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WO2011060557A1 - Procédé de sélection d'aptamères - Google Patents

Procédé de sélection d'aptamères Download PDF

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Publication number
WO2011060557A1
WO2011060557A1 PCT/CA2010/001887 CA2010001887W WO2011060557A1 WO 2011060557 A1 WO2011060557 A1 WO 2011060557A1 CA 2010001887 W CA2010001887 W CA 2010001887W WO 2011060557 A1 WO2011060557 A1 WO 2011060557A1
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Prior art keywords
aptamers
membrane
dna
aptamer
target
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Xing-fang LI
Jing Zhang
Yanming Liu
X. Chris Le
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University of Alberta
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University of Alberta
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised
    • C12N2330/31—Libraries, arrays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the present invention relates to a method for efficient and simultaneous selection of aptamers which specifically bind to target molecules.
  • 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).
  • aptamers are easier to produce and inexpensive since the generation process occurs in vitro without the need for animals.
  • aptamers can be generated against any target protein, and the binding target site of the protein can be determined.
  • 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.
  • SELEX systematic evolution of ligands by exponential enrichment
  • target-specific aptamers are selected and synthesized in vitro from a random aptamer library
  • 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).
  • 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.
  • the present invention relates to a method for efficient and simultaneous selection of aptamers which specifically bind to target molecules.
  • 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;
  • 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.
  • the aptamer comprises single-stranded DNA (ss-DNA) or single stranded RNA (ss-RNA).
  • the method further comprises amplifying uneluted ss-DNA remaining on the membrane, and determining the sequences of the uneluted ss-DNA.
  • the gel electrophoresis is sodium dodecyl sulfate-polyacrylamide gel
  • the membrane is a polyvinyldifluoride membrane.
  • the target molecules are bacterial proteins.
  • the bacterial protein is E. coli protein formamidopyrimidine DNA glycosylase.
  • the bacterial proteins are extracted from cell lysate and separated by 2D gel electrophoresis followed by western blotting.
  • the target molecules are viral proteins.
  • the viral protein is the core protein of hepatitis B virus.
  • the target molecules are mammalian cell proteins.
  • the mammalian cell proteins are extracted from cell lysate and separated by 2D gel electrophoresis followed by western blotting.
  • the washing of step (d) is conducted at least twenty-five times.
  • the elution of step (f) is conducted at least fifteen times.
  • the elution of step (f) comprises gradient elution with stepwise increases of urea concentration.
  • the amplification is conducted for 19 to 20 cycles.
  • the amplification includes adding dimethyl sulfoxide to a PC mixture.
  • the amplification is conducted at an annealing temperature of 56°C.
  • 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:
  • the physical support may comprise a nitrocellulose or polyvinylidene difluoride membrane.
  • the eluted aptamers may be characterized by amplification and sequencing.
  • 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 ⁇ g for lanes 3 to 5; 15 ⁇ g 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 ⁇ g for lanes 2 to 4; 15 ⁇ g 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
  • 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 pCRTM 4-TOPOTM 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.
  • 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.
  • 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.
  • binding means an interaction or complexation between a target and an aptamer (ss-DNA or ss-RNA), resulting in a sufficiently stable complex.
  • 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.
  • 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.
  • the invention is directed to a method of selecting aptamers which specifically bind to target molecules comprising the steps of:
  • 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
  • the target molecules may be initially separated by gel electrophoresis, such as by SDS-
  • Suitable physical supports include nitrocellulose or polyvinylidene difluoride (PVDF) membranes.
  • PVDF polyvinylidene difluoride
  • test proteins thrombin, lysozyme, formamidopyrimidine DNA glycosylase (Fpg), bovine serum albumin (BSA), and ovalbumin
  • 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 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
  • 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.
  • 2D gel electrophoresis Two-dimensional
  • the target molecules are viral proteins.
  • 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.
  • 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.
  • the membrane may be blocked with BSA.
  • 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.
  • 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).
  • 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.
  • elution step the ssDNA/RNA bound to the target proteins are eluted from the complexes.
  • 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.
  • the five target proteins resulted in five groups of ten elution fractions.
  • the PCR conditions i.e., the cycle number, PCR mixture, and annealing temperature
  • the cycle number i.e., the cycle number, PCR mixture, and annealing temperature
  • 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.
  • DNA with high GC contents may form stem-loop secondary structures and induce the generation of shorter sequences of products.
  • DMSO dimethyl sulfoxide
  • UV absorbance detection of OD 2 6o is commonly used to estimate DNA concentration. To determine the best dynamic range of this detection (SmartspecTM 3000; BioRad), DNA samples with known concentrations were tested. Based on the OD3 ⁇ 4o values (1 unit of OD 2 6o equals 33 pg/mL ssDNA), detected ssDNA concentrations were calculated for comparison. The calculated concentrations match the actual concentrations when OD 26 o is in the range of 0.05 to 0.2 (Au), corresponding to concentrations of DNA from 1.65 to 6.6 ⁇ g 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.
  • FIGS. 9 to 10 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.
  • FIGS. 1 1 to 15 show the PCR products from the elution solutions 5-10 for the five proteins.
  • 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.
  • the bands of washing solutions 29 and 30 (lanes 7 and 8) are stronger than those of washing solutions 27 and 28.
  • 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.
  • elution solutions 1-15 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.
  • 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.
  • three elution buffer solutions were used:
  • sequences of ssDNA which may specifically bind to their target proteins 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
  • 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).
  • 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).
  • K d binding affinity
  • Tm melting temperature
  • AG change in Gibbs free energy
  • 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 TATM kit), BSA, dNTP kit (Invitrogen);
  • MinEluteTM PCR purification kit (Qiagen, Mississauga, ON); TweenTM 20 (Fisher Scientific, Nepean, ON); TEMED and EDTA (EMD Chemical Inc., Gibbstown, NJ, USA).
  • 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 ⁇ of 10% SDS, 40 ⁇ 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
  • 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.
  • 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.
  • 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:
  • 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)
  • 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 TATM kit, Invitrogen).
  • 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 MgCl 2 , 5 mM KC1, 1 mM CaCl 2 , 0.02% TweenTM 20; Stoltenburg et al, 2006).
  • binding buffer 100 mM NaCl, 20 mM Tris-HCl at pH 7.6, 2mM MgCl 2 , 5 mM KC1, 1 mM CaCl 2 , 0.02% TweenTM 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.
  • 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 4 40 (approximate to 10 24 ) random sequences.
  • most aptamer libraries contain no more than lO 16 random sequences due to limitations of synthesis.
  • 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% TweenTM 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.
  • elution buffer 40 mM Tris-HCl at pH 8.0, 10 mM EDTA, 3.5M urea, 0.02% TweenTM 20; Stoltenburg et al, 2006
  • the optimized PCR mixture consisted of 10 ⁇ , of 1 Ox PCR buffer, 5.6 ⁇ , of 50 mM MgCl 2 , 5 ⁇ , of 20 mM forward primer (SEQ ID NO: 62), 5 ⁇ of 20 mM reverse primer (SEQ ID NO: 63), 4 ⁇ of 10 mM dNTPs, 63 ⁇ , of autoclaved deionized water, 5 of DMSO, 0.5 ⁇ . of 5 unit ⁇ L Taq polymerase and 2 ⁇ , of template DNA.
  • 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.
  • the amplified 80-bp PCR products were purified by a MinEluteTM 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
  • 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).
  • sequence DNA library by DMSO and betaine application to in vitro combinatorial selection of aptamers. J. Biochem. Biophys. Methods 2005, 64, 147-151.

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Abstract

L'invention concerne un procédé de sélection d'aptamères se liant spécifiquement à des molécules cibles. Le procédé comprend : la séparation des molécules cibles par électrophorèse sur gel et leur transfert sur une membrane ; l'incubation de la membrane avec un mélange randomisé d'aptamères, en particulier des ADN monocaténaires, dans des conditions permettant de former des complexes aptamère-molécule cible sur la membrane ; le lavage de la membrane ou sa mise en contact avec l'ADNase I pour éliminer les aptamères non liés et les aptamères faiblement liés ; puis l'élution des aptamères liés à partir des complexes aptamère-molécule cible, et l'amplification et le séquençage des aptamères élués.
PCT/CA2010/001887 2009-11-20 2010-11-22 Procédé de sélection d'aptamères Ceased WO2011060557A1 (fr)

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US20140148356A1 (en) * 2012-08-28 2014-05-29 The Governors Of The University Of Alberta Apparatus and method for single-cycle selection of aptamers
WO2015041909A1 (fr) * 2013-09-18 2015-03-26 General Electric Company Procédés de sélection d'éléments de liaison et leurs utilisations
WO2016018934A1 (fr) * 2014-07-29 2016-02-04 Companion Dx Reference Lab, Llc (Texas) Biomarqueurs et sélection d'aptamères basée sur leur morphologie
EP3056901A1 (fr) * 2015-02-13 2016-08-17 Technische Universität Berlin Procédé de contrôle de protéines à l'aide d'aptamères
WO2018053185A1 (fr) 2016-09-14 2018-03-22 Alios Biopharma, Inc. Oligonucléotides modifiés et méthodes d'utilisation
US10359419B2 (en) 2013-10-02 2019-07-23 General Electric Company Methods for detection of target using affinity binding
WO2020018578A1 (fr) * 2018-07-17 2020-01-23 University Of Washington Compositions et procédés associés à une sélection de cellules réversible fondée sur un aptamère
EP3604554A1 (fr) * 2018-08-02 2020-02-05 Rheinische Friedrich-Wilhelms-Universität Bonn Procédé d'identification ou de production d'un aptamère pour un peptide dénaturé ou une protéine dénaturée
WO2024009960A1 (fr) * 2022-07-05 2024-01-11 Eisai R&D Management Co., Ltd. Technologie d'enrichissement de banque de ligands de biofluide et ses utilisations

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WO1997038134A1 (fr) * 1996-04-05 1997-10-16 Nexstar Pharmaceuticals, Inc. Methode de detection de compose cible au moyen d'un ligand d'acide nucleique
WO2002024954A1 (fr) * 2000-09-22 2002-03-28 Somalogic, Inc. Traitements selex modifiés sans purification de protéines

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WO1997038134A1 (fr) * 1996-04-05 1997-10-16 Nexstar Pharmaceuticals, Inc. Methode de detection de compose cible au moyen d'un ligand d'acide nucleique
WO2002024954A1 (fr) * 2000-09-22 2002-03-28 Somalogic, Inc. Traitements selex modifiés sans purification de protéines

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9150866B2 (en) * 2012-08-28 2015-10-06 The Governors Of The University Of Alberta Apparatus and method for single-cycle selection of aptamers
US20140148356A1 (en) * 2012-08-28 2014-05-29 The Governors Of The University Of Alberta Apparatus and method for single-cycle selection of aptamers
WO2015041909A1 (fr) * 2013-09-18 2015-03-26 General Electric Company Procédés de sélection d'éléments de liaison et leurs utilisations
US9828599B2 (en) 2013-09-18 2017-11-28 General Electric Company Methods of selecting binding-elements and uses thereof
US10359419B2 (en) 2013-10-02 2019-07-23 General Electric Company Methods for detection of target using affinity binding
WO2016018934A1 (fr) * 2014-07-29 2016-02-04 Companion Dx Reference Lab, Llc (Texas) Biomarqueurs et sélection d'aptamères basée sur leur morphologie
EP3056901A1 (fr) * 2015-02-13 2016-08-17 Technische Universität Berlin Procédé de contrôle de protéines à l'aide d'aptamères
US10267798B2 (en) 2015-02-13 2019-04-23 Technische Universität Berlin Method for detecting proteins by means of aptamers
WO2018053185A1 (fr) 2016-09-14 2018-03-22 Alios Biopharma, Inc. Oligonucléotides modifiés et méthodes d'utilisation
US10793859B2 (en) 2016-09-14 2020-10-06 Janssen Biopharma, Inc. Modified oligonucleotides and methods of use
US12077757B2 (en) 2016-09-14 2024-09-03 Janssen Pharmaceutica Nv Modified oligonucleotides and methods of use
WO2020018578A1 (fr) * 2018-07-17 2020-01-23 University Of Washington Compositions et procédés associés à une sélection de cellules réversible fondée sur un aptamère
CN112805384A (zh) * 2018-07-17 2021-05-14 华盛顿大学 涉及基于适体的可逆细胞选择的组合物和方法
US12509661B2 (en) 2018-07-17 2025-12-30 University Of Washington Compositions and methods related to aptamer-based reversible cell selection
EP3604554A1 (fr) * 2018-08-02 2020-02-05 Rheinische Friedrich-Wilhelms-Universität Bonn Procédé d'identification ou de production d'un aptamère pour un peptide dénaturé ou une protéine dénaturée
WO2020025539A1 (fr) * 2018-08-02 2020-02-06 Rheinische Friedrich-Wilhelms Universität Bonn Méthode d'identification ou de production d'un aptamère pour un une protéine ou un peptide dénaturé
WO2024009960A1 (fr) * 2022-07-05 2024-01-11 Eisai R&D Management Co., Ltd. Technologie d'enrichissement de banque de ligands de biofluide et ses utilisations

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