGC AGC ACA GAG GTC AGA TG GTT GAC TAA AGC ACA CAC 31 TAT CCA CCC TCC CGC GGG TC CA CC TAT GCG TGC TAC CGT

GAA-3'

5'-TTC ACG GTA GCA CGC ATA GG AGC CCA TCC CCG ATT AGC 32 AAA CCC TTA AAA TAT TGT AA GT CA TCT GAC CTC TGT GCT

GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG GTA CTA AGC GCT GCG TTC 33 TGT GAC CAC GGG ATT ACT GT TT CA TCT GAC CTC TGT GCT GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG CGC CTC GCT AGG AGA GAG 34 GAA GGC GTC AAA TCG AAC GA AG CA TCT GAC CTC TGT GCT GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG TAA GGT ATA CGT AGC TTA 35 GTA TGA GAT GCT GGA TGA TT CA CA TCT GAC CTC TGT GCT

GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG GCA AAC AAT AGG CTC GAA 36 TTA AAT CAC TGG CAT CGA GG TT CA TCT GAC CTC TGT GCT GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG TGT TTC CCG CTA CTA ACG 37 ACA GTC AAG GTA TGA ACT CC TG CA TCT GAC CTC TGT GCT

GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG AAA AGA TAC TGG CGC GAT 38 GTT TTA CAC AGA ATT TGC GG AA CA TCT GAC CTC TGT GCT GCT-3'

5' -TTC ACG GTA GCA CGC ATA GG TCA GAC CCA GCC ACC GCG 39 AAG GGA GTT AAG TTA ACC AC GT CA TCT GAC CTC TGT GCT

GCT-3' 5'-TTC ACG GTA GCA CGC ATA GG TGG CAG AAC CGC GCG TAA 40 CTG TGT CCC GTA GCG TGA AG AA CA TCT GAC CTC TGT GCT

GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG TGA CAT CTA GCA CGA TAA 41 TCA ATA TAC TGG TCG CTT GC TC CA TCT GAC CTC TGT GCT

GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG TGA CCC GGC CAG CCC CCG 42 CCC AAC GCA GTC TTA AAT GC TA CA TCT GAC CTC TGT GCT

GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG CCA GAG TTC ATT GCG CTC 43 CAT AAG TGT TCT TGA GTG AA AT CA TCT GAC CTC TGT GCT

GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG CCA AGA GCA GGT CCA GAC 44 GGA GCC CGC GCC AGT CAT TC AG CA TCT GAC CTC TGT GCT GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG GAC ATC CAC CGC AAG GGC 45 CGC GGA TAG TGG TGA CTA TC GG CA TCT GAC CTC TGT GCT GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG CGG GCC AAG TGC GTC AAG 46 ATA AGG AGC GTG AAG TTA CG TA CA TCT GAC CTC TGT GCT GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG CGA TCA GAT AGA CCG ACA 47 TCG GTG CAT AGA GTT GAG CC AC CA TCT GAC CTC TGT GCT

GCT-3'

5'-TTC ACG GTA GCA CGC ATA GG TGG ACC CGC GGG AGG GTG 48 GAT AGT GTG TGC TTT AGT CA AC CA TCT GAC CTC TGT GCT

GCT-3'

Sequences of ssDNA which may specifically bind to Fpg protein are set out in Table 3. All the sequences were grouped by their primer sequences. Common bases were compared for groups 1 (FIG. 32) and 2 (FIG. 33).

Table 3. Sequences of ssDNA which may specifically bind to Fpg protein

Group Sequence SEQ ID NO

1 5'-AGC AGC ACA GAG GTC AGA TG CGG CGG AGT AGC GCG TTG 49 ATT ATG CCA AGT CGG TCC GT GC CCT ATG CGT GCT ACC GTG

AA-3'

5' -AGC AGC ACA GAG GTC AGA TG CTG GAT TTC ATG ATC GCC 50 ATG TCT CGA TTG TGG GCC GA AA CCT ATG CGT GCT ACC GTG

AA-3'

5' -AGC AGC ACA GAG GTC AGA TG ACT CGG GGA ACC GGG GAG 51 AGT GAA GCC TCG CGC AAT CA TA CCT ATG CGT GCT ACC GTG AA-3'

5'-AGC AGC ACA GAG GTC AGA TG TAC AGC GGC GCA ACG ATA 52 ACG GCT CAC TAA ACC CGA CG AT CCT ATG CGT GCT ACC GTG

AA-3'

5'-AGC AGC ACA GAG GTC AGA TG AGA GAT TAG CGT AGG GAA 53 GCC GAT GCC GAG CCT CGC CC AA CCT ATG CGT GCT ACC GTG AA-3'

5' -AGC AGC ACA GAG GTC AGA TG AGT TGG CCG AAG CCA ACC 54 TTC AAA GCG CAG ACG CCA TC AT CCT ATG CGT GCT ACC GTG

AA-3'

2 5' -TTC ACG GTA GCA CGC ATA GG GCA CGG ACC GAC TTG GCA 55 TAA TCA ACG CGC TAC TCC GC CG CAT CTG ACC TCT GTG CTG CT-3'

5' -TTC ACG GTA GCA CGC ATA GG TTT CGG CCC ACA ATC GAG 56 ACA TGG CGA TCA TGA AAT CC AG CAT CTG ACC TCT GTG CTG

CT-3'

5' -TTC ACG GTA GCA CGC ATA GG TAT GAT TGC GCG AGG CTT 57 CAC TCT CCC CGG TTC CCC GA GT CAT CTG ACC TCT GTG CTG

CT-3'

5' -TTC ACG GTA GCA CGC ATA GG ATC GTC GGG TTT AGT GAG 58 CCG TTA TCG TTG CGC CGC TG TA CAT CTG ACC TCT GTG CTG

CT-3'

5' -TTC ACG GTA GCA CGC ATA GG TTG GGC GAG GCT CGG CAT 59 CGG CTT CCC TAC GCT AAT CT CT CAT CTG ACC TCT GTG CTG

CT-3'

5'-TTC ACG GTA GCA CGC ATA GG ATG ATG GCG TCT GCG CTT 60 TGA AGG TTG GCT TCG GCC AA CT CAT CTG ACC TCT GTG CTG CT-3'

The secondary structures of sequences identified as above may be analyzed and compared to identify and characterize aptamers having desirable parameters such as, for example, binding affinity (K_d), melting temperature (Tm), and change in Gibbs free energy (AG).

Exemplary embodiments of the present invention are described in the following

Examples, which are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter. Example 1 - Test proteins

Five standard proteins were used as test target molecules: the E. coli protein

formamidopyrimidine DNA glycosylase or Fpg (mw 30.2 kDa); thrombin (mw 36.7 kDa) and lysozyme (mw 14.7 kDa) against which aptamers have been generated to provide nM level binding affinity; and bovine serum albumin (BSA) and ovalbumin against which aptamers have not been generated (Invitrogen, Burlington, ON).

Example 2 - SDS-PAGE and Western Blot

The following reagents were used: 30% acrylamide mix solution, 40% acrylamide mix solution, ammonium persulfate, low range SDS-PAGE molecular weight standards, lOx Tris- glycine buffer, PVDF membrane (BioRad Laboratories, Mississauga, ON); SDS, Tris-base, PCR kit, DNA cloning and sequencing kit (TOPO TA™ kit), BSA, dNTP kit (Invitrogen);

MinElute™ PCR purification kit (Qiagen, Mississauga, ON); Tween™ 20 (Fisher Scientific, Nepean, ON); TEMED and EDTA (EMD Chemical Inc., Gibbstown, NJ, USA).

Proteins were separated on 5% stacking gels and 12% resolving gels. The 5% stacking gel consisted of 4 mL of 5% stacking gel mixture (2.7 mL deionized water, 0.67 mL of 30% acrylamide mixture, 0.5 mL of 1 M Tris-HCl at pH 7.8, 40 ί of 10% SDS, 40 μΕ of 10% ammonium persulfate, 4 ih of TEMED). The 12% resolving gel consisted of 10 mL of 12% resolving gel mixture (3.3 mL deionized water, 4.0 mL of 30%o acrylamide mixture, 2.5 mL of 1.5M Tris-HCl at pH 8.8, 0.1 mL of 10% SDS, 0.1 mL of 10% ammonium persulfate, and 4 xL of TEMED). The ammonium persulfate solution was freshly prepared for each set of experiments. The total amount of protein for SDS-PAGE was 0.36 nmole (0.03 nmole BSA, 0.04 nmole ovalbumin, 0.1 nmole Fpg, 0.05 nmole thrombin and 0.14 nmole lysozyme). The protein samples were heated at 95°C for five minutes before loading into wells with loading buffer (200 mg SDS, 2 mL glycerol, 0.5% 2-mercaptoethanol, 0.5 mL Tris-HCl at pH 6.8, 0.1% bromophenol blue, and deionized water to a final volume of 10 mL). The volume ratio of protein and loading buffer was 1 : 1. A potential of 8 V/cm was applied when protein samples were still in the stacking gel. After the dye front moved to the resolving gel, the potential was increased to 15 V/cm. Running buffer (1000 mL) consisted of 100 mL of lOx Tris-glycine buffer, 1% SDS, and 900 ml deionized water. The proteins were transferred to PVDF membrane overnight under 125 mAmp at 4°C. The blotting buffer (1000 mL) consisted of 200 mL methanol, 100 mL of lOx Tris-glycine buffer, and 700 mL deionized water. The bands containing the target proteins were excised to improve DNA binding and to minimize interference from the blocking solution (3% BSA). 36 nmole of DNA library is needed to ensure a 100:1 ratio of ssDNA molecules and protein molecules. However, the efficiency of transfer of protein from the gel to the PVDF membrane is around 50-60%. 70-80% efficiency is seldom achieved. Based on the transfer efficiency, the amount of DNA library was estimated as more than 20 nmole but less than 28.8 nmole. 30 nmole of aptamer library was subsequently used.

Example 3 - Random DNA library and primers

The DNA library and primers for PCR amplification of the library were synthesized by Integrated DNA Technologies (Coralville, IA, USA). The DNA library consists of 40 random bases in the middle region and 20 bases at the 5' and 3' ends:

5' -AGC AGC ACA GAG GTC AGA TG (N:25252525)(N) (N)( ) (N)(N) (N)(N) (N)(N) (N)(N) (N)(N) (N)(N) (N)(N) (N)( ) (N)(N) (N)(N) (N)(N) (N)(N) (N)( ) (N)(N) (N)(N) (N)(N) (N)(N) (N)(N) CC TAT GCG TGC TAC CGT GAA-3' (SEQ ID NO: 61)

The following primers for PCR amplification of the DNA library were used:

Primer 1 : 5' -AGC AGC ACA GAG GTC AGA TG-3' (no label) (SEQ ID NO: 62) Primer 2: 5' -TTC ACG GTA GCA CGC ATA GG-3' (no label) (SEQ ID NO: 63)

The following primers for DNA sequencing were used (TOPO TA™ kit, Invitrogen):

M13 Forward primer: 5' -GTA AAA CGA CGG CC AG-3' (SEQ ID NO: 64)

M13 Reverse primer: 5' -CAG GAA ACA GCT ATG AC-3' (SEQ ID NO: 65)

T3 primer: 5' -ATT AAC CCT CAC TAA AGG GA-3' (SEQ ID NO: 66) T7 primer: 5' -TAA TAC GAC TCA CTA TAG GG-3' (SEQ ID NO: 67)

The sequence of the plasmid DNA is shown in FIG. 34 (SEQ ID NO: 68; TOPO TA™ kit, Invitrogen).

Example 4 - Single cycle selection method

The single cycle selection method includes the steps of incubation or binding, partitioning comprising washing and elution, and PCR amplification of the selected DNA (FIG. 2). 30 nmole of an 80-nt-long ssDNA randomized library (SEQ ID NO: 61) was dissolved with binding buffer (100 mM NaCl, 20 mM Tris-HCl at pH 7.6, 2mM MgCl₂, 5 mM KC1, 1 mM CaCl₂, 0.02% Tween™ 20; Stoltenburg et al, 2006). The PVDF membrane pieces containing the five target proteins were incubated for forty-five minutes in the DNA library/binding buffer solution. Based on molar concentrations, the ratio of ssDNA molecules to the target protein was 100:1 to allow excess ssDNA molecules to bind to the target proteins. A length of 40 random bases in ssDNA should provide 4⁴⁰ (approximate to 10²⁴) random sequences. However, most aptamer libraries contain no more than lO¹⁶ random sequences due to limitations of synthesis. The amount of 1 mole ssDNA contains 6.02 X 10²³ molecules; thus the amount of 30 nmole ssDNA I contains 30 ^χ 10^"9 6.02 ^χ 10²³ = 1.806 x 10¹⁶ (or approximately 2 ^χ 10¹⁶) molecules, which ensures that at least each random sequence has one molecule during the binding step.

After incubation, the PVDF membrane pieces were washed twenty times with clean binding buffer. Washing solutions were collected separately and precipitated with ethanol. The amounts of DNA in the washing solutions were determined by UV absorbance detection. After washing, the PVDF membrane pieces containing the five target-aptamer complexes were each separated into a tube. The bound ssDNA were eluted from the complexes on the specific membrane with elution buffer (40 mM Tris-HCl at pH 8.0, 10 mM EDTA, 3.5M urea, 0.02% Tween™ 20; Stoltenburg et al, 2006) ten times and collected in ten fractions. DNA in each elution solution was purified with ethanol precipitation and amplified by PCR. Parameters that may affect PCR amplification were examined and optimized. The optimized PCR mixture consisted of 10 μΐ, of 1 Ox PCR buffer, 5.6 μΐ, of 50 mM MgCl₂, 5 μΐ, of 20 mM forward primer (SEQ ID NO: 62), 5 μΐ of 20 mM reverse primer (SEQ ID NO: 63), 4 μΐ of 10 mM dNTPs, 63 μΐ, of autoclaved deionized water, 5 of DMSO, 0.5 μΐ. of 5 unit^L Taq polymerase and 2 μΐ, of template DNA.

After nineteen PCR cycles, amplified PCR products were analyzed with 12% DNA PAGE gel at 120 volts for one hour. The 12% DNA PAGE gel consisted of 2.5 mL of 40% acrylamide mixture, 5.5 mL deionized water, 2 mL of 5x TBE buffer, 62 iL of 10% ammonium persulfate, and 10 L of TEMED. The DNA PAGE gels were stained with ethidium bromide to confirm that only full-length (80 bp) PCR products were obtained. Example 5 - DNA cloning and sequencing

Before cloning, the amplified 80-bp PCR products were purified by a MinElute™ PCR purification kit (Qiagen) to eliminate interference from left primers, self-dimers, and unspecific PCR products. The PCR purification was performed according to the manufacturer's

instructions. Fragments ranging from 70 bp to 4 kb were retained in the column, while primers, polymerase, and salts were removed. The desired PCR products were eluted from the column. Purified PCR products were precipitated from the buffer and dissolved in autoclaved deionized water. Cloning was performed using a TOPO TA™ kit (Invitrogen). Taq polymerase-amplified PCR products were directly inserted into plasmid vectors. The linear plasmid vector contained single 3' thymidine (T) overhangs, and Taq polymerase-amplified PCR products had a single deoxy adenosine (A) on each 3' end. Based on the principle of complementary base pairing, PCR products were ligated with the vector efficiently. DNA cloning and sequencing were performed according to the manufacturer's instructions.

To perform the ligation, 1 μΐ, of purified PCR product, 1 xl, of salt solution (1.2 M NaCl, 0.06 M MgCl₂), 3 μΐ. of autoclaved deionized water, and 1 μL of TOPO™ vector (10 ng/μΕ plasmid DNA in 50% glycerol, 50 nM Tris-HCl at pH 7.4, 1 mM EDTA, 2mM DTT, 0.1 % Triton™ X-100, 100 μg/mL BSA, 30 μΜ phenol red) were mixed gently and incubated for five minutes at room temperature. 2 μΐ, of the recombinant plasmid vectors with inserted target DNA fragments was added to one vial of DH5a-Tl™ chemically competent cells (Invitrogen) and then incubated for thirty minutes on ice. The cells were heat-shocked for thirty seconds in a 42°C water bath, and transferred to ice. 250 μΐ, of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 niM KC1, 10 mM MgCl₂, 10 niM MgS0₄, 20 mM glucose) was added to each tube. The tubes were capped and shaken at 200 rpm horizontally at 37°C for one hour. Selective plates (LB medium containing 50 μg/mL ampicillin) were pre-warmed in a 37°C incubator for thirty minutes. 20-50 μΐ. of the transformed bacterial cells were cultured on each selective plate. The plates were incubated at 37°C for twelve to sixteen hours. After overnight culturing, single colonies were selected from the plates and cultured in LB broth medium with 50 μg/mL ampicillin for another overnight culturing at 37°C for twelve to fourteen hours. When the broth medium became turbid, the cultured cells were ready for plasmid DNA isolation and

purification.

The cells in broth medium were collected after centrifugation at 13,000 rpm for five minutes. The plasmid DNA containing the inserted fragment was precipitated and purified (PureLink™ Quick Plasmid Miniprep Kit, Invitrogen). Plasmid DNA was eluted from the membrane in the column by TE buffer (10 mM Tris-HCl at pH 8.0, 0.1 mM EDTA). DNA samples were precipitated using ethanol precipitation, and dried DNA samples were dissolved in autoclaved deionized water. Sequences were analyzed using T3 primer (SEQ ID NO: 66), T7 primer (SEQ ID NO: 67), or Ml 3 forward/reverse primer (SEQ ID NO: 64 / SEQ ID NO: 65).

As will be apparent to those skilled in the art, various modifications, adaptations and variations of the foregoing specific disclosure can be made without departing from the scope of the invention claimed herein.

REFERENCES

The following references are incorporated herein by reference (where permitted) as if reproduced in their entirety. All references are indicative of the level of skill of those skilled in the art to which this invention pertains. Clark, S.L.; Remcho, V.T. Aptamers as analytical reagents. Electrophoresis 2002, 23, 1335- 1340. Ellington, A.D.; Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346, 818-22.

Jayasena, S. Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin.

Chem. 1999, 45, 1628-1650.

Jension, R.D.; Gill, S.C.; Pardi, A.; Polisky, B. High-resolution molecular discrimination by RNA. Science 1994, 263, 1425-1429.

Kang, J.; Lee, M.S.; Gorenstein, D.G. The enhancement of PCR amplification of a random

sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers. J. Biochem. Biophys. Methods 2005, 64, 147-151.

Luzi, E.; Minunni, M.; Tombelli, S.; Mascini, M. New trends in affinity sensing: aptamers for ligand binding. Trends Anal. Chem. 2003, 22, 810-818.

Mann, D.; Reinemann, C; Stoltenburg, R.; Strehlitz, B. In vitro selection of DNA aptamers binding ethanolamine. Biochemical and Biophysical Research Communications 2005, 338, 1928-1934.

Musheev, M. U.; Krylov, S. N. Selection of aptamers by systematic evolution of ligands by exponential enrichment: addressing the polymerase chain reaction issue. Analytica Chemica Acta 2006, 564, 91-96.

Nimjee, S.M.; Rusconi, CP.; Sullenger, B.A. Aptamers: an emerging class of therapeutics.

Annu. Rev. Med. 2005, 56:555-83.

Patel, D.J.; Suri, A.K. Structure, recognition and discrimination in RNA aptamer complexes with cofactors, amino acids, drugs and aminoglycoside antibiotics. Rev. Mol. Biotechnol. 2000, 74, 39-60.

Stoltenburg, R.; Reinemann, C; Strehlitz, B. DNA aptamer selection using a ligand evolution process. American Biotechnology Laboratory 2006, 24/1, 18-20.

Tuerk, C; Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990, 249, 505-10.

You, K. M.; Lee, S. H.; Im, A.; Lee, S. B. Aptamers as functional nucleic acids: in vitro selection and biotechnological applications. Biotechnol. Bioprocess. Eng. 2003, 8, 64-75.

Claims

1. A method of selecting aptamers which specifically bind to target molecules comprising the steps of:

(a) transferring the target molecules onto a membrane;

(b) incubating the membrane with a randomized mixture of aptamers under conditions to form aptamer-target molecule complexes on the membrane;

(c) washing the membrane to remove unbound aptamers and weakly bound aptamers;

(e) eluting the bound aptamers from the aptamer-target molecule complexes;

(f) amplifying the eluted aptamers; and

(g) determining the sequences of the eluted aptamers.

2. The method of claim 1 , wherein an aptamer specific to the target molecule may be selected with a single cycle of steps (b) to (f).

3. The method of claim 1, wherein the target molecules are first separated from a mixture by gel electrophoresis.

4. The method of claim 2, wherein the aptamer comprises single-stranded DNA (ss-DNA).

5. The method of claim 2, further comprising amplifying uneluted ss-DNA remaining on the membrane, and determining the sequences of the uneluted ss-DNA.

6. The method of claim 3, wherein the gel electrophoresis is sodium dodecyl sulfate- polyacrylamide gel electrophoresis.

7. The method of claim 1 , wherein the membrane is a polyvinyldifluoride membrane.

8 The method of claim 1, wherein the target molecules are bacterial proteins.

9. The method of claim 8, wherein the bacterial protein is E. coli protein

formamidopyrimidine DNA glycosylase.

10. The method of claim 8, wherein the bacterial proteins are first extracted from cell lysate and separated by 2D gel electrophoresis, followed by western blotting.

11. The method of claim 1 , wherein the target molecules are a viral protein.

12. The method of claim 11, wherein the viral protein is the core protein of hepatitis B virus.

13. The method of claim 1 , wherein the washing of step (c) is conducted at least twenty- five times.

14. The method of claim 1, wherein the elution of step (e) is conducted at least fifteen times.

15. The method of claim 1, wherein the elution of step (e) comprises gradient elution with stepwise increases of urea concentration.

16. The method of claim 1, wherein the amplification of step (f) is conducted for 19 to 20 cycles.

17. The method of claim 1, wherein the amplification of step (g) includes adding dimethyl sulfoxide to a PCR mixture.

18. The method of claim 1 , wherein the amplification of step (g) is conducted at an

annealing temperature of 56°C.

19. The method of claim 1 , wherein the aptamer comprises single stranded RNA.

20. A method of selecting and identifying an aptamer which specifically binds to a target molecule, comprising a single cycle of the steps of:

(a) transferring the target molecules onto a physical support;

(c) washing the membrane to remove unbound aptamers and weakly bound aptamers;

(e) eluting the bound aptamers from the aptamer-target molecule complexes; and

(f) characterizing the eluted aptamers.

21. The method of claim 20 wherein the physical support comprises a nitrocellulose or polyvinylidene difluoride membrane.

22. The method of claim 20 wherein the eluted aptamers are characterized by amplification and sequencing.