METHOD OF IDENTIFYING OR PRODUCING AN APTAMER FOR A DENATURED PEPTIDE OR PROTEIN

Abstract

The invention relates to a method of identifying or producing an aptamer, the method comprising the steps of: a) Providing a candidate mixture of nucleic acids; b) Contacting the candidate mixture with a target sample; c) Partitioning nucleic acids that bind to the target sample from those that do not bind; d) Amplifying the binding nucleic acids to produce a population of nucleic acids that bind the target sample; e) Single strand displacement of the amplified nucleic acids, f) optionally repeating the steps a) to e), thereby identifying or producing an aptamer, wherein the target sample provides one or more peptides and/or proteins in a denatured form.

Claims

1. A method of identifying or producing an aptamer, the method comprising the steps of: a) Providing a candidate mixture of nucleic acids; b) Contacting the candidate mixture with a target sample; c) Partitioning nucleic acids that bind to the target sample from those that do not bind; d) Amplifying the binding nucleic acids to produce a population of nucleic acids that bind the target sample; e) Single strand displacement of the amplified nucleic acids, f) optionally repeating the steps a) to e), thereby identifying or producing an aptamer, characterized in that the target sample provides one or more peptides and/or proteins in a denatured form.

2. The method according to claim 1, wherein an aptamer recognizing a denatured target peptide or protein is identified or produced.

3. The method according to claim 1, wherein in step b) the target sample is provided on a Western blot.

4. The method according to claim 1, wherein in step b) the target sample is provided as an isolated peptide or protein, particularly as a single protein band on a Western blot membrane.

5. The method according to claim 1, wherein in step b) the target sample is provided as a multitude of peptides or proteins, preferably a fraction of peptides or proteins or a (whole) cell lysate, separated on a Western blot membrane.

6. The method according to claim 1, wherein in step c) a single protein band or a plurality of protein bands on a Western blot membrane are selected for recovery of binding nucleic acids.

7. The method according to claim 5, wherein in step c) for an initial number of SELEX rounds a plurality of protein bands on the Western blot membrane are selected for recovery and for a following number of SELEX rounds a single protein band is selected for recovery of binding nucleic acids.

8. The method according to claim 1, wherein in step c) the nucleic acids binding to target peptide(s) or protein(s) are recovered from a Western blot membrane using a buffer containing urea and sodium dodecyl sulfate.

9. The method according to claim 1, wherein in step c) the recovered nucleic acids are purified, preferably by spin column-based nucleic acid purification, phenol-chloroform extraction and/or nucleic acid precipitation.

10. The method according to claim 1, wherein in step b) in addition to the target sample a sample for negative-selection containing denatured peptides and/or proteins is provided on a Western blot.

11. The method according to claim 1, wherein in step a) the candidate mixture of nucleic acids comprises at least one nucleotide that is modified to comprise a functionalization introduced by click chemistry.

12. The method according to claim 11, wherein the modified nucleotide comprises an alkyne-modified nucleobase that is further modified via a 1,3 dipolar cycloaddition of an azide to yield an azide-alkyne modified nucleobase, wherein the azide preferably comprises a group selected from benzyl and indole.

13. A method of identifying or producing an aptamer for the identification of a denatured peptide or protein using the method of claim 1.

14. The method of claim 13, wherein the aptamer is usable in a Western blot assay.

15. An aptamer, wherein the aptamer has a nucleotide sequence selected from the group comprising: TABLE-US-00018 (SEQ ID NO: 1) 5′-AACAAGANANACACGANGGGGGCACACCAAAGCACCGNNCGAAA-3′ and (SEQ ID NO: 2) 5′-NNAACACCCACNCACGCCAANCCCACCACCCCNACACNCCCAC-3′, wherein N is selected from T or ethynyl-dU.

Description

[0051] The Figures and Examples which follow serve to illustrate the invention in more detail but do not constitute a limitation thereof.

[0052] The figures show:

[0053] FIG. 1 the results of a fluorescent binding test of the benzyl-functionalized monoclone of SEQ ID NO: 1 selected according to one embodiment of the method, shown on the right, and the starting library (“Bibliothek”) on the left.

[0054] FIG. 2 the results of binding studies of the 7.sup.th and 10.sup.th SELEX round visualized with IR-fluorescence according to a second embodiment of the method of the invention.

[0055] FIG. 3 the results of a fluorescent binding test of the indol-functionalized monoclone of SEQ ID NO: 2 selected according to the second embodiment of the method, in PC3 cells and THP-1 cell line.

[0056] FIG. 4 the results of purified DNA recovered using different recovery buffers after 24 cycles of amplification on an ethidium bromide stained 4% agarose gel.

MATERIALS

Nucleic Acids:

[0057]

TABLE-US-00001 name specification supplier Library (OW1_Cy5): 5′-CY5-AGC CAC GGA Library Ella Biotech AGA ACC AGA NNN NNN NNN NNN NNN NNN GmbH, NNN NNN NNN NNN NNN NNN NNN NNN NNG Germany CAG AAG CGA CAG CAA CA-3′(SEQ ID NO: 3) N: dA, dC, dG, Ethynyl-dU (EdU) (1:1:1:1) (DNA) Panacea: 5′-NNN NNN NNN NNN NNN NNN Click-competitor Ella Biotech NNN NNN NNN NNN NNN NNN NNN A-3′(SEQ ID NO: 4) GmbH, N: dA, dC, dG, Ethynyl-dU (EdU) (1:1:1:1) (DNA) Germany 5′-Biotin- X*AG CCA CGG AAG AAC CAG A -3′ forward primer Ella Biotech (SEQ ID NO: 5) (OW1 fw bio) GmbH, *X = C18 Spacer Germany 5′-IR-800CW- X*AG CCA CGG AAG AAC CAG forward primer Ella Biotech A-3′ (SEQ ID NO: 5) (OW1 fw IR-800) GmbH, Germany Phosphate-TGT TGC TGT CGC TTC TGC (SEQ ID reverse primer Ella Biotech NO: 6) (OW1 rev P) GmbH, Germany

Proteins:

[0058]

TABLE-US-00002 Catalogue name specification supplier no. Pwo DNA polymerase 2.5 U/μl Genaxxon, M3002.0100 Germany λ-Exonuclease 10 U/μl Thermo EN0562 Scientific Recombinant human Abcam, UK Ab43615 GST-p53 Bovine Serum AppliChem, 232-963-2 Albumine (BSA) Germany Streptavidin- GE-Healthcare RPN-1231-2ML Horseradish Amersham, UK peroxidase

Cell Lines:

[0059]

TABLE-US-00003 name supplier Catalogue no. PC3-LN ProQinase, Germany LNCaP DSMZ, Germany ACC 256

Chemicals:

[0060]

TABLE-US-00004 Catalogue name supplier no. Tris(4-(3-hydroxy-propoyl)- Baseclick, Germany BCMI-006-5 [1,2,3]triazol-1-ylmethyl) amine (THPTA) Copper sulphate Sigma-Aldrich 451657-10G  5-Ethynyl-5′-O-triphosphate- Baseclick, Germany BCT-08 2′-deoxyuridine (Ethinyl-dUTP) dATP Genaxxon bioscience, M3018.0020 Germany dCTP Genaxxon bioscience, M3019.0020 Germany dGTP Genaxxon bioscience, M3020.0020 Germany TWEEN ® 20 Merck Millipore, 8170721000 Germany TRIS Carl ROTH, Germany 201-064-4 Glycin Carl ROTH, Germany 200-272-2 NaCl Labochem    .sup. LC-5932.1 international, Germany MgCl.sub.2 Carl ROTH, Germany 2189.2 CaCl.sub.2 Carl ROTH, Germany 5239.1 NaHCO.sub.3 Carl ROTH, Germany 8551.1 UREA Carl ROTH, Germany 3941.1 EDTA AppliChem, Germany 200-449-4 SDS Carl ROTH, Germany 2326.2 Dextrane sulfate Sigma-Aldrich 9011-18-1 Agarose Carl ROTH, Germany K297.3

Miscellaneous:

[0061]

TABLE-US-00005 Catalogue name supplier no. Amicon 10 K MWCO Columns Merck Millipore, UFC501096 Germany NucleoSpin ® Gel and Macherey-Nagel, 740609.250 PCR Clean-up Germany ECL-substrates A and B Thermo Scientific 32106 Pierce, USA TOPO TA cloning kit Thermo Fisher K4575J10 Scientific, USA NucleoSpin Plasmid kit Macherey-Nagel, 740588.250 Germany NucleoSpin ® Gel and Macherey-Nagel, 740609.250 PCR Clean-up Germany BCA Protein Assay Kit Thermo Scientific 23225 Pierce, USA Pierce ® ECL Western Thermo Fisher K4575J10 Blotting Substrate Scientific, USA

Buffers Example 1

[0062]

TABLE-US-00006 name specification TBST 20 mM TRIS, 137 mM NaCl, 0.1% TWEEN ® 20, pH 7.6 TBST-MC 20 mM TRIS, 137 mM NaCl, 1 mM MgCl.sub.2, 1 mM CaCl.sub.2, 0.1% TWEEN ® 20, pH 7.6 Recovery Buffer 50 mM NaHCO.sub.3, 1.25M UREA, 6.25 mM EDTA, 0.5% SDS WetBlot Buffer 25 mM TRIS, 192 mM Glycine SDS-PAGE running 25 mM TRIS, 192 mM Glycine, 3.4 mM SDS Buffer

Buffers Example 2

[0063]

TABLE-US-00007 name specification Homogenizing buffer 5 mM Tris, 300 mM Sucrose, 0.1M EDTA, 1 mM PMSF, pH 7.4 4x Laemmli buffer 78.1 mM Tris, 12.5% Glycerol, 6.3% 2-Mercaptoethanol, 0.003% Bromophenol Blue, 1% SDS, pH 6.8 TBS-T 20 mM Tris, 137 mM NaCl, 1 mM MgCl.sub.2, 1 mM CaCl.sub.2, 0.1% TWEEN ® 20, pH 7.6 Recovery Buffer 0.1M NaHCO.sub.3, 1% SDS, pH 8.0 Transferring Buffer 2.5 mM Tris, 2% Glycine SDS-PAGE Running 25 mM Tris, 200 mM Glycine, 0.1% SDS Buffer

Example 1

The Method Targeting GST-p53 and Employing a Benzyl-Modification

1.1—Preparing a Candidate Mixture of Nucleic Acids

[0064] The single stranded DNA starting library comprising 44 wobble positions (OW1_Cy5: 5′-CY5-AGC CAC GGA AGA ACC AGA NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNG CAG AAG CGA CAG CAA CA-3; N: dA, dC, dG, Ethynyl-dU (1:1:1:1)) was synthesized and HPLC purified Ella Biotech, Martinsried, Germany.

[0065] The library was further functionalized with the Benzyl-azide in a Cu(I) catalysed azide-alkyne cycloaddition (CuAAC reaction) as follows: 5 μl of a 100 μM DNA solution were mixed with 65 μl H.sub.2O and 10 μL 10×Phosphate Buffer, pH 7.0. Then 10 μl 10 mM azide solution were added and the reaction was started by addition of 10 μl freshly prepared catalyst solution (4 mM tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 1 mM CuSO.sub.4, 25 mM Sodium ascorbate, incubated for 10 min at room temperature before addition to the DNA mixture). The reaction mixture was incubated for 1 h at 37° C., 350 rpm and the functionalized ssDNA was purified with an Amicon 10 K MWCO column according to the manufacturers recommendation. Washing was performed once with 400 μL H.sub.2O. The DNA candidate mixture was heated up to 95° C. for 5 min and let cool down to room temperature.

1.2—Preparation of the Target Sample

[0066] 1 μg recombinant human GST-p53 expressed and purified by abcam, UK and 20 μg of HEK (Human Embryonic Kidney) cell lysate for negative-SELEX as well as a molecular weight marker were loaded onto a 12.5% TRIS-Glycin SDS-PAGE gel and gelelectrophoresis was performed for 15 min at 100 V followed by 60 min at 170 V. A Western blot using a nitrocellulose membrane was prepared using the Mini-Trans-Blot cell (BioRad) according to the manufacturers recommendation. Afterwards the membrane was blocked with 5% BSA in TBST for 1 h at room temperature on a shaking platform. The membrane was washed 3 times for 5 min with TBST while shaking and the piece containing endogenous p53 was cut out from the HEK lysate lane.

1.3—Contacting the Candidate Mixture with the Target Sample and Partitioning the Nucleic Acids that Bind to GST-p53 from Those that do not Bind

[0067] All of the DNA candidate mixture was added to 5 mL of TBST-MC and incubated with the membrane for 3 h at room temperature while shaking. The membrane was washed 3 times for 5 min with TBST-MC while shaking before the piece containing GST-p53 was cut out and added to a tube containing 200 μL recovery buffer. The sample was heated to 99° C. for 10 min while shaking at 850 rpm. Purification was performed with an Amicon 10 K MWCO column according to the manufacturers recommendation. Washing was performed 3 times with 400 μL H.sub.2O and the last centrifugation step was prolonged to 10 min to reduce the final volume. The resulting sample was used for the subsequent PCR Amplification.

1.4—Amplification of the Binding Nucleic Acids

[0068] The purified DNA (enriched library) was used in a 100 μL PCR reaction that was prepared on ice according to the scheme displayed in table 1. 0.1 pmol of OW1-Cy5 was used as positive control and H.sub.2O without DNA as negative control. The samples were cycled in a Mastercycler® nexus (Eppendorf) for 10 cycles.

TABLE-US-00008 TABLE 1 PCR scheme Reagent Stock-Conc. Final-Conc. Water DNA solution PWO-Buffer 10X 1X dN*TPs 25 mM each 250 μM each F-Primer (biotinylated) 100 μM 1 μM R-Primer (−P) 100 μM 1 μM PWO-Pol. 2.5 U/μl 2.5 U dN*TPs: dATP/dCTP/dGTP/Ethynyl-dUTP

[0069] The cycling scheme was as follows: 2 min at 95° C., followed by 30 sec at 95° C./30 sec at 62° C./1 min at 72° C. for X cycles, 2 min at 72° C., storage at 10° C. After 10 cycles the PCR reaction was split into 10 PCR tubes (10 μL each), 10 μL of the controls were also transferred into fresh PCR tubes. 12 fresh 100 μL PCR reactions were prepared with these on ice according to the scheme in table 1. The samples were cycled until the desired product band could be detected (after another 20 cycles) on an ethidium bromide stained 4% agarose gel. The dsDNA PCR products were purified with the NucleoSpin® Gel and PCR Clean-up kit according to the manufacturers recommendation. 2 PCR samples were pooled on one column and the DNA was eluted 2 times 5 min with 25 μL H.sub.2O at 65° C. The purified samples were pooled (250 μL total) and a backup of this SELEX round (10% of the purified dsDNA) was stored at −20° C.

1.5—Single Strand Displacement

[0070] Purified dsDNA PCR product was supplemented with 25 μl 10× exonuclease reaction buffer and 10 μl lambda exonuclease were added. The mixture was incubated for 60 min at 37° C., 350 rpm and checked on an ethidium bromide stained 4% agarose gel for remaining dsDNA. If there was no dsDNA left, the ssDNA digestion product was purified with the NucleoSpin® Gel and PCR Clean-up kit according to the manufacturers recommendation. The DNA was eluted 2 times 5 min with 25 μL H.sub.2O at 65° C.

1.6—Functionalization of the Enriched Library

[0071] For the next SELEX round the enriched single stranded library was again further functionalized with the Benzyl-azide in a CuAAC reaction as follows: 50 μl of the ssDNA solution were mixed with 20 μl H.sub.2O and 10 μL 10× Phosphate Buffer, pH 7.0. Then 10 μl 10 mM azide solution were added and the reaction was started by addition of 10 μl freshly prepared catalyst solution (4 mM THPTA, 1 mM CuSO.sub.4, 25 mM Sodium ascorbate, incubated for 10 min at room temperature before addition to the DNA mixture). The reaction mixture was incubated for 1 h at 37° C., 350 rpm and the functionalized ssDNA was purified with an Amicon 10 K MWCO column according to the manufacturers recommendation. Washing was performed once with 400 μL H.sub.2O. The DNA candidate mixture was heated up to 95° C. for 5 min and let cool down to room temperature. The ssDNA concentration was determined (see table 2) via A260 on a Nanodrop (Thermo Fisher) and all of the ssDNA sample was afterwards used in the next SELEX round.

1.7-2.sup.nd to 5.sup.th SELEX Round

[0072] The next SELEX round was performed as described for the first round (chapters 1.2-1.6). In total 5 rounds of SELEX were performed. To increase the selection pressure the washing times were increased, the binding time was reduced and a click competitor (Panacea) functionalized with the Benzyl-azide in the same way as the candidate mixture was added as summarized in the following table 2:

TABLE-US-00009 TABLE 2 Summary of SELEX strategy Amount of # of PCR Amount of click cycles SELEX funcitionalized Binding Washing with competitor needed for round ssDNA (pmol) time (h) TBST-MC (pmol) amplification 1 500 3 3 × 5 min — 10 + 20 2 16.8 3 3 × 5 min — 10 + 20 3 4.2 3 3 × 5 min — 10 + 15 4 2.6 3  3 × 10 min 500 10 + 15 5 2.4 1  6 × 10 min 500 10 + 10

1.8—Amplification of Enriched Library and OW1-Cy5

[0073] The enriched library after round 5 as well as the OW1-Cy5 library were amplified in ten 100 μL PCR reactions each as described in chapter 1.4. 1 μL of the sample resulting from the 5.sup.th SELEX round was used per PCR reaction and 0.1 pmol of OW1-Cy5, respectively. After 15 PCR cycles the dsDNA PCR products were visualized on an ethidium bromide stained 4% agarose gel and afterwards purified with the NucleoSpin® Gel and PCR Clean-up kit according to the manufacturers recommendation. The DNA was eluted 2 times 5 min with 25 μL H.sub.2O at 65° C.

1.9—Chemiluminescent Binding Assay

[0074] Single strand displacement and functionalization were performed as described in chapters 1.5 and 1.6 and three GST-p53 Western blots were prepared as described in chapter 1.2 except that the piece containing endogenous p53 was not cut out from the HEK lysate lane this time. 10 pmol of functionalized and non-functionalized enriched library as well as functionalized OW1, respectively, were added to 3×5 mL TBST-MC containing 0.1 mg/mL Dextrane sulphate and 500 pmole functionalized Panacea click competitor, each. The mixtures were added to one Western blot each and incubated on a shaking platform for 1 h at room temperature. The membranes were washed 3 times for 5 min with TBST-MC while shaking before 5 mL per blot of 1:1000 Streptavidin-HRP in TBST-MC were added. After 30 min of incubation at room temperature on the shaker the membranes were washed again for 3×2 min, 1×5 min, 1×10 min and 1×15 min. 7.5 mL of ECL-substrate A and 7.5 mL of ECL-substrate B were mixed and 5 mL of the mixture were added per Western blot. After one minute of incubation the ECL-substrate mixture was exchanged for 5 mL TBST-MC and the chemiluminescence was detected on a VersaDoc (BioRad).

[0075] After 5 SELEX rounds the binding of the starting library OW1 was compared to the binding of the benzyl-functionalized and non-functionalized enriched library and a stronger binding of the functionalized enriched library was seen. This result demonstrates that nucleic acids that bind to the GST-p53 target were successfully enriched. Since a benzyl-modification was used for the selection, without the benzyl-modification no binding was detected.

1.10—Cloning and Sequencing of the Enriched SELEX Pool

[0076] Before cloning the sequences into a bacterial vector, a poly-A-overhang needs to be added by Taq-PCR.

Sample: 1 μl enriched SELEX pool+19 μl ddH.sub.2O
PTC: 1 μl library+19 μl ddH.sub.2O
NTC: 20 μl ddH.sub.2O

TABLE-US-00010 TABLE 3 Buffer conditions Taq PCR: Reagent Stock-Conc. Final-Conc. Water DNA solution Taq-Buffer 10X 1X dN*TPs 25 mM each 250 μM each F-Primer (biotinylated) 100 μM 1 μM R-Primer (−P) 100 μM 1 μM Taq-Pol. 2.5 U/μl 2.5 U

[0077] The temperature steps were the same as for the Pwo-PCR described before. 10 cycles were done.

[0078] Cloning was performed according to standard protocol provided by the manufacturer with the TOPO TA cloning kit (Thermo Fisher).

[0079] Transformation: One aliquot TOP10 E. coli cells (provided by Thermo Fisher) was thawn on ice. To 25 μl bacteria suspension, 2 μl of cloning reaction were added and incubated for 10 min on ice. A heat shock was induced at 42° C. for 30 sec. Cells were put on ice for 5 min before 250 μl LB medium was added and the bacteria were incubated at 37° C. for 1 h, shaking at 300 rpm. Then, they were spread on agar plates containing ampicillin as a selection antibiotic and grown over night at 37° C. On the next day, small amounts of the grown colonies were used as samples for a Taq PCR to control for successful cloning and transformation. Sample preparation: A small fraction was taken from each colony, resuspended in 5 μl ddH.sub.2O, incubated at 95° C. for 10 min and centrifuged for 10 min at 21130 rcf. The supernatant was used as PCR template. In the cases in which the colony PCR lead to an amplification product of the correct length, small amounts of the colonies were taken and incubated overnight at 37° C. shaking at 120 rpm in 5 ml LB-medium containing 100 μg/ml ampicillin each. On the next day, the plasmids were purified according to standard protocol of the NucleoSpin Plasmid kit (Macherey Nagel) and sent to GATC for Sanger sequencing.

1.11—Fluorescent Binding Assay of Monoclone/Single Sequence

[0080] An IRD800CW-labelled forward-Primer was used for amplification of the DNA as described in chapter 1.8 and 1.4, respectively. Afterwards single strand displacement and functionalization was performed as described in chapters 1.5 and 1.6 and three GST-p53 Western blots were prepared as described in chapter 1.2 except that the piece containing endogenous p53 was not cut out from the HEK lysate lane this time. 10 pmole of functionalized and non-functionalized enriched library as well as functionalized OW1, respectively, can be added to 3×5 mL TBST-MC containing 0.1 mg/mL Dextrane sulphate and 500 pmole functionalized Panacea click competitor, each. The mixtures were then added to one Western blot each and incubated on a shaking platform for 1 h at room temperature. The membranes were washed 3 times for 5 min with TBST-MC while shaking before the fluorescence was visualized using an Odyssey Scanner (Li-Cor).

[0081] The FIG. 1 shows the results of the fluorescent binding test of the monoclone of the aptamer of SEQ ID NO: 1. The benzyl-functionalized Sequence (SEQ ID NO: 1), shown on the right, was labeled with the fluorescent dye IRD800CW and showed a stronger binding to GST-P53 than the starting library (“Bibliothek”), shown on the left, as can be taken from the FIG. 1.

Example 2

The Method Targeting Membrane Proteins of PC3 Cells

2.1—Preparing a Candidate Mixture of Nucleic Acids

[0082] The single stranded DNA starting library comprising 44 wobble positions (OW1: 5′-AGC CAC GGA AGA ACC AGA-N44-GCA GAA GCG ACA GCA ACA-3′; N: dA, dC, dG, Ethynyl-dU (1:1:1:1)) was synthesized and HPLC purified Ella Biotech, Martinsried, Germany.

[0083] The library was further functionalized with the Indole-azide in a Cu(I) catalysed azide-alkyne cycloaddition (CuAAC reaction) as follows: 5 μl of a 100 μM DNA solution were mixed with 65 μl H.sub.2O and 10 μL 10× Phosphate Buffer, pH 7.0. Then 10 μl 10 mM azide solution were added and the reaction was started by addition of 10 μl freshly prepared catalyst solution (4 mM THPTA, 1 mM CuSO.sub.4, 25 mM Sodium ascorbate, incubated for 10 min at room temperature before addition to the DNA mixture). The reaction mixture was incubated for 1 h at 37° C., 650 rpm and the functionalized ssDNA was purified with a NucleoSpin® Gel and PCR Clean-up column according to the manufacturers recommendation. Elution was performed four times with 15 μL ddH.sub.2O. The DNA candidate mixture was heated up to 95° C. for 3 min and let cool down to room temperature.

2.2—Preparation of the Target Sample

[0084] PC3 and LNCaP cells were fractionated to the membrane and cytosolic fraction. At least 2 million cells were used for the subcellular fractionation, cultured in three to four T75 flasks. First, the media was discarded and cells washed with cold DPBS. Subsequently, the cells were detached from the surface by adding cold DPBS, pipetting and scraping. Detached cells in DPBS were spun down at 1000× g and RT for 3 min. The supernatant was discarded and pellet was resuspended in 750 μl ice cold and freshly prepared homogenizing buffer containing 0.3 M sucrose, 5 mM Tris-HCl, 0.1 mM EDTA, 1 mM PMSF, pH 7.4. Cells were homogenized on ice, applying 100 strokes with the homogenizing potter. Then, the solution was transferred to a 2-mL vial and centrifuged two times at 800× g for 5 min at 4° C. Pellet, containing intact cells and nuclei, was discarded and the supernatant was centrifuged at 20,000×g for 2 hours at 4° C., yielding cytosolic fraction in the supernatant and enriched membrane fraction in the pellet. To dilute cytosolic proteins in the pellet as much as possible, the pellet was washed with cold DPBS, resuspended in 500 μl homogenizing buffer and centrifuged at 20,000×g for 1 hour at 4° C., again. The last centrifugation step was repeated and, finally, the pellet was resuspended in 250 μl 5 mM Tris and 1 mM PMSF, pH 7.4. Concentration of the protein fractions was determined with Pierce® BCA Protein Assay Kit according to the manufacturers recommendation.

[0085] 1 μg of each protein fraction (PC3.PM as a target, and PC3.CY, LNCaP.PM, LNCaP.CY for negative-SELEX) was mixed with 4× Laemmli buffer and heated up at 95° C. for 5 minutes. Denaturized protein samples and additional PageRuler Prestain Protein Ladder were loaded onto a 10% Tris-Glycin SDS-PAGE gel and electrophoresis was performed at 50 V for 5 min followed at 150 V until the lowest protein ladder band came to the end of the chamber. Semi-dry transfer of proteins was performed with the Semi-dry transfer cell (BioRad) to the nitrocellulose membrane. Afterwards, the membrane was blocked in 5% BSA in TBS-T buffer for 1 hour at RT on a rotator. Finally, the membrane was washed three times with TBS-T for 5 min on a rotator.

2.3—Contacting the Candidate Mixture with the Target Sample and Partitioning the Nucleic Acids that Bind to Membrane Proteins of PC3 Cells (PC3.PM) from Those that do not Bind 1 nmole of the DNA candidate mixture was added to 2.5 mL of TBS-T with 1 nmole of indole-modified click-competitor and 0.01 mg/ml of dextran sulphate and incubated with the membrane for 3 h at RT on a rotator. The membrane was washed 3 times for 5 min with TBS-T while rotating before the lane with membrane proteins of PC3 cells was cut out and added to a tube contacting 1 mL of recovery solution. The sample was incubated at 99° C. for 10 min and 1,400 rpm. The sample was purified with EtOH precipitation and the pellet resuspended in 80 μl of ddH.sub.2O. The resulting sample was used for the subsequent PCR Amplification.

2.4—Amplification of the Binding Nucleic Acids

[0086] Recovered and purified sequences were amplified before going to the next round. PCR reaction volume was 800 μl. Additionally, a no template control (only ddH.sub.2O instead of the sample) and a positive control (OW1 library) were amplified with the PCR reaction. The PCR master mix was prepared on ice using freshly prepared ddH.sub.2O. During the selection, thymidine in the dNTPs mixture was replaced with EdU and instead of usual primers, a biotinylated forward and phosphorylated reverse primers were used. 100 μl of the reaction mixture were aliquoted into 0.5 ml vials and transferred from ice directly to a preheated PCR cycler.

TABLE-US-00011 TABLE 4 PCR scheme. Reagent Stock C Final C 1x 10x PWO buffer 10x 1x 10 μl fwd primer (bio) 100 nM 1 μM 1 μl rev primer (pho) 100 nM 1 μM 1 μl dNTPs (each) 25 mM 1.25 μM 1 μl PWO Polymerase 2.5 U/μl 0.025 U/μl 1 μl ddH.sub.2O 76 μl Sample 10 μl

TABLE-US-00012 TABLE 5 Settings used in the PCR reaction. Step no Step Temperature Time 1 Initial denaturation 95° C. 2 min 2 Denaturation 95° C. 30 s 3 Annealing 62° C. 30 s 4 Elongation 72° C. 1 min 5 Repetition of step 2-4 6 Final elongation 72° C. 2 min 7 Cooling down 4° C. ∞

[0087] The samples were cycled until the desired product band could be detected on an ethidium bromide stained 4% agarose gel. The dsDNA PCR products were purified with the NucleoSpin® Gel and PCR Clean-up kit according to the manufacturers recommendation. The DNA was eluted 4 times with 25 μl of ddH.sub.2O.

2.5—Single Strand Displacement

[0088] After purification, 10 μL of Lambda Exonuclease buffer and 1 μL of Lambda Exonuclease were added to 90 μL dsDNA. The mixture was incubated at 37° C. for 15 min and 1,000×g in a thermomixer. The completeness of digestion was checked with the agarose gel electrophoresis, where the band at 80 bp should not be visible anymore, but only the band of ssDNA at 50 bp. After digesting one strand of DNA, the solution was purified with the NucleoSpin® Gel and PCR Clean-up kit according to the manufacturers recommendation. The DNA was eluted 4 times with 15 μl of ddH.sub.2O.

2.6—Functionalization of the Enriched Library

[0089] For the next SELEX round all of the enriched single stranded library was again further functionalized with the Indole-azide in a CuAAC reaction as follows: 60 μl of the ssDNA solution were mixed with 10 μl ddH.sub.2O and 10 μL 10× Phosphate Buffer, pH 7.0. Then, 10 μl 10 mM azide solution were added and the reaction was started by addition of 10 μl freshly prepared catalyst solution (4 mM THPTA, 1 mM CuSO.sub.4, 25 mM Sodium ascorbate, incubated for 10 min at room temperature before addition to the DNA mixture). The reaction mixture was incubated for 1 h at 37° C., 650 rpm and the functionalized ssDNA was purified with a NucleoSpin® Gel and PCR Clean-up kit according to the manufacturers recommendation. Elution was performed 4 times with 25 μL ddH.sub.2O. The DNA candidate mixture was heated up to 95° C. for 3 min and let cool down to room temperature. The ssDNA concentration was determined via A260 on a Nanodrop (Thermo Fisher) and all of the ssDNA sample was afterwards used in the next SELEX round.

2.7-2.sup.nd to 7.sup.th SELEX Round

[0090] The next SELEX round was performed as described for the first round (chapters 2.2-2.6). In total 7 rounds of SELEX were performed. To increase the selection pressure the washing times were increased, the binding time was reduced and the amount of negative selection target (LNCaP.PM) was increased.

TABLE-US-00013 TABLE 6 Summary of SELEX strategy. Incubation Washing PCR SELEX Target pmole Time & Number of round Selection μg (OW1) Competitors Time Volume cycles volume 1 POSITIVE: PC3.PM: 1 1,000 1:1 click- 3 h 3 × 5 min  18 800 μl 2 PC3.PM PC3.CY: 1 15 competitor 3 ml 16 800 μl 3 NEGATIVE: LNCaP.CY: 1 14 & 0.01 mg/ml 2 h 3 × 10 min 16 800 μl PC3.CY dextran 5 ml LNCaP.PM sulphate 4 +NEGATIVE: +LNCaP.PM: 2 28 16 800 μl 5 LNCaP.PM 24 16 800 μl 6 10 1 h 3 × 15 min 18 1000 μl 7 +LNCaP.PM: 10 10 7 ml 16 1000 μl

2.8—Amplification of Enriched Pools and Initial OW1 Library

[0091] Sequences, obtained after the second, fourth, sixth, and seventh round as well as the OW1 library were amplified in 800-1000 μl of PCR reaction mix, for which approximately 0.1 pmole of selected sequences per 100 μl were used. After 8 PCR cycles, the dsDNA PCR products were purified with the NucleoSpin® Gel and PCR Clean-up kit according to the manufacturers recommendation. Single strand displaced and modification of ssDNA with the Indole followed as described in chapter 2.5 and 2.6.

2.9—Chemiluminescent Binding Assay

[0092] The membrane was prepared with 1 μg of each protein fraction as described in chapter 2.2. Each membrane was incubated in 2.5 mL of TBS-T with 10 pmol of ssDNA sequences (OW1 library, 2.sup.nd, 4.sup.th, 6.sup.th, or 7.sup.th round pool), 1:1 click-competitor and 0.01 mg/ml dextran sulphate. Subsequently, tubes with the membranes were rotated for 1 hour at RT. After incubation, the membranes were washed three times with 5 ml of TBS-T for 10 minutes. Streptavidin-HRP was incubated with the membrane and, consequently, strong interaction between biotin and streptavidin was formed. For each membrane 2 ml of 1:1000 diluted Streptavidin-HRP in TBS-T were used and incubated with the membrane for 30 min at RT while rotating. Afterwards, the membrane was washed with TBS-T 3×1 ml (2 min), 1×2 ml (5 min), 1×3 ml (10 min) and 1×4 nil (15 min). Finally, the reagent with peroxidase (1 mL) and luminol (1 mL) was added and incubated for 1 min. The chemiluminescence was detected on a VersaDoc (BioRad).

2.10-8.sup.th to 10.sup.th SELEX Round

[0093] The last three SELEX rounds were performed as described for the first round (chapters 2.2-2.6) with an exception that only band 2 (FIG. 2) was cut out and the recovered sequences used for the next round. The cut membrane piece was recovered in 100 μl of recovery solution for 10 min at 99° C. and 1,400 rpm. The sample was purified with EtOH precipitation and the pellet resuspended in 10 μl of ddH.sub.2O. The resulting sample was used for the subsequent PCR amplification in 100 μl of PCR reaction mix using fluorophore-labelled forward primer instead of biotinylated one. Afterwards, amplified DNA was used for further PCR in 800 μl of PCR reaction mix (1 μl of PCR product/100 μl PCR reaction mix) as described in chapter 2.4.

[0094] The FIG. 2 shows the binding studies of the 7.sup.th and 10.sup.th SELEX round visualized with IR-fluorescence. As can be seen in the FIG. 2, after the seventh round, only band 2 was cut out and recovered sequences were used for the next SELEX rounds.

Other SELEX Steps were Done the Same as Described Previously. To Increase the Selection pressure the washing times and volume were increased and the amount of the ssDNA was decreased.

TABLE-US-00014 TABLE 7 Summary of SELEX strategy for 8.sup.th to 10.sup.th SELEX round. Incubation Washing PCR SELEX Target pmole Time & Number of round Selection μg (OW1) Competitors Time Volume cycles volume 8 POSITIVE: 2.sup.nd PC3.PM: 1 10 1:1 click- 1 h 3 × 10 min 20 100 μl 9 band-PC3.PM PC3.CY: 1 5 competitor 5 ml 16 100 μl 10 NEGATIVE: LNCaP.CY: 1 5 & 0.01 2 h 5 × 10 min 15 100 μl PC3.CY LNCaP.PM: 1 mg/ml 5 ml LNCaP.PM dextran LNCaP.PM sulphate

[0095] 2.11—IR-Fluorescence Binding Studies

[0096] The last three rounds were performed and binding visualized using IR-800CW-labelled forward primer, as the visualization needed to be directly performed for each round. For the binding studies, 10 pmol (8.sup.th round) or 5 pmol (9.sup.th and 10.sup.th round) of Indole-modified ssDNA was used in 2.5 mL of TBS-T with additional 1:1 click-competitor and 0.01 mg/mL of dextran sulphate. The membrane was incubated for 1 h at RT while rotating, and afterwards washed 3 times with 5 mL of TBS-T for 10 min. Finally, the bound sequences were visualized using an Odyssey Scanner (Li-Cor).

2.12—Cloning and Sequencing of the Enriched SELEX Pool

[0097] Before cloning the sequences into a bacterial vector, a poly-A-overhang needs to be added by Taq-PCR. Additional sample no-template control was prepared, where only water was used instead of the template (10.sup.th round SELEX pool).

TABLE-US-00015 TABLE 8 PCR scheme for amplification with Taq-polymerase. Substance Stock Conc. Final Conc. Volume Taq-Buffer 5x 1x 20 μL MgCl.sub.2 25 mM 2 mM (+1.5 mM) 8 μl dNTPs 25 mM 250 μM 1 μL fw-Primer 100 μM 0.5 μM 1 μL rv-Primer (without 5′-P!) 100 μM 0.5 μM 1 μL Taq-Polymerase 5 U/μL 0.025 U/μL 1 μL ddH.sub.2O 67 μL Template/water 1 μl

TABLE-US-00016 TABLE 9 Settings used in the PCR reaction with the longer final elongation time. Step no Step Temperature Time 1 Initial denaturation 95° C. 2 min 2 Denaturation 95° C. 30 s 3 Annealing 62° C. 30 s 4 Elongation 72° C. 1 min 5 Repetition of step 2-4 6 Final elongation 72° C. 5 min 7 Cooling down 4° C. ∞

[0098] In total, 11 PCR cycles were performed.

[0099] Cloning was performed according to standard protocol with the TOPO TA cloning kit (Thermo Fisher).

[0100] Transformation: One aliquot TOP10 E. coli cells (provided by Thermo Fisher) was thawn on ice. To 25 μl bacteria suspension, 2 μl of cloning reaction were added and incubated for 10 min on ice. A heat shock was induced at 42° C. for 30 sec. Cells were put on ice for 5 min before 250 μl LB medium was added and the bacteria were incubated at 37° C. for 1 h, shaking at 300 rpm. Then, they were spread on agar plates containing ampicillin as a selection antibiotic and grown over night at 37° C.

[0101] On the next day, 50 single clones were picked with a tip and resuspended in 5 mL of LB-Amp-Medium (50 μg/ml Amp) for each sample. The samples were incubated over night at 37° C. and 120 rpm. On the next day, 2 mL of E. coli LB-Medium were purified according to standard protocol of the NucleoSpin Plasmid kit (Macherey Nagel). After purification, 1 μl of each sample was taken and used for the PCR. The samples for which the PCR lead to an amplification product of the correct length were sent to GATC for Sanger sequencing.

2.13—IR-Fluorescence Binding Studies of Monoclone/Single Sequence

[0102] The most enriched sequences obtained by Sanger sequencing were used for the binding assay. Clones prepared in chapter 2.12 were amplified in 3 rounds, each time taking 1 μl of the template for 100 μl PCR reaction (see Table 1). Instead of using biotinylated forward primer, an IRD800CW-labelled forward-primer was used. The amplification was done after 8 cycles. In the third round of amplification, no clones were seen on the agarose gel and the PCR was performed in 800 μl to have a sufficient amount of DNA for further binding studies. After the purification with NucleoSpin® Gel and PCR Clean-up kit, single strand displacement and functionalization was performed as described in chapters 2.5 and 2.6. The binding was tested on the membrane with 1 μg of all four proteins fractions, prepared as described in chapter 2.2. 0.5 pmol of Indole-modified ssDNA of each clone was used during 1-hour incubation with the membrane in 2.5 mL of TBS-T with additional 0.5 pmol of Indole-modified click-competitor and 0.1 mg/ml of dextran sulphate. After incubation, the membrane was washed 3 times with 5 mL of TBS-T for 10 min while rotating. The fluorescence was visualized using an Odyssey Scanner (Li-Cor).

[0103] The FIG. 3 shows the results of the fluorescent binding test of the monoclone of of the aptamer of SEQ ID NO: 2. The indol-functionalized Sequence (SEQ ID NO: 2), was labeled with the fluorescent dys IRD800CW. As can be taken from the FIG. 3, the THP-1 cell line (THP-1, on the right) as well as the PC3 cells (on the left) with which the selection was performed, showed an intense signal in the region of band 2, thus probably the same protein. Other cell lines tested also showed other bands that could possibly be splice variants of the target.

Example 3

Comparison of Different Buffers in Recovering Nucleic Acids Binding to a Target Sample

[0104] In step c) the nucleic acids that bind to the target sample are partitioned from those that do not bind. This includes recovering the nucleic acids binding to a target, for example by recovering from a Western blot membrane. The recovered nucleic acids may be purified before being used in further SELEX cycles to remove proteins and/or residual buffer components which otherwise may interfere with a PCR reaction. Preferred purification methods are phenol-chloroform extraction and nucleic acid precipitation.

[0105] Various recovery conditions were tested for a recovery of single stranded DNA from 1 μg of protein on a Western blot. As a model nucleic acid a single stranded DNA library comprising 42 wooble positions (FT2: CAC GAC GCA AGG GAC CAC AGG NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN CAG CAC GAC ACC GCA GAG GCA; N: dA, dC, dG, C8-Alkyne-dU (1:1:1:1)) (SEQ ID NO: 7) was used, which was synthesized and PAGE purified by ATDBio (Southampton, United Kingdom). The library was further functionalized with indole-azide in a Cu(I) catalysed azide-alkyne cycloaddition as described in example 2.1. 1 μg of protein fraction PC3.PM as described in example 2.2 was used as the protein.

3.1 Recovery from a Western Blot

[0106] 1 μg per lane of PC3.PM proteins were loaded onto a 10% Tris-Glycin SDS-PAGE gel and after electrophoresis proteins were transferred onto a nitrocellulose membrane as described in example 2.2. The Western blot was cut into sections of three columns each, which were incubated with 20 pmol of the indole-modified FT2 library, 20 pmol of click competitor and 0.01 mg/ml dextran sulfate for 3 h at room temperature on a rotator. Thereafter the membranes were washed 3 times for 5 min with TBS-T, each lane was separated and added to a tube containing 1 mL of different recovery solutions. The samples were incubated at 99° C. for 40 min and 1400 rpm for recovery. Table 10 summarises the respective recovery solutions.

TABLE-US-00017 TABLE 10 Summary of recovery solutions No name specification 1 ddH.sub.2O 2 0.1M ammonium acetate 3 1M NaCl 4 1 × UREA 1.25M Urea, 12.5 mM Na.sub.2EDTA 5 4 × UREA 9M Urea, 50 mM Na.sub.2EDTA 6 mild SDS buffer (WB) 200 mM glycine pH 2.2, 1% Tween 20, 0.1% SDS 7 Recovery Buffer 0.1M NaHCO.sub.3, 1% SDS, pH 8.0 Example 2 (ChlP) 8 Recovery Buffer 50 mM NaHCO.sub.3, 1.25M UREA, Example 1 (UR + Ch) 6.25 mM EDTA, 0.5% SDS

[0107] After the respective recovery, the DNA remaining on the membrane was visualized by chemiluminescence using Streptavidin-HRP. Chemiluminescence was detected on a VersaDoc (BioRad). A membrane without recovery was used as control. The comparison of the membranes showed that recovery using a buffer was generally more effective than heating the membrane in ddH.sub.2O, ammonium acetate or 1 M NaCl, as the intensity of the bands remaining on the membranes incubated in buffer was greatly reduced compared to the intensity of the bands on the control.

[0108] This shows that using urea buffers 1×UREA and 4×UREA, mild SDS buffer and the recovery buffers as used in examples 1 and 2 allowed more effective recovery of the DNA. These recovered DNA sample were therefore further examined in view of purification and amplification.

3.2 Purification and Amplification Via PCR

[0109] The DNA samples recovered using using urea buffers 1×UREA and 4×UREA, and the recovery buffers ChlP and UR+Ch were purified using phenol/chloroform extraction followed by ethanol precipitation (PC) or via ethanol precipitation alone (EtOH). The DNA of the samples was then amplified via PCR using 24 cycles and PCR products were visualized on an ethidium bromide stained 4% agarose gel.

[0110] The FIG. 4 shows the PCR products of recovered, purified DNA after 24 cycles of amplification on an ethidium bromide stained 4% agarose gel. As control a sample without DNA denoted “NTC”=no template control was used. As can be taken from the FIG. 4, the urea buffers 1×UREA and 4×UREA, and the recovery buffers UR+Ch and ChlP used in examples 1 and 2, respectively, showed good recovery of DNA after purification and PCR. Particularly the recovery buffer of Example 1 (UR+Ch) containing urea and SDS provided good recovery results in combination with phenol-chloroform extraction and with ethanol precipitation. Also recovery buffer of Example 2 (ChlP) provided good recovery results in combination with ethanol precipitation alone.