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WO2019067383A1 - Criblage d'aptamères - Google Patents

Criblage d'aptamères Download PDF

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Publication number
WO2019067383A1
WO2019067383A1 PCT/US2018/052525 US2018052525W WO2019067383A1 WO 2019067383 A1 WO2019067383 A1 WO 2019067383A1 US 2018052525 W US2018052525 W US 2018052525W WO 2019067383 A1 WO2019067383 A1 WO 2019067383A1
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WO
WIPO (PCT)
Prior art keywords
aptamer
aptamers
sequence
nucleotides
adapter
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Application number
PCT/US2018/052525
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English (en)
Inventor
Alexander Wei
Chongli YUAN
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Purdue Research Foundation
Sumitomo Chemical Company Limited
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Application filed by Purdue Research Foundation, Sumitomo Chemical Company Limited filed Critical Purdue Research Foundation
Publication of WO2019067383A1 publication Critical patent/WO2019067383A1/fr

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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/13—Applications; Uses in screening processes in a process of directed evolution, e.g. SELEX, acquiring a new function

Definitions

  • the present disclosure relates to aptamers for the detection of an analyte in a sample; a substrate having attached thereto the aptamers according to the invention; a kit of parts for the detection of an analyte in a sample comprising said aptamers; and a method for the detection of an analyte in a sample comprising the use of said aptamers.
  • Aptamers are synthetic receptors that can adopt local or global conformations that enable them to bind molecular analytes with high affinity.
  • Aptamers are generally considered as biopolymers and can be made from natural or unnatural oligonucleotides, amino acids, or hybrid structures Currently, a large number of generated aptamers can bind various targets, ranging from simple inorganic molecules to large protein complexes, and entire cells. In fact, aptamers are generally nucleotide analogues of antibodies, but aptamer generation is significantly easier and cheaper than the production of antibodies.
  • a common feature or strategy in aptamer generation, and to identify high affinity aptamer candidates is the ability to identify high-affinity molecules within a large library of structures by "directed evolution," in which candidate aptamers are subjected to multiple cycles of chemical selection steps, followed by self-amplification and further screening.
  • the process, systematic evolution of ligands by exponential enrichment (SELEX) involves use of a library of randomly generated oligonucleotides which undergoes a process of sequential selection (typically 15-20 iterations) to generate aptamers having high affinity for target molecules 1 ' 2 .
  • an aptamer library of randomly generated sequences of fixed length flanked by constant 5' and 3' primer ends (known as adapter sequences, figure 1 ) are typically bound to a solid substrate via base pairing of the adapter sequence with surface-tethered oligonucleotide primers.
  • the annealed complex is then washed with the target molecules of interest, with any oligomers that do not bind to the aptamer released into the wash.
  • the bound sequences l are then eluted and amplified by PCR to prepare for subsequent rounds of selection.
  • aptamers of high affinity can be identified based on consensus sequences found in members of the final aptamer library via DNA sequencing.
  • a method for identifying an aptamer molecule capable of binding a target analyte in a sample comprising: providing a plurality of test aptamer molecules each comprising a sequence of base units having at a first end a first adapter sequence common to all or a majority of the aptamers and at a second end a second adapter sequence common to all or a majority of the aptamers, providing a substrate comprising surface tethered sequences complementary or substantially complementary to said first and/or said second adapter sequence(s); and wherein said method comprises the steps of: i) hybridising the test aptamer molecules to said surface tethered sequences complementary to said first adapter sequence; ii) exposing said hybridised aptamers to a sample under conditions whereby target analytes in said sample that have target analyte binding sites for an aptamer, bind any one or more of said test hybridised aptamer molecules; iii)
  • Reference herein to an analyte is any selected target the aptamer can recognise but most typically is selected from the group comprising: protein, enzyme, antigen, receptor, hormone, metabolite, an organic molecule and carbohydrate.
  • the purpose of the invention is to identify aptamer molecules from a number of test aptamer molecules to determine those with the best binding affinity for a given one or more target analyte(s) in a sample. Therefore, preferably, said test aptamer molecules form part of a library of aptamers suitable for screening according to the method of the invention. Accordingly, ideally, steps i) - iii) and/or iv) - vi); or i) - vi) are repeated a number of times, such as 5-20 times, including all intervals therein and ideally, 10-15 times.
  • said adapter sequences at either end of said aptamer are different from each other.
  • substrate refers to any material or coating to which sequences can be tethered such as, but not limited to, silver, gold, aluminium, copper, platinum, iron and silica metals/metalloids or an alloy thereof or a polymer-coated version thereof, or indeed a polymer material.
  • the method disclosed herein relies upon the sequential binding of a pair of adapter sequences of the test aptamers to surface tethered sequences, and therefore requires the sequential hybridisation of said adapter sequences to the surface tethered sequences.
  • this can be achieved by numerous means such as the provision of different adapter sequences that vary in their sequences such that their hybridisation properties differ whereby sequential hybridisation of said adapter sequences to the surface tethered sequences is controlled by sequential changes in reaction conditions.
  • first and second surface tethered sequences are provided on separate substrates thus the method steps i) and iv) involve the use of a first substrate comprising a first set of surface tethered sequences complementary or substantially complementary to the first adapter sequence and a second substrate comprising a second set of surface tethered sequences complementary or substantially complementary to the second adapter sequence, respectively.
  • said first and said second substrates are made from the same material.
  • said first and second substrates are made from different materials.
  • a dissociation step is provided following the elution or extraction step of iii) and/or vi) above whereby the eluted or extracted aptamer is separated from the bound target analyte(s).
  • an amplification step is provided following the elution or extraction step of iii) and/or vi) whereby, in the former case, the eluted or extracted aptamer(s) are amplified before the subsequent hybridisation.
  • said base units are nucleotides, ideally, selected from the group comprising: ssDNA and ssRNA, more ideally still comprising natural and/or unnatural nucleotides.
  • said sequence of base units preferably comprises biopolymers known to bind DNA or RNA such as, but not limited to, oligonucleotides, peptide nucleic acid, locked nucleic acid and oligodeoxynucleotides such as deoxyribonucleic acid or ribonucleic acid.
  • both the sequence of base units and the adapter sequences By utilising both adapter sequences for target analyte recognition one can achieve increased target diversity and also permit the possibility of binding multiple sites on the same target, such that they interact to form secondary structures.
  • this can, in part, be influenced by the size of the sequence of base units forming the aptamer; where a short sequence of base units is used, both adapter ends may hybridise and bind the same target molecule leading to multiple binding of the same target analyte at the same site or in close proximity to one another. Therefore, according to this preferred embodiment said base units are between 5-40 nucleotides in length and most ideally 15-35 nucleotides in length and even more ideally 20-30 nucleotides in length.
  • each adapter sequence may lead to the binding of two or more different sites on the same target.
  • said base units are between 25-100 nucleotides in length and most ideally 30-75 nucleotides in length and even more ideally 35-50 nucleotides in length.
  • the methodology disclosed herein uses a double hybridisation selection step to identify the best aptamer molecules wherein each adapter sequence is, in turn, hybridised to surface tethered sequences (and/or substrate) and the aptamer is tested for binding to the target analyte.
  • each adapter sequence is, in turn, hybridised to surface tethered sequences (and/or substrate) and the aptamer is tested for binding to the target analyte.
  • this raises the further possibility of using a different target analyte site in the first and second hybridisation steps such that the first adapter sequence is tested for its ability to bind a first target analyte site and the second adapter sequence is tested for its ability to bind a second target analyte site, or even a different target analyte.
  • This will thus lead to the identification of aptamer molecules capable of simultaneously binding two target sites of interest (within the same target or two different targets) giving rise to bifunctional aptamer molecules.
  • said method comprises identifying an aptamer molecule capable of binding at least two target analytes in a sample and/or at least two target analyte sites in a single target in a sample wherein the target analyte or the target analyte site of step ii) differs from the target analyte or the target analyte site of step v).
  • said target analyte(s) or said target analyte site(s) is/are any binding partner the aptamer can recognise but most typically is selected from the group comprising: protein, enzyme, antigen, receptor, hormone, metabolite, an organic molecule and carbohydrate.
  • the invention concerns the identification of aptamers with the best binding affinity/specificity for at least one target analyte or analyte site
  • the invention employs the sequential binding of different parts of the aptamer, i.e. using at least a part of the adapter sequences at both ends of the aptamer, thus leading to the identification of aptamers with superior affinity for target analytes or analyte sites.
  • aptamers utilising at least a part of the adapter sequences at both end of the aptamer has been found to increase the diversity of base sequences involved in analyte binding leading to isolation of aptamers with unique recognition sequences not otherwise obtainable by conventional SELEX. Without wishing to be bound by theory, it is thought that this process maximises utilisation of the oligonucleotide adapter space to create unique recognition sequences, some of which may be superior in binding affinity versus those generated by standard protocols. Further, it is thought that by adopting this process aptamers can be found that are capable of binding multiple target sites thereby promoting formation of secondary structure that leads to multiple binding motifs that are multivariant in nature, thus offering aptamers with superior binding capabilities.
  • an aptamer comprising: a) a sequence of base units
  • said base units are between 5-40 nucleotides in length and most ideally 15-35 nucleotides in length and even more ideally 20-30 nucleotides in length.
  • said base units are between 25-100 nucleotides in length and most ideally 30-75 nucleotides in length and even more ideally 35-50 nucleotides in length.
  • said first adapter sequence and said second different adapter are different from each other, ideally but not exclusively, in terms of sequence.
  • a library of aptamers comprising: a plurality of test aptamers at least one of which, and preferably a number of which such as a majority or even all, has/have a different sequence of base units with respect to other aptamers in said library; but common to all or a majority of the aptamers is a first adapter sequence at a first end of said aptamer and a second adapter sequence at a second end of said aptamer.
  • said first and second adapter sequences are placed at the same end of each of the same or similar aptamers or at different/opposite ends of the same or similar aptamers.
  • said first adapter sequence and said second different adapter are different from each other, ideally, but not exclusively, in terms of sequence.
  • a kit of parts for identifying an aptamer molecule capable of binding a target analyte in a sample comprising: a plurality of test aptamer molecules each comprising a sequence of base units having at a first end a first adapter sequence common to all or a majority of the aptamers and at a second end a second adapter sequence common to all or a majority of the aptamers; and/or a plurality of test aptamers at least one of which, and preferably a number of which, has/have a different sequence of base units with respect to other aptamers in said library; but common to all or a majority of the aptamers is a first adapter sequence at a first end of said aptamer and a second adapter sequence at a second end of said aptamer; and at least one substrate comprising at least one surface tethered sequence complementary or substantially complementary to said first and/or said second adapter sequence.
  • any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.
  • FIG. 1 Schematic illustration of an aptamer according to the invention comprising a first and a second outer adapter sequence and positioned therebetween a random sequence of nucleotides whose binding affinity for a target analyte is to be tested.
  • Figure 2. Schematic illustration of the selection method according to the invention wherein aptamers according to the invention and so comprising a first and a second outer adapter sequence are exposed to test or target material after which they are eluted or extracted from their substrate before being bound to a second substrate and then exposed to the same or a different test or target material, after which the process is repeated using test or target material that is less and less concentrated thus effectively increasing the stringency of the assay until only those aptamers with the strongest binding affinity will recognise test or target material.
  • Beads 1 and 2 provide two alternative anchoring surfaces for aptamer libraries. These two types of beads are used in alternating orders during SELEX to encompass both ends of aptamers (5' and 3') in target recognition. Although not shown, PCR amplification of aptamers that bound test or target material was undertaken prior to sequential binding to beads 1 or 2.
  • Figure 3 (a). Shows a consensus sequence of random regions (52 bp) of aptamers.
  • Figure 3 (b). Shows alignment results of 10 randomly selected colonies.
  • the consensus sequence binding affinities to Cortisol can be determined using BLI (Bio- layer Interferometry).
  • SELEX systematic evolution of ligands by exponential enrichment
  • ssDNA single- stranded DNA
  • Binding sequences are selected and amplified by PCR, e.g. using biotin-labelled primers. This is followed by removal of antisense strands to generate an ssDNA pool for subsequent rounds of selection.
  • the enrichment of the selected pools can be monitored by flow cytometry binding assays, with selected pools having increased fluorescence compared with the unselected DNA library.
  • the random aptamer library (N30) and primers were purchased from Trilinkbiotech.
  • the structure of the aptamer library attached to the beads is: 5' TAG GGA AGA GAA GGA CAT ATG AT- N30-TTG ACT AGT ACA TGA CCA CTT GA 3'
  • the buffers used were:
  • Wash/Binding (W&B) buffer (10mM Tris-HC!, pH 7.5, 1 mM EDTA and 2M NaCI);
  • TE buffer (10m Tris-HCl, 1 mM EDTA).
  • Streptavidin labelled magnetic Dynabeads !Vl-270 were used to immobilize the aptamer librar via streptavidin-biotin interactions for each round of the SELEX; with the use of a strong magnet to separate the beads by pull downs.
  • Cortisol Sigma-Aldrich, >98%) and progesterone (Sigma-Aldrich, >99%) were used as the positive and negative analytes to be used in the rounds of the process.
  • a 1 .5 mL eppedndorff tube containing a 500 ⁇ aliquot of M-270 Dynabeads was placed over a magnet until said Dynabeads have been pulled down to the bottom of the tube. Then the 500 ⁇ of storage buffer was pipetted off the Dynabeads, retaining the Dyanbeads within the tube. The Dynabeads were then washed with W&B buffer. Specifically, 500 ⁇ _ of W&B buffer was applied and the tube vortexed until the solution is a homogenous orange/brown colour and there was no sediment remaining in the tube. The beads were then pulled down again using a magnet. This wash process was carried out in total 3 times.
  • Biotinylated reverse primer (1 nmole) was added to the 500 ⁇ washed Dynabead solution and the mixture was incubated for 10 minutes with gentle agitation at room temperature. The beads were then washed a total of three times using with W&B buffer using the same method as described above. Following the final application of fresh W&B buffer to this point the beads and primers can be produced in bulk and stored at 4°C.
  • the selection rounds used were both positive (Cortisol) and negative (progesterone).
  • the positive was the main analyte used within this process, as it represents the target substrate.
  • concentration of Cortisol was initially high and steadily decreased before a negative selection round (progesterone) was completed followed by further positive selection rounds in which the concentration of the target substrate (Cortisol) was steadily increased.
  • the use of such a gradient allows for the total specific binding affinity of the aptamer library to increase with every round.
  • the table below displays the concentration of Cortisol (positive) or progesterone (negative) used as screening substrate for each of SELEX selection rounds 1 to 1 1 .
  • the hybridized library was washed with SELEX buffer twice to remove any free DNA fragments.
  • the sample is subjected to gentle agitation for 5 minutes per wash to ensure that the DNA is not perturbed (e.g., sheared) by, e.g. vortexing;
  • the incubated mixture is then subjected to a magnetic pull down and the supernatant is removed and collected to use for the PCR (PCR template).
  • the beads are then washed 3 times with SELEX buffer.
  • PCR step used for this process was primarily high fidelity PCR using Phusion polymerase (NEB). Later a mutagenic step was added to increase the diversity of aptamer library.
  • NEB Phusion polymerase
  • a mutagenic step was added to increase the diversity of aptamer library.
  • Each reaction included 5X PCR reaction Buffer, 10mM dNTPs, 10 nmoles primers (reverse biotinylated for ease of separation), 4 % PCR reaction volume for the DNA template volume and 0.5 L Phusion polymerase.
  • the PCR was run over 36 cycles with initial hold at 98°C (30s), denaturing at 98°C (10s), annealing at 60°C (30s), extension at 72°C (30s), final extension at 72°C (60s) and then finally hold at 4°C.
  • the PCR product was then analysed using gel electrophoresis (15% DNA-PAGE gel). Two control samples will be loaded simultaneously: 1 . a DNA template free PCR sample and 2. DNA template.
  • Dynabeads 100 ⁇ is washed 3 times using W&B buffer.
  • the Dynabeads are then incubated with 40 L of the PCR product that includes the reverse biotinylated primer.
  • the sample is then gently vortexed until the solution is homogenous.
  • the sample is then incubated with gentle agitation for 15 minutes at room temperature.
  • the sample is then washed with W&B buffer three times.
  • the volume of the pooled supernatant is noted and an equal volume of isopropanol is added to the tube followed by centrifugation for 15 mins at 15,00rpm, 4°C.
  • centrifugation for 15 mins at 15,00rpm, 4°C.
  • most of the solution is removed and one equal volume of ice-cooled 70% ethanol is added to the tube before a further centrifugation step (5 mins at 15,000 rpm, 4°C).
  • the ethanol is then pipetted from the tube and the tube is placed in a heated plate (45°C) for 15 mins to remove any remaining ethanol.
  • the next generation DNA library can be resuspeneded in 50 ⁇ of TE buffer.
  • the DNA library concentration is then measured using UV-Vis and adjusted to 5 ⁇ .
  • the library is then ready for a further round of SELEX.
  • the SELEX selection round may be repeated several times (e.g. 15-20 times) before the library is annealed to T vectors (T-easy, Promega), transformed into E coli (XL Blue Ultra II, Stratagene) and the selected aptamer sequences subjected to Sanger sequencing to determine the sequence of the aptamers.

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Abstract

La présente invention concerne des aptamères pour la détection d'un analyte dans un échantillon ; un substrat auquel sont attachés les aptamères selon l'invention ; un kit d'éléments pour la détection d'un analyte dans un échantillon ; et un procédé de détection d'un analyte dans un échantillon comprenant l'utilisation desdits aptamères.
PCT/US2018/052525 2017-09-30 2018-09-25 Criblage d'aptamères WO2019067383A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2020069565A1 (fr) 2018-10-02 2020-04-09 WearOptimo Pty Ltd Système de mesure

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WO2011068909A2 (fr) * 2009-12-01 2011-06-09 Brigham And Women's Hospital, Inc. Compositions de cellule d'aptamères
WO2014100434A1 (fr) * 2012-12-19 2014-06-26 Caris Science, Inc. Compositions et procédés pour le criblage d'aptamères
US20150064696A1 (en) * 2012-03-28 2015-03-05 Nec Corporation Method for detecting target substance, assay kit, and detection apparatus

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Publication number Priority date Publication date Assignee Title
WO2011068909A2 (fr) * 2009-12-01 2011-06-09 Brigham And Women's Hospital, Inc. Compositions de cellule d'aptamères
US20150064696A1 (en) * 2012-03-28 2015-03-05 Nec Corporation Method for detecting target substance, assay kit, and detection apparatus
WO2014100434A1 (fr) * 2012-12-19 2014-06-26 Caris Science, Inc. Compositions et procédés pour le criblage d'aptamères

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Title
ABDELSAYED, MM ET AL.: "Multiplex Aptamer Discovery through Apta-Seq and Its Application to ATP Aptamers Derived from Human- Genomic SELEX", ACS CHEMICAL BIOLOGY, vol. 12, no. 8, 11 July 2017 (2017-07-11), pages 2149 - 2156, XP055586176, ISSN: 1554-8929, DOI: 10.1021/acschembio.7b00001 *
LIU, Q ET AL.: "Aptamer-Conjugated Nanomaterials for Specific Cancer Cell Recognition and Targeted Cancer Therapy", NPG ASIA MATERIALS, vol. 6, no. 2, 11 April 2014 (2014-04-11), pages 1 - 10, XP055586152 *
RAY, P ET AL.: "Cell-SELEX Identifies a (Sticky) RNA Aptamer Sequence", JOURNAL OF NUCLEIC ACIDS, vol. 2017, no. 4943072, 17 January 2017 (2017-01-17), pages 1 - 10, XP055586116, ISSN: 2090-0201, DOI: 10.1155/2017/4943072 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020069565A1 (fr) 2018-10-02 2020-04-09 WearOptimo Pty Ltd Système de mesure
US12048558B2 (en) 2018-10-02 2024-07-30 WearOptimo Pty Ltd System for determining fluid level in a biological subject

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