Detection of modifications of polynucleotides by proximity assays

Abstract

The present invention relates to the detection of modifications in polynucleotides by binding an affinity binding element to a nucleotide sequence. The affinity binding element may include a compound that binds to a modified nucleotide base and a nucleotide sequence complementary to a target nucleotide sequence, an oligonucleotide primer complementary to the target nucleotide sequence, or a nucleotide sequence complementary to an oligonucleotide linker. The present invention provides methods for detecting one or more modifications in a nucleotide sequence. The invention also provides affinity binding elements, and optionally, oligonucleotide primers and / or oligonucleotide probes, and methods of using the affinity binding elements in assays to detect modified bases. The methods of the invention enable the detection of modified nucleotides associated with cancer, psychiatric disorders or metabolic diseases.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 529,794, filed on July 31, 2023, the entire disclosure of which is incorporated herein by reference, including all figures, tables and amino acid or nucleic acid sequences. Background Technology

[0003] Modifications on polynucleotides have been identified as being involved in a variety of cellular processes and human diseases, including, for example, cancer, mental disorders, and metabolic diseases. Therefore, the detection of nucleic acid modifications is currently attracting attention in the medical field, particularly in the diagnosis and treatment of diseases associated with polynucleotide modifications.

[0004] Traditional methods for identifying polynucleotide modifications involve sodium bisulfite conversion and sequencing, or affinity capture and sequencing of modified polynucleotides; however, sequencing requires specialized instruments and is time-consuming. Furthermore, existing methods are used to reveal modification sites but are difficult to use for quantitatively measuring the proportion of modified polynucleotides.

[0005] Therefore, there is still a need for a method that can detect nucleotide modifications with sufficient specificity and efficiency without using sequencing, thereby reliably distinguishing modified nucleotide bases from unmodified nucleotide bases. Summary of the Invention

[0006] This invention relates to the detection of polynucleotide modifications, such as DNA or RNA modifications, including, for example, methylation of adenosine. The invention provides at least one oligonucleotide and at least one affinity-binding element, such as an antibody or aptamer. In some embodiments, the oligonucleotide is linked to the affinity-binding element and serves as an oligonucleotide primer. In some embodiments, if the modification is an RNA modification, cDNA is generated via reverse transcription. The invention also relates to a method for detecting polynucleotide modifications by binding the affinity-binding element to the modified polynucleotide base. In some embodiments, the detection of polynucleotide modifications includes, for example, using immunoaffinity assays based on proximity linkage assay (PLA) or immunoaffinity assays based on proximity extension assay (PEA).

[0007] In certain embodiments, the binding of the affinity binding element to the modified polynucleotide, and the hybridization of the oligonucleotide to the polynucleotide sequence proximal to the site of the modified polynucleotide, can be used to detect the modified polynucleotide, for example, by nucleic acid amplification, more specifically, by PCR, preferably by real-time PCR (qPCR), digital PCR (dPCR) or digital PCR in droplets (dPCRd). In certain embodiments, multiplexed PCR can be advantageously used to detect at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different modifications in a polynucleotide by using at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different PCR assays. In certain embodiments, multiplexed PCR can be preferably used to detect at least two identical modifications in a polynucleotide in a single sample or in 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different samples. In certain embodiments, sample barcoding is included in the nucleic acid tail.

[0008] The present application also relates to the oligonucleotides and affinity binding elements, as well as compositions, biological compositions, detection kits and diagnostic kits thereof. BRIEF DESCRIPTION OF DRAWINGS

[0009] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0010] Figures 1A-1H Steps of an exemplary immunoaffinity RNA modification assay using the affinity binding element and oligonucleotide primers are shown.

[0011] Figure 2 Steps of an exemplary proximity ligation assay (PLA)-based immunoaffinity RNA modification assay using the affinity binding element and oligonucleotides, with modification-specific antibodies binding to modified RNA bases are shown. This figure also provides an example of when the target RNA does not contain a modified RNA base, and the modification-specific antibody does not bind.

[0012] Figure 3 Proximity ligation assay (PLA)-based immunoaffinity RNA modification assay using the affinity binding element and oligonucleotides, with modification-specific antibodies binding to modified RNA bases are shown. This figure also provides an example of quantitatively detecting the proportion of RNA modifications for a specific RNA sequence.

[0013] Figures 4A-4B Detection of single N 6 -methyladenosine bases. DETAILED DESCRIPTION

[0014] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms "comprises", "comprising", "including", "having" and "contains" or variations thereof, are intended to mean a non-exclusive inclusion, such that a process, method, article, composition or apparatus that comprises, includes or contains a list of elements is not necessarily limited to those elements, but can include other elements not expressly listed or inherent to such process, method, article, composition or apparatus. The transitional terms / phrases "comprises", "comprising", "containing", "having", "consisting essentially of, "consisting essentially of", "consisting of and "consisting of" (and any grammatical variations thereof) are used interchangeably.

[0015] The phrase "consisting essentially of or "consisting essentially of means that the implementation includes the specified materials or steps, and those that do not materially affect the basic and novel characteristics of the implementation.

[0016] The term "about" denotes an acceptable error range over determined by one of skill in the art for the particular numeric value, which is dependent on how the value is measured or determined, i.e., limitations of the measurement system. In the context of polynucleotide lengths and where the term "about" is used, the polynucleotide comprises the number of bases or base pairs specified, varying by 0-10% (X ± 10%) of that value. In the context of compositions comprising amounts of components and where the terms "about" or "approximately" are used, the composition comprises the amount of the component specified, varying by 0-10% (X ± 10%) of that value (error range). In the context of pH, the terms "about" or "approximately" are intended to include ± 0.2 units of the pH value.

[0017] In the present disclosure, ranges are used as shorthand for describing a group of values. Whenever a numerical range is given, it should be understood that every number within that range and to the nearest tenth...hundredth...etc. is also specifically envisioned within the range. For example, the range of 1.0-10.0 should be read to include the end values 1.0 and 10.0, as well as any number between and including 1.1, 1.2, 1.3, 1.4, and so forth, to 10.0, to the nearest tenth. The same logic is applied to any range that is given.

[0018] The term "antibody" refers to an immunoglobulin or a fragment form thereof. The term includes, but is not limited to, polyclonal or monoclonal antibodies of the IgA, IgD, IgE, IgG, and IgM classes, derived from human or other mammalian cell lines, including natural forms or genetically modified forms, such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. "Antibody" encompasses complex forms, including but not limited to fusion proteins containing an immunoglobulin portion. "Antibody" also includes antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAb, Fc, heavy chain single variable domains (VHH), and other compositions, whether or not they retain antigen binding function.

[0019] As used herein, the term "positive" when referring to a result or signal indicates the presence of the analyte or substance being detected in a sample. The term "negative" when referring to a result or signal indicates the absence of the analyte or substance being detected in a sample. Positives and negatives are typically determined by comparison to at least one control, such as a threshold level required for a sample to be positive, or a negative control, such as a known blank. A "control" sample or value refers to a sample used as a reference, typically a known reference, for comparison to a test sample. For example, a test sample can be taken under test conditions, such as in the presence of a test compound (e.g., in the absence of a test compound (negative control), or in the presence of a known compound (positive control)), and compared to a sample from known conditions. A control can also represent an average value collected from a plurality of tests or results. Those skilled in the art will recognize that controls can be designed to evaluate any number of parameters, and will understand which controls are valuable in a given situation and are capable of analyzing data based on comparison to the control values. Controls can also be used to determine the significance of data. For example, if there is a variation in the value of a parameter in a control, then a variation in a test sample would not be considered significant.

[0020] As used herein, a "calibration control" is similar to a positive control in that it contains a known amount of a known analyte. In the case of a real-time PCR assay, a calibration control can be designed to contain known amounts of multiple known analytes. The amount of analyte in a calibration control can be set to the lowest cutoff amount, for example, such that a sample above that amount would be considered "positive" for that analyte, and a sample below that amount would be considered "negative" for that analyte. In some cases, a multi-level calibration control can be used in order to more accurately determine the range of amounts of an analyte. For example, an assay can include calibration controls at known low and high amounts, or at known minimum, intermediate, and maximum amounts.

[0021] As used herein, "subject," "patient," "individual," and their grammatical equivalents are used interchangeably and refer to a mammal, such as a human and non-human primates, as well as rabbits, cats, dogs, mice, murines, squirrels, goats, pigs, deer, and other mammalian species, unless otherwise indicated. The term "patient" does not necessarily indicate that the subject has been diagnosed with a particular disease, but generally refers to an individual under the medical or veterinary supervision. A "patient" can be an individual who is seeking treatment, monitoring, adjustment or modification of an existing treatment regimen, etc.

[0022] The term "biological sample," "sample from an individual," "sample from a patient," or "sample from a subject" encompasses a variety of sample types obtained from an organism. The organism can include, for example, eukaryotic organisms, including, for example, fungi, plants, and animals; bacteria; or archaea. The term encompasses body fluids from eukaryotic organisms, such as blood, blood components, saliva, nasal mucus, serum, plasma, cerebrospinal fluid (CSF), urine, and other liquid samples of biological origin, as well as solid tissue biopsy, tissue cultures, lysates or supernatants from cultured patient cells. In the context of the present disclosure, the biological sample is typically a body fluid containing detectable amounts of nucleotide sequences, such as sequences from a virus, or preferably sequences from the genome of a subject, such as a tissue sample, blood or a blood component (e.g., plasma or serum), saliva, oropharyngeal, nasopharyngeal, or nasal secretions (mucus). The biological sample can be treated, such as by removing cells or cell debris, prior to assay. The term also encompasses samples that are manipulated after their procurement, such as by reagent treatment, lysis, sedimentation, or enrichment for particular components.

[0023] As used herein, the term "nucleic acid," "oligonucleotide," or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof, which can be in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids comprising known analogues of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term "nucleic acid" is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0024] As used herein, the term "isolated nucleic acid" molecule refers to a nucleic acid molecule that is separated from other nucleic acid molecules that normally associate with it in its natural environment. Thus, an "isolated nucleic acid molecule" includes, but is not limited to, a nucleic acid molecule that is free of the nucleotides that naturally flank the nucleic acid molecule (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction digestion) at either or both ends of the nucleic acid molecule. In addition, an isolated nucleic acid molecule can further include engineered nucleic acid molecules, such as recombinant nucleic acid molecules or synthetic nucleic acid molecules. For example, a nucleic acid molecule that is present in a nucleic acid library (e.g., a cDNA library or a genomic library) or a gel (e.g., an agarose or polyacrylamide gel) along with hundreds to millions of other nucleic acid molecules, or present in a gel containing restriction-digested genomic DNA, is not an "isolated nucleic acid."

[0025] As used herein, the term "gene" refers to a DNA segment that is involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) involved in the transcription / translation of the gene product, as well as intervening sequences (introns) between individual coding segments (exons) that are involved in the regulation of transcription / translation.

[0026] As used herein, the term "identical" or percent "identity," in the context of two or more polynucleotide sequences or amino acid sequences, refers to two or more sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (for example, the nucleotide probe used in the methods of the application has at least 70% sequence identity, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the target sequence or its complement), when compared and aligned for maximum correspondence as measured using one of the following sequence comparison algorithms, or by visual inspection and eye. Such sequences are subsequently referred to as "substantially identical." For polynucleotide sequences, the definition also pertains to the complement of the test sequence.

[0027] The term "hybridizes to," in reference to two sequences, indicates that the two sequences have sufficient complementarity to each other that nucleotide base pairing occurs between the two sequences. Sequences that hybridize to each other can be completely complementary, but can also exist with some degree of mismatch. Depending on the stringency of the hybridization, mismatches between two complementary sequences of up to about 5% to 20% are still allowed to hybridize between the two sequences. Generally, high stringency conditions have higher temperatures and lower salt concentrations, while low stringency conditions have lower temperatures and higher salt concentrations. High stringency conditions for hybridization are preferred.

[0028] Affinity binding elements and primer design and detection

[0029] In certain embodiments, the present application provides one or more affinity binding elements capable of binding to a modified nucleotide base. In certain embodiments, the modified nucleotide base is a modified DNA base or a modified RNA base. In certain embodiments, the modified nucleotide is methylated adenosine, methylated adenine, methylated cytosine, acetylated cytidine, pseudouridine, inosine, oxidation products of methylated cytosine (e.g., 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC)). In certain embodiments, known nucleotide modifications can be found in reference databases, such as RMbase (see World Wide Web: rna.sysu.edu.cn / rmbase / ), Modomics (see World Wide Web: genesilico.pl / modomics / ), EWAS Data Hub (see World Wide Web: hngdc.cncb.ac.cn / ewas / datahub / index), and the National Genomic Data Center (see World Wide Web: ngdc.cncb.ac.cn).

[0030] In certain embodiments, the affinity binding element can comprise a protein, peptide, or aptamer that binds to a modified nucleotide base. In certain embodiments, the protein is an antibody, such as an anti-N6-methyladenosine (m6A) antibody, an anti-inosine (I) antibody, an anti-pseudouridine (Ψ) antibody, an anti-1-methyladenosine (mlA) antibody, an anti-2-methyladenosine (m2A) antibody, an anti-N6,N6-dimethyladenosine (m6,6A) antibody, an anti-8-methyladenosine (m8A) antibody, an anti-5-methylcytosine antibody, an anti-5-hydroxymethylcytosine antibody, an anti-5-formylcytosine antibody, or an anti-5-carboxylcytosine antibody. In certain embodiments, the protein of the affinity binding element can be an RNA binding protein, such as a YT521-B homology domain (YTH) family protein. In certain embodiments, the YTH domain family protein can be YTHDF1, YTHDF2, or YTHDF3. Polyclonal and monoclonal antibodies that bind to modified nucleotides, such as methylated adenosines, are available from commercial suppliers, such as Synaptic Systems GmbH (Goettingen, Germany), Epigentek Group Inc. (Farmingdale, NY, USA), Novus Biologicals LLC (Centennial, CO, USA), or Abeam PLC (Boston, MA, USA).

[0031] In certain embodiments, the affinity binding element can comprise a nucleotide sequence. The nucleotide sequence can be linked to the protein or peptide of the affinity binding element. In certain embodiments, the nucleotide sequence of the affinity binding element can be designed to hybridize to upstream or downstream of, or a portion of, a target nucleic acid sequence comprising a modified base. Alternatively or additionally, the nucleotide sequence of the affinity binding element can be designed to hybridize to a different oligonucleotide molecule, such as an oligonucleotide linker or an oligonucleotide primer that is complementary to a nucleotide sequence upstream or downstream of a target nucleic acid sequence comprising a modified base. In certain embodiments, the oligonucleotide linker can link the nucleotide sequence of the affinity binding element to an oligonucleotide that is complementary to a nucleotide sequence located upstream or downstream of a target nucleic acid sequence comprising a modified base. In certain embodiments, the affinity binding element can bind to at least 1, 2, 3, 4, 5, or more different modified nucleotide bases.

[0032] In some embodiments, the nucleotide sequence of the affinity-binding element or the sequence of the oligonucleotide primer is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, about 30, about 40, about 45, about 50, about 60, about 68, about 75, about 80, about 90, or about 100 bases in length. In some embodiments, the target nucleotide sequence containing the modified bases to which the affinity-binding element hybridizes is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, about 30, about 40, about 50, or about 100 bases in length. In some embodiments, the oligonucleotide may have an anchoring structure, a loop structure, a foot structure, or any combination thereof, such as a SuperSelective primer-type oligonucleotide. In some embodiments, the length of the nucleotide sequence upstream or downstream of the target nucleotide sequence containing the modified bases and hybridizing with the affinity-binding element or the oligonucleotide primer (i.e., the complementary region upstream or downstream of the target nucleotide sequence) is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, about 30, about 40, about 50 bases, or longer. In a preferred embodiment, the complementary region upstream or downstream of the target nucleotide sequence is about 15 to about 60 bases, preferably about 16 to about 50 bases, more preferably about 17 to about 40 bases, more preferably about 17 to about 35 bases, and even more preferably about 15 to about 30 bases. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, about 50, about 75, about 100, about 150, about 200, about 250, about 300, about 350, or about 384 or more oligonucleotide primers may be used with affinity-binding elements in a single reaction, wherein each oligonucleotide primer is complementary to a different region upstream or downstream of the target nucleotide sequence, and each oligonucleotide hybridizes to a nucleotide sequence adjacent to a polynucleotide modification site. In some embodiments, the nucleotide sequence of the oligonucleotide or the nucleotide sequence of the affinity-binding element binds immediately adjacent to the modification site (e.g., at 0 nucleotides upstream or downstream of the modification site), or binds to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, about 30, about 40, about 50, about 60, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350 or more nucleotides upstream or downstream of the modification site.

[0033] In certain embodiments, a sample specific identifier (also referred to in the art as an index, barcode, zip code, aptamer, etc.) can be incorporated into the nucleotide sequence of the affinity binding element or the sequence of the oligonucleotide primer, such that when the cDNA or proximity sequence is generated, different samples can be mixed and detected by using the barcode specific primers (e.g., by DNA sequencing).

[0034] In certain embodiments, the nucleotide sequence of the oligonucleotide primer or the affinity binding element can be 100% complementary to the sequence upstream or downstream of the modification site, or have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence complementarity. In certain embodiments, the sequence of the oligonucleotide primer or the affinity binding element can also have a plurality of possible alternative nucleotides represented by IUPAC notation, such as R, Y, S, W, K, M, B, D, H, V, N, or a gap ("-" or ".") nucleotide. In certain embodiments, the sequence of the affinity binding element can also include a universal base, such as 5-nitroindole.

[0035] In certain embodiments, the oligonucleotide can be complementary to a region upstream or downstream of the modification site, and can be linked to an oligonucleotide linker or directly linked to the nucleotide sequence of the affinity binding element. The linked oligonucleotide can be used as a basis for synthesizing a DNA template, such as a cDNA molecule and / or a PCR detectable reporter. In certain embodiments, the PCR detectable reporter can be a site to which an oligonucleotide probe can bind. In certain embodiments, the nucleotide sequence of the affinity binding element can prime a reverse transcription reaction to generate a single stranded cDNA.

[0036] In certain embodiments, the present application provides one or more primers for amplifying cDNA produced by reverse transcription of a target nucleic acid, or for amplifying a DNA template produced by ligation of the oligonucleotide primer to the oligonucleotide adaptor, or to the nucleotide sequence of the region of the affinity binding member (sometimes also referred to as a target nucleotide sequence), the amplified region (or amplicon) of which is about 10 bp to about 5000 bp, about 20 bp to about 4000 bp, about 30 bp to about 3000 bp, about 40 bp to about 2000 bp, about 50 bp to about 1000 bp, about 60 bp to about 750 bp, about 70 bp to about 500 bp, about 100 bp to about 1000 bp, about 100 bp to about 500 bp, or about 300 to about 500 bp. Primers for use in amplification reactions can be designed according to known algorithms or by one of skill in the art. For example, algorithms implemented in commercially available or custom software can be used to design primers for amplifying a target sequence based on the complementarity and stringency of the primer to the target region. Stringency refers to hybridization conditions selected to optimize binding of polynucleotide sequences with varying degrees of complementarity. Stringency is influenced by temperature, ionic strength, the presence of organic solvents in the hybridization mixture, and the length and base composition of the sequences to be hybridized, as well as the degree of base mismatch, among other factors. The combination of parameters is more important than the absolute measurement of any one factor.

[0037] Generally, the primers or primer sequences used to amplify DNA can be at least 12 bases in length, more commonly about 15, about 18, about 20, about 21, about 22, about 23, about 24, about 25, or about 30 base pairs in length. Primers or primer sequences used to amplify DNA are generally designed so that the melting temperatures of all primers involved in a particular reaction differ from one another by no more than 5°C, and most preferably differ from one another by no more than 2°C. Primers or primer sequences used to amplify DNA are also designed to avoid priming for extension on themselves or one another. The concentration of primers should be sufficient to bind to the number of target sequences being amplified to provide an accurate assessment of the number of amplified sequences. Those of skill will recognize that the amount of primer concentration will vary depending on the binding affinity of the primer and the amount of sequence to be bound. Typical primer concentrations range from about 0.1 μM to about 1 μM, about 0.2 μM to about 0.8 μM, about 0.3 μM to about 0.7 μM, about 0.4 μM to about 0.6 μM, or about 0.4 μM to about 0.5 μM.

[0038] In certain embodiments, oligonucleotide probes can be designed to hybridize to a nucleic acid sequence or portion thereof. In certain embodiments, the complementary nucleotide segment of a probe is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or 100 base pairs or longer. In preferred embodiments, the complementary nucleotide segment of a probe is about 15 to about 60 base pairs, preferably about 16 to about 50 base pairs, more preferably about 17 to about 40 base pairs, more preferably about 17 to about 35 base pairs, more preferably about 18 to about 25 base pairs. In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 125, or more probes can be used in a single reaction, along with oligonucleotide primers complementary to regions upstream or downstream of a modification site and an affinity binding element or a nucleotide sequence of said affinity binding element. In certain embodiments, there are multiple oligonucleotide primers complementary to multiple regions upstream or downstream of a modification site. In addition, probes can be labeled with a fluorescent label (e.g., used with a quencher label). The concentration of a probe can be optimized to facilitate an amplification reaction. In certain embodiments, the probe can be 100% complementary to the target sequence, or have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence complementarity. In certain embodiments, the sequence of the probe can also have multiple possible alternative nucleotides represented by IUPAC notation, e.g., R, Y, S, W, K, M, B, D, H, V, N, or a gap (“-” or “.”) nucleotide.

[0039] There are a variety of formats that utilize fluorescent probes. These formats are generally based on fluorescence resonance energy transfer (FRET) and include molecular beacons, Scorpion® probes, and TaqMan® probes. FRET is a distance-dependent interaction between a donor and an acceptor molecule. The donor and acceptor molecules are fluorophores. If the excitation and emission spectra of the fluorophores overlap, excitation of the donor fluorophore is transferred to the acceptor fluorophore in very close proximity (typically about 10-100 angstroms). As a result, the lifetime of the donor molecule is shortened and its fluorescence is quenched, while the fluorescence intensity of the acceptor molecule is enhanced and depolarized. When the excitation state energy of the donor is transferred to a non-fluorophore acceptor, the fluorescence of the donor is quenched and the acceptor will not subsequently emit fluorescence. In this case, the acceptor acts as a quencher.

[0040] A FRET-based format for real-time PCR uses a DNA probe called a "molecular beacon" (see, e.g., Tyagi et al., Nat. Biotech. 16:49-53, 1998; U.S. Patent No. 5,925,517). The molecular beacon has a hairpin structure with a quencher dye and a reporter dye in close proximity to each other at the end of the hairpin stem. Upon hybridization to a complementary sequence, the loop of the hairpin structure becomes double-stranded and forces the quencher and reporter dyes apart, resulting in a fluorescent signal. A related detection method uses a hairpin primer as a fluorescence generating probe (Nazarenko et al., Nucl. Acid Res. 25:2516-2521, 1997; U.S. Patent No. 5,866,336; U.S. Patent No. 5,958,700). The PCR primer can be designed to produce a fluorescent signal only when the primer is in a linear structure, i.e., incorporated into the PCR product.

[0041] The amplification product can also be detected in solution using a fluorescence-generating 5' nuclease assay (TaqMan assay). See, Holland et al., Proc. Natl. Acad. Sci. U.S.A. 88:7276-7280, 1991; U.S. Patent Nos. 5,538,848, 5,723,591, and 5,876,930. The TaqMan probe is designed to hybridize to a sequence in the desired PCR product. The 5' end of the TaqMan probe contains a fluorescent reporter dye. The 3' end of the probe is blocked to prevent extension of the probe and contains a dye that quenches the fluorescence of the 5' fluorophore. During the subsequent amplification, if a polymerase with 5' exonuclease activity is present in the reaction, the 5' fluorescent label is cleaved off. Cleavage of the 5' fluorophore results in an increase in detectable fluorescence.

[0042] Illustratively, the probe can have the following formula: 5' fluorophore-probe-quencher 3' or 5' quencher-probe-fluorophore 3'.

[0043] In addition to the hairpin structure and the 5'-nuclease PCR assay, other formats using the FRET mechanism have been developed. For example, a single-stranded signal primer has been modified by attachment to two dyes to form a donor / acceptor dye pair such that the fluorescence of the first dye is quenched by the second dye. The signal primer contains a restriction site (U.S. Patent No. 5,846,726) that, when hybridized to the target sequence, allows a proper restriction enzyme to nick the primer. The cleavage separates the two dyes and a fluorescent change is observed due to the decrease in quenching. Non-nucleotide linking reagents for coupling oligonucleotides to ligands have also been described (U.S. Patent No. 5,696,251).

[0044] Other fluorescent probes include inorganic molecules, multi-molecular mixtures of organic and / or inorganic molecules, crystals, heteropolymers, and the like. For example, CdSe-CdS core-shell nanocrystals encapsulated in a silica shell can be readily derivatized for coupling to biomolecules (Bruchez et al. (1998) Science, 281 :2013-2016). Similarly, high fluorescent quantum dots (cadmium selenide capped with zinc sulfide) have been covalently coupled to biomolecules for ultrasensitive bio-detection (Warren and Nie (1998) Science, 281 :2016-2018).

[0045] In certain embodiments, multiplex PCR can be used in the methods of the application. Multiplex PCR makes it possible to detect multiple polynucleotide fragments in the same reaction. See, e.g., PCR PRIMER, A LABORATORY MANUAL (Dieffenbach, ed., 1995) Cold Spring Harbor Press, pp. 157-171. For example, different oligonucleotide primers complementary to regions upstream or downstream of the modification site, different affinity binding elements, or different probes targeting PCR-detectable reporters can be added in parallel in the same reaction vessel. Multiplex assays can involve the use of different fluorescent labels to detect different target sequences that are amplified. In preferred embodiments, a single pair of primers is used to amplify a cDNA sequence, so that fluorescent probes annealing to the sample nucleic acid sequence are degraded.

[0046] In certain embodiments, the probes described herein can comprise any useful label at any useful position in the nucleic acid sequence, including fluorescent labels and quencher labels, e.g., at the 3’ end and / or 5’ end. Exemplary fluorescent labels include quantum dots or fluorophores. Examples of fluorescent labels for use in the present methods include fluorescein, 6-FAM™ (Applied Biosystems, Carlsbad, CA, USA), TET™ (Applied Biosystems, Carlsbad, CA, USA), VIC™ (Applied Biosystems, Carlsbad, CA, USA), MAX, HEX™ (Applied Biosystems, Carlsbad, CA, USA), TYE™ (ThermoFisher Scientific, Waltham, MA, USA) dyes (TYE 665, TYE 705, TEX, JOE), Cy™ (Amersham Biosciences, Piscataway, NJ, USA) dyes (Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7), Texas Red® (Molecular Probes, Inc., Eugene, OR, USA), Texas Red-X, Alexa Fluor® (Molecular Probes, Inc., Eugene, OR, USA) dyes (Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750), DyLight™ (ThermoFisher Scientific, Waltham, MA, USA) dyes (DyLight 350, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight 633, DyLight 649, DyLight 755), ATTO™ (ATTO-TEC GmbH, Siegen, Germany) dyes (ATTO 495, ATTO 488, ATTO 495, ATTO 532, ATTO 550, ATTO 565, ATTO 568, ATTO 647N, ATTO 655, ATTO 680, ATTO 700, ATTO 725, ATTO 748, ATTO 750, ATTO 756, ATTO 770, ATTO 925), BODIPY® (Life Technologies, Carlsbad, CA, USA) dyes (BODIPY FL, BODIPY R6G, BODIPY TR, BODIPY 493 / 503, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665), and IR® (Li-Cor Biosciences, Lincoln, NE, USA) dyes (IR 700, IR 800, IR 807).Siegen, Germany) dyes (ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 520, ATTO 532, ATTO 550, ATTO 565, ATTO Rhol01, ATTO 590, ATTO 594, ATTO 610, ATTO 620, ATTO 633, ATTO 635, ATTO 637, ATTO 647, ATTO 647N, ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO725, ATTO 740), BODIPY® (Molecular Probes, Eugene, OR, USA) dyes (BODI FLPY, BODIPYR6G, BODIPY TMR, BOPDIPY 530 / 550、BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665), HiLyte Fluor™ (AnaSpec, Fremont, California, USA) dyes (HiLyte Fluor 488, HiLyte Fluor 555, HiLyte Fluor 594, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750), AMCA, AMCA-S, Cascade® Blue (Molecular Probes, Eugene, Oregon, USA), Cascade Yellow, Coumarin, Hydroxycoumarin, Rhodamine Green™-X (Molecular Probes, Eugene, Oregon, USA), Rhodamine Red™-X (Molecular Probes, Eugene, Oregon, USA), Rhodamine 6G, TMR, ABY™ (Applied Biosystems, Carlsbad, California, USA), TAMRA™ (Applied Biosystems, Carlsbad, California, USA), 5-TAMRA, JUN™ (Applied Biosystems, Carlsbad, California, USA), ROX™ (Applied Biosystems, Carlsbad, California, USA), Oregon Green® (Life Technologies, Grand Island, NY, USA), Oregon Green 500, IRDye® 700 (Li-Cor Biosciences, USA)IRDye 800, WellRED D2, WellRED D3, WellRED D4, and Lightcycler® 640 (Roche Diagnostics GmbH, Mannheim, Germany). In some embodiments, bright fluorophores with an extinction coefficient of > 50,000 M-1cm-1and appropriate spectra matching the fluorescence detection channel can be used. -1 cm -1

[0047] ​In certain embodiments, the reaction mixture comprises a fluorescently labeled probe and produces a fluorescently labeled reaction product. In embodiments of the methods and compositions of the application, the fluorophore used as a label to produce a fluorescently labeled probe can be any of a variety of fluorophores, including but not limited to: 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid; acridine and its derivatives, such as acridine and acridine isothiocyanate; 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate, Lucifer Yellow VS; N-(4-aminophenyl)-1-naphthyl)maleimide; anthranilamide, Brilliant Yellow; BIODIPY fluorophores (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene); coumarin and its derivatives, such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanosine; DAPDXYL sulfonyl chloride; 4',6-diamidino-2-phenylindole (DAPI); 5',5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetic acid; 4,4'-dihydrostilbene-2,2'-disulfonic acid disothiocyanate; 4,4'-stilbene-2,2'-disulfonic acid disothiocyanate; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-4'-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); EDANS (5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid), eosin and its derivatives, such as eosin isothiocyanate; erythrosin and its derivatives, such as erythrosin B and erythrosin isothiocyanate; ethidium bromide and other ethidium salts; fluorescein and its derivatives, such as 5-carboxyfluorescein (FAM), hexachlorofluorescein, 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), and fluorescein isothiocyanate (FITC); fluorescamine; green fluorescent protein and its derivatives, such as EBFP, EBFP2, ECFP, and YFP; IAEDANS (5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid), malachite green isothiocyanate; 4-methylumbelliferone; o-cresolphthalein; nitrotyrosine; pararosaniline; phenol red; B-phycoerythrin; o-phenylenediamine malonate; pyrene and its derivatives, such as pyrene butyrate, 1-pyrenesulfonyl chloride, and succinimidyl 1-pyrene butyrate; QSY 7; QSY 9; Reactive Red 4 (Cibacron® Brilliant Red 3B-A);Rhodamines and their derivatives, such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (Rhodamine 6G), rhodamine isothiocyanate, lissamine rhodamine B sulfonyl chloride, rhodamine B, rhodamine 123, sulfo-rhodamine B, sulfo-rhodamine 101, and the sulfonyl chloride derivative of sulfo-rhodamine 101 (Texas Red); N,N,N',N-tetramethyl-carboxyrhodamine (TAMRA); tetramethylrhodamine; tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rose bengal; and terbium chelate derivatives. In certain embodiments, the concentration of the fluorescent probe in the compositions and methods of use is about 0.01 μΜ to about 100 μΜ, about 0.1 μΜ to about 100 μΜ, about 0.1 μΜ to about 50 μΜ, about 0.1 μΜ to about 10 μΜ, or about 0.11 μΜ to about 1 μΜ. In certain embodiments, the concentration of the fluorescent probe is about 0.01 μΜ, about 0.1 μΜ, 0.2 μΜ, about 0.25 μΜ, about 0.3 μΜ, about 0.4 μΜ, or about 0.5 μΜ.

[0048] Exemplary quencher labels include fluorophores, quantum dots, metal nanoparticles, and other related labels. Suitable quenchers include Black Hole Quencher®-1 (Biosearch Technologies, Novato, CA), BHQ-2, Dabcyl, Iowa Black® FQ (Integrated DNA Technologies, Coralville, IA), Iowa Black RQ, QXL™ (Anaspec, Inc., Fremont, CA), QSY 7, QSY 9, QSY 21, QSY 35, IRDye QC, BBQ-650, Atto 540Q, Atto 575Q, Atto 575Q, MGB 3' CDPI3, and MGB-5' CDPI3. In one instance, the term "quencher" refers to a substance that reduces emission from a fluorescent donor when in close proximity to the donor. In preferred embodiments, the quencher is located within 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotide bases of the fluorescent label. Fluorescence is quenched when the fluorescence emitted from a fluorophore is detectably reduced, e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more.

[0049] In certain embodiments, each probe used in a single reaction can have a different fluorophore. In certain embodiments, the quencher for each probe can be the same or different. In specific embodiments, the fluorophore used for each probe can be 6-FAM, HEX, Texas Red-X, ATTO 647N, and Cy5.5, while the quencher is Iowa Black FQ or Iowa Black RQ.

[0050] In certain embodiments, the method comprises calculating the proportion of modified polynucleotide molecules, for example by measuring the total amount of a particular polynucleotide sequence (for DNA targets) or measuring the total amount of a particular polynucleotide sequence (for RNA targets) using qPCR or dPCR on the same sample, and measuring the amount of modified polynucleotide molecules. The proportion of polynucleotide modification is the modified polynucleotide divided by the total amount of polynucleotide.

[0051] The present application also provides kits comprising at least a single affinity binding member and, optionally, oligonucleotide primers and oligonucleotide probes as described above, and packaged in suitable packaging material, optionally in combination with instructions for using the kit components, for example instructions for performing the method of the application. In one embodiment, the kit comprises at least a single affinity binding member in an amount and, optionally, oligonucleotide probes and primers, and instructions for performing the assay are provided in a label or packaging. In a further embodiment, the kit comprises a manufactured product for performing the assay. Preferably, the kit according to the present application comprises at least a single affinity binding member and, optionally, oligonucleotide probes and primers. The kit comprises more than one affinity binding member, for example at least two, at least three, at least four, at least five, at least six, at least 7, at least 8, at least 9 or at least 10 different affinity binding members, in particular when the kit is intended to distinguish between polynucleotide sequences having a modification and polynucleotide sequences not having a modification, for example a methylated adenosine. The kit comprises more than one probe or oligonucleotide primer complementary to a nucleotide sequence upstream or downstream of a target nucleic acid sequence comprising a modified base, for example at least two, at least three, at least four, at least five, at least six, at least 7, at least 8, at least 9 or at least 10 different probes and / or primers, in particular when the kit is intended to distinguish between polynucleotide sequences having a modification and polynucleotide sequences not having a modification, for example a methylated adenosine.

[0052] In the kit according to the present application, the affinity binding members and / or the oligonucleotides (primers, probes, linkers) can be stored separately from each other, or partially mixed, or completely mixed.

[0053] The oligonucleotides or affinity binding elements can be provided in dry form, or dissolved in an appropriate solvent, as judged by one skilled in the art. Appropriate solvents include TE, PCR grade water, and the like.

[0054] In preferred embodiments, the kits according to the application can further comprise additional reagents suitable for the PCR or reverse transcriptase PCR (RT-PCR) step.

[0055] Such reagents are known to those skilled in the art and include water, such as nuclease-free water, RNase-free water, DNase-free water, PCR grade water; salts, such as magnesium, magnesium chloride, potassium; buffers, such as Tris; enzymes, including polymerases, such as Taq, Vent, Pfu (all trademarks), activatable polymerases, reverse transcriptases, and the like; nucleotides, such as deoxynucleotides, dideoxynucleotides, dNTPs, dATP, dTTP, dCTP, dGTP, dUTP; additional reagents, such as DTT and / or RNAse inhibitors; and polynucleotides, such as polyT, polydT, and other oligonucleotides, such as primers.

[0056] In another preferred embodiment, the kits according to the application comprise a PCR control. Such controls are known in the art and include a qualitative control, a positive control, a negative control, an internal control, a quantitative control, an internal quantitative control, and a calibration range. The internal control for the PCR step can be a template that is not related to the target template in the PCR step. Such controls can also comprise control primers and / or control probes. In certain embodiments, a negative control is included that comprises a polynucleotide sequence associated with the target nucleotide sequence, such as an unmodified portion of the target nucleotide sequence (or amplicon). As an example, the unmodified portion of the target nucleotide sequence does not comprise a methylated adenosine. In certain embodiments, the positive control is of the same sequence with the same modification and in the same location and of a known quantity; it can also be used to generate a calibration curve to quantify the modification.

[0057] In preferred embodiments, the kits according to the application comprise means for extracting and / or purifying nucleic acids from a biological sample, such as from blood, serum, plasma, saliva, or nasal secretions. Such means are well known to those skilled in the art.

[0058] In preferred embodiments, the kit according to the application comprises instructions for its use. The instructions can preferably be a brochure, a card or the like. The instructions can also be in two forms: one in a detailed form, gathering exhaustive information about the kit and its use, possibly including bibliographic data; the other in a quick guide form or a memo, for example in card form, gathering the key information needed to use the kit. Thus, the instructions can include instructions for carrying out any of the methods of the application described herein. For example, a composition can be included in a container, package or dispenser with instructions for carrying out a methylated nucleotide detection assay. The instructions can additionally include storage information, expiration date or any information required by regulatory agencies (e.g., the U.S. Food and Drug Administration or the European Medicines Agency) for use on human or animal subjects. The instructions can be "printed material", for example on paper or cardboard within the kit, on a label attached to the kit or to the packaging material, or attached to a vial or tube containing a component of the kit. The instructions can be included on an audio or video tape, and can also be included on a computer readable medium, such as a magnetic disk (floppy disk or hard disk), an optical CD (e.g., CD- or DVD-ROM / RAM), a magnetic tape, a flash memory, an electronic storage medium (e.g., RAM and ROM), and hybrids thereof (e.g., magnetic / optical storage medium).

[0059] The present application also relates to the field of diagnosis, prognosis and drug / therapy efficiency monitoring.

[0060] Detection of polynucleotide modifications

[0061] The methods of the application use an affinity binding element to recognize and attach to a modification of interest on a polynucleotide molecule. In preferred embodiments, the modification is methylated adenosine. In certain embodiments, the polynucleotide molecule can be derived from a single cell.

[0062] In certain embodiments, the method of detecting a polynucleotide modification uses the binding of two separate molecules. In certain embodiments, the first molecule can be an affinity binding element comprising a nucleotide sequence, and the second molecule can be an oligonucleotide that hybridizes to a region of the polynucleotide molecule that is located in the vicinity of the first binding event of the affinity binding element and on the same polynucleotide molecule.

[0063] In certain embodiments, the first molecule and the second molecule can interact to generate a PCR detectable reporter. In certain embodiments, the first molecule and the second molecule can hybridize directly, or the first molecule and the second molecule can interact indirectly, for example, through at least one intermediate molecule, such as an oligonucleotide linker, where the first molecule hybridizes to a first region of the oligonucleotide linker and the second molecule hybridizes to a second region of the oligonucleotide linker, thereby generating a ligation site between the first molecule and the second molecule, which can then be ligated using a ligase. In certain embodiments, the oligonucleotide linker is about 6 to about 50 bases, about 10 to about 50 bases, about 15 to about 25 bases, or preferably about 20 bases in length. In certain embodiments, the first region of the oligonucleotide linker is about half of the oligonucleotide linker, and the second region of the oligonucleotide linker is about half of the oligonucleotide linker. In preferred embodiments, the first region of the oligonucleotide linker is about 10 bases homologous to the 5' end of the first molecule and 10 bases homologous to the 3' end of the second molecule.

[0064] In certain embodiments of the ligation step, a ligase is provided. In preferred embodiments, the ligase is a DNA ligase. DNA ligases are enzymes that are capable of catalyzing the formation of a phosphodiester bond between two polynucleotide strands (ends) when joined at adjacent sites on complementary strands. DNA ligases generally require ATP (EC 6.5.1.1) or NAD (EC 6.5.1.2) as a cofactor to seal nicks in double-stranded DNA. DNA ligases that can be used in the ligation step include T4 DNA ligase, E. coli DNA ligase, Thermus aquaticus (Taq) ligase, Thermus thermophilus DNA ligase, or Pyrococcus furiosus DNA ligase. Other ligases suitable for use in the methods disclosed herein are also known in the art, and such embodiments are within the scope of the present application.

[0065] In certain embodiments, if the target polynucleotide molecule is double-stranded DNA, the DNA molecule can be denatured, for example, by heating the DNA to about 70 °C to about 99 °C, or about 95 °C, or by chemical denaturation. In certain embodiments, sodium hydroxide or a salt can be added to the DNA for chemical denaturation. In certain embodiments, denaturation of the DNA can be performed prior to the affinity binding element recognizing and attaching to the modification of interest on the polynucleotide molecule.

[0066] In certain embodiments, the oligonucleotide linker can comprise a nucleic acid sequence. In certain embodiments, the oligonucleotide linker can diffuse freely in the sample, or hybridize or otherwise bind to the first molecule or the second molecule. In certain embodiments, after the first molecule and the second molecule hybridize directly, primer template extension occurs to form a complementary sequence of either or both of the first molecule and the second molecule. In certain embodiments, the PCR detectable reporter can be a nucleic acid sequence that allows primer binding and optionally allows probe binding. Once the nucleic acid sequence comprising the first molecule, the second molecule, and optionally the oligonucleotide linker is formed, it can be used in enzyme-based nucleic acid amplification to generate a signal for detection and quantification of RNA modification.

[0067] In certain embodiments, the method of detecting a polynucleotide modification uses a single molecule binding. In certain embodiments, the single molecule can be an affinity binding element comprising an oligonucleotide sequence that can hybridize to a region on the target polynucleotide that is proximal to the modified base site and on the same polynucleotide molecule. In certain embodiments, high temperature does not allow efficient hybridization of the oligonucleotide to the target polynucleotide. In certain embodiments, the high temperature that results in low hybridization efficiency is at least about 5 °C to about 20 °C higher than the melting temperature of the oligonucleotide. In certain embodiments, low ionic conditions do not allow efficient hybridization of the oligonucleotide to the target polynucleotide. In certain embodiments, the low ionic conditions can be, for example, KCl concentration of about 20 mM to about 50 mM and / or MgCl2concentration of about 1.5 mM to about 2.5 mM. In certain embodiments, once the affinity binding element is attached to the modified nucleotide base, the kinetics of the oligonucleotide sequence binding is driven by the proximity effect. In certain embodiments, once the oligonucleotide primer hybridizes to the complementary sequence on the polynucleotide, a reverse transcription reaction can be initiated. If the polynucleotide is RNA, the reverse transcription can synthesize a specific cDNA of the region proximal to the modified base site. In certain embodiments, the cDNA can be amplified by enzyme-based nucleic acid amplification using a single DNA primer or a pair of primers. In certain embodiments, the amplification can be sequence specific and allow detection and quantification of the modified base. In certain embodiments, the hybridization of the oligonucleotide to the target polynucleotide occurs by cooperativity of the affinity binding element binding to at least one modified nucleotide base and the transient hybridization of the oligonucleotide primer sequence to the target nucleotide sequence.

[0068] In certain embodiments, the first molecule is double stranded in regions that are not homologous to the oligonucleotide adaptor and the second molecule is double stranded in regions that are not homologous to the target polynucleotide. In certain embodiments, the double stranded portions can inhibit the formation of secondary structures and non-specific interactions of the probes with the polynucleotide target. In certain embodiments, the polynucleotide sample can be mixed with the second molecule and heated together, then cooled and the first molecule added. In certain embodiments, the pre-heating step can inhibit the formation of secondary structures and increase the specific interaction of the probes with the polynucleotide target.

[0069] Any detection method or system capable of detecting nucleotides can be used in the methods according to embodiments of the present application, and such suitable detection methods and systems are well known in the art. In certain embodiments, next generation sequencing, mass spectrometry, microarrays, nanoarrays, microbead / particle binding coupled to flow cytometry, protein arrays, or fluorescent imaging can be used.

[0070] In preferred embodiments, the mixture of the polynucleotide sample or control and the second molecule is incubated at about 60 °C to about 70 °C or about 65 °C for about 3 minutes to about 15 minutes or about 5 minutes, cooled to about 30 °C to about 45 °C or about 37 °C, and then mixed with the first molecule. The binding reaction can be incubated at about 4 °C to about 45 °C or about 37 °C for about 15 minutes to about 12 hours or about 1 hour, and then cooled at about 1 °C to about 10 °C or about 4 °C. In certain embodiments, a mixture comprising a DNA ligase and oligonucleotide adaptor in ligation buffer is added to the target polynucleotide and the first and second molecules and incubated at about 30 °C to about 45 °C or about 37 °C for about 5 minutes to about 60 minutes or about 10 minutes, and then incubated at about 1 °C to about 10 °C or about 4 °C for up to about 10 minutes. In certain embodiments, the template nucleotides resulting from ligation can be quantified.

[0071] In certain embodiments, fluorescence can be detected using a fluorescence microscope, a fluorescence scanner, a spectrofluorometer and a microplate reader, a flow cytometer, a real-time PCR machine, or a digital PCR machine.

[0072] In certain embodiments, detection of at least one single- or double-stranded nucleic acid, such as specific synthesis of cDNA or PCR detection of a reporter proximal to a modified base site region, is performed by an enzyme-based nucleic acid amplification method.

[0073] As used herein, "enzyme-based nucleic acid amplification method" means any method in which enzyme-catalyzed nucleic acid synthesis occurs.

[0074] Such enzyme-based nucleic acid amplification methods can preferably be selected from the group consisting of LCR, Q-beta replication, NASBA, LLA (ligation linear amplification), TMA, 3SR, polymerase chain reaction (PCR), wherein especially all PCR-based methods known in the art are included, such as reverse transcription PCR (RT-PCR), singleplex and multiplex PCR, real-time PCR (qPCR), end-point PCR, digital PCR (dPCRd) performed in droplets, such as the Droplet Digital™ PCR technology provided by Bio-Rad Laboratories, Inc. (Hercules, CA, USA); see Worldwide Website: bio-rad.com / en-us / life-science / learning-center / introduction-to-digital-pcr / what-is-droplet-digital-pcr?ID=MDV31M4VY#Hindson), digital PCR (dPCR), quantitative or qualitative PCR, and combinations thereof. These enzyme-based nucleic acid amplification methods are well known to the person skilled in the art and are especially described in Saiki et al. (1988) Science 239:487, EP 200 362 and EP 201 184 (PCR); Fahy et al. (1991) PCR Meth. Appl. 1 :25-33 (3SR, self-sustained sequence replication); EP 329 822 (NASBA, nucleic acid sequence-based amplification); U.S. Pat. No. 5,399,491 (TMA, transcription-mediated amplification); Walker et al. (1992) Proc. Natl. Acad. Sci. USA 89:392-396 (SDA, strand displacement amplification); EP 0 320 308 (LCR, ligase chain reaction); Bustin and Mueller (2005) Clin. Sci. (London) 109:365-379 (real-time reverse transcription PCR).

[0075] In some embodiments, the enzyme-based nucleic acid amplification method is selected from polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR), dPCR, dPCRd, multiplex PCR or RT-PCR, and real-time PCR or RT-PCR. In other embodiments, the enzyme-based nucleic acid amplification method is a real-time, optionally multiplexed, quantitative PCR or RT-PCR method.

[0076] As used herein, "multiplexing" refers to the detection of at least two different modified nucleotide bases in a single sample or in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different samples by the use of at least two oligonucleotide probes, wherein each of the modified nucleotide bases can be detected by at least one of the probes. Preferably, the signals emitted by different probes bound to their target nucleic acids (e.g., PCR detectable reporters) can be detected separately by labeling each probe with a different fluorescent donor. In preferred embodiments, at least three oligonucleotide probes are used to detect at least three different modified bases. In more preferred embodiments, at least four oligonucleotide probes are used to detect at least four different modified bases. In even more preferred embodiments, at least five oligonucleotide probes are used to detect at least five different nucleotide bases. Typically, the target polynucleotide sequence comprises one or more modified bases, enabling the probes to distinguish between polynucleotide sequences based on the presence or absence of a modified base.

[0077] Exemplary PCR reaction conditions typically include two-step or three-step cycling. Two-step cycling includes a denaturation step followed by a hybridization / elongation step. Three-step cycling includes a denaturation step followed by a hybridization step followed by a separate elongation step. The polymerase reaction is incubated under conditions in which the primers hybridize to the target sequence and are extended by the polymerase. The cycling conditions of the amplification reaction are chosen so that the primers specifically hybridize to the target sequence and are extended.

[0078] Successful PCR amplification requires high yield, high selectivity, and controlled reaction rates at each step. Yield, selectivity, and reaction rates are typically dependent on temperature, and the optimal temperature depends on the composition and length of the polynucleotides, enzymes, and other components in the reaction mixture. Furthermore, the optimal temperature for each step can be different. The optimal reaction conditions can vary depending on the target sequence and the composition of the primers. Thermal cyclers, such as real-time PCR systems, provide the necessary control over the reaction conditions to optimize the PCR process for a particular assay. For example, a real-time PCR system can be programmed by selecting the temperatures that need to be maintained, the length of time for each cycle, the number of cycles, etc. In some embodiments, a temperature gradient can be programmed so that different sample wells are maintained at different temperatures, etc.

[0079] In certain embodiments, the target polynucleotide sequence can be RNA or DNA. The RNA or DNA can be artificially synthesized or isolated from a natural source. In some embodiments, the RNA target nucleic acid sequence can be a ribonucleic acid, such as RNA, mRNA, piRNA, tRNA, rRNA, ncRNA, gRNA, shRNA, siRNA, snRNA, miRNA, and snoRNA. The DNA or RNA template can be present in any useful amount.

[0080] The reverse transcriptase useful in this invention can be any polymerase exhibiting reverse transcriptase activity. Preferred enzymes include those exhibiting reduced RNase H activity. Several reverse transcriptases are known in the art and are commercially available (e.g., from BioRadiation Laboratories, Hercules, CA; Boehringer Ingelheim Corp., Indianapolis, Indiana; Life Sciences, Rockville, Maryland; New England Biolabs, Inc., Beverly, MA; Perkin Elmer Corp., Novak, Connecticut; Pharmacia LKB Biotechnology, Inc., Piscataway, NJ; Qiagen, Inc., Valencia, CA; Stratagene, La Jolla, CA). In some embodiments, the reverse transcriptase may be avian myeloblastosis virus reverse transcriptase (AMV-RT), Moroni murine leukemia virus reverse transcriptase (M-MLV-RT), human immune virus reverse transcriptase (HIV-RT), EIAV-RT, RAV2-RT, or thermophilic bacteria with carbon dioxide (COD). C. hydrogenoformans DNA polymerase, rTth DNA polymerase, SUPERSCRIPT I, SUPERSCRIPT II, ​​and their mutants, variants, and derivatives. It should be understood that various reverse transcriptases, including those not specifically disclosed above, may be used in this invention without departing from the scope of this disclosure or preferred embodiments.

[0081] The DNA polymerase used in this invention can be any polymerase capable of replicating DNA molecules. Preferred DNA polymerases are thermostable polymerases and polymerases with exonuclease activity, which are particularly useful in PCR. Thermostable polymerases are isolated from a variety of thermophilic bacteria, such as *Thermophyton aquatilis*. Thermus aquaticus (Taq), Thermomycosis brevicornu ( Thermus brockianus (Tbr), Feverella fulva ( Thermus flavus (Tfl), Red-crowned Thermophyton ( Thermus ruber (Tru), thermophilic bacteria ( Thermus thermophilus (Tth), thermophilic cocci ( Thermococcus litoralis (Tli) and thermococci ( Thermococcus Other species of *genus*, *Acidophilus thermophilus* ( Thermoplasma acidophilum (Tac), Neoapollosporium ( Thermotoga neapolitana(Tne), Thermophyton floccosum ( Thermotoga maritime (Tma) and Thermomyces genus ( Thermotoga Other species of *genus*, including *Fireballococcus faecalis* (…). Pyrococcus furiosus (Pfu), Warwickia volcanotherae ( Pyrococcus woesei ) (Pwo) and genus Pyrococcus ( Pyrococcus Other species of *Bacillus thermophilus* (genus), including *Bacillus thermophilus* (genus). Bacillus sterothemophilus (Bst), acidophilic thermosulfuric leaf fungus ( Sulfolobus acidocaldarius (Sac), sulfur-bearing sulfide fungi ( Sulfolobus solfataricus (Sso), cryptic heat network bacteria ( Pyrodictium occultum (Poc), Arbidol ( Pyrodictium abyssi ) (Pab) and thermophilic autotrophic methanobacteria ( Methanobacterium thermoautotrophicum ) (Mth), and its mutants, variants or derivatives.

[0082] Many DNA polymerases are known in the art and are commercially available (e.g., from BioLoop Laboratories, Hercules, CA; Boehringer Ingelheim Corp., Indianapolis, Indiana; LifeScience, Inc., Rockville, MD; New England Biolabs, Inc., Beverly, MA; Perkin Elmer Corp., Novak, Connecticut; Pharmacia LKB Biotechnology, Inc., Piscataway, NJ; Qiagen, Inc., Valencia, CA; Stratagene, Inc., La Yola, CA). In some embodiments, the DNA polymerase may be Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, or VENT. TM DEEPVENT TM And its active mutants, variants or derivatives. It should be understood that various reverse DNA polymerases can be used in this invention, including DNA polymerases not specifically disclosed above, without departing from the scope of the invention or its preferred embodiments.

[0083] The reverse transcriptase enzyme can be present in any suitable ratio to the DNA polymerase enzyme. In some embodiments, the ratio of reverse transcriptase to DNA polymerase in a unit of activity is greater than or equal to 3. Other ratios of reverse transcriptase to DNA polymerase can be used in the present application, as will be appreciated by one skilled in the art.

[0084] In preferred embodiments, the reactions according to the present application can also comprise other reagents suitable for PCR steps.

[0085] Such reagents are known to those skilled in the art and include water, such as nuclease-free water, RNase-free water, DNase-free water, PCR grade water; salts, such as magnesium, magnesium chloride, potassium; buffers, for example Tris; enzymes; nucleotides such as deoxynucleotides, dideoxynucleotides, dNTPs, dATP, dTTP, dCTP, dGTP, dUTP and modified nucleotides such as deaza-, locked nucleic acids and peptide nucleic acids; other reagents, such as DTT and / or RNAse inhibitors; and polynucleotides such as poly-T and poly-dT.

[0086] Certain methods and compositions for amplifying and / or detecting nucleic acids are described in U.S. Patent No. 9,493,824, U.S. Patent No. 10,988,762, U.S. Patent No. 10,053,676, U.S. Patent No. 6,627,424, U.S. Patent No. 7,541,170, U.S. Patent No. 7,666,645, U.S. Patent No. 7,560,260, U.S. Patent No. 8,367,376, U.S. Patent No. 9,145,550, U.S. Patent No. 9,688,969, U.S. Patent No. 10,577,593, U.S. Patent No. 10,301,675, U.S. Patent No. 8,338,094, and U.S. Patent No. 9,200,318, each of which is incorporated herein by reference in its entirety. U.S. Patent No. 9,493,824 describes nucleic acid amplification / detection reaction mixtures and uses thereof; U.S. Patent No. 10,988,762 describes reverse transcriptases and uses thereof; U.S. Patent No. 10,053,676 describes polymerase storage compositions and uses thereof; U.S. Patent No. 6,627,424 describes DNA polymerases and uses thereof; U.S. Patent No. 7,541,170 describes polymerases and uses thereof; U.S. Patent No. 7,666,645 describes polymerases and uses thereof; U.S. Patent No. 7,560,260 describes polymerases, particularly Pfu polymerase, and uses thereof; U.S. Patent No. 8,367,376 describes polymerases, particularly Pfu polymerase, and uses thereof; U.S. Patent No. 9,145,550 describes polymerases, particularly Pfu polymerase, and uses thereof; U.S. Patent No. 9,688,969 describes polymerases, particularly Pfu polymerase, and uses thereof; U.S. Patent No. 10,577,593 describes polymerases, particularly Pfu polymerase, and uses thereof; U.S. Patent No. 10,301,675 describes compositions and methods for synthesizing cDNA from an RNA template and replicating the cDNA and kits thereof; U.S. Patent No. 8,338,094 describes methods for synthesizing cDNA from an RNA template and replicating the cDNA and kits thereof; and U.S. Patent No. 9,200,318 describes methods for synthesizing cDNA from an RNA template and replicating the cDNA and kits thereof.

[0087] Targets of polynucleotide detection

[0088] In certain embodiments, the methods provided herein can be used to detect one or more modifications of a polynucleotide (also referred to herein as a“target sequence,”“target nucleic acid sequence,”“target nucleotide sequence,” or“target polynucleotide sequence,” and used interchangeably) in a sample. In certain embodiments, the modification can be a methylated nucleobase, such as an adenosine. In certain embodiments, one modified base can be detected in a polynucleotide sequence, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different modified bases can be detected in a polynucleotide sequence. In preferred embodiments, the one, two, or more modifications described above are located in a target sequence that is about 60 to about 5000 nucleotides in length.

[0089] In certain embodiments, the methods of the application can be used to detect polynucleotide modifications that are found to be associated with a variety of diseases or other maladies in a subject, such as cancer, mental disorders, and metabolic diseases, as described in the Modomics database (World Wide Web: [https: / / genesilico.pl / modomics / diseases) or m 6 A database of modifications (see World Wide Web: m6add.edbc.org, both of which are incorporated by reference herein in their entirety) In certain embodiments, the cancer is caused by abnormal or blocked differentiation of cancer stem cells, such as leukemia (e.g., acute myeloid leukemia), Hodgkin’s lymphoma, colorectal cancer, gastric cancer, lung cancer, renal cell carcinoma, melanoma, ovarian cancer, breast cancer, glioblastoma, bladder cancer, osteosarcoma, hepatocellular carcinoma, pancreatic cancer, prostate cancer, or cervical squamous cell carcinoma. In certain embodiments, the mental disorder is, for example, major depressive disorder, attention deficit / hyperactivity disorder, Alzheimer’s disease, or Parkinson’s disease. In certain embodiments, the metabolic disease is, for example, obesity, cardiovascular disease, hypertension, or diabetes. In certain embodiments, modifications of polynucleotides are found to be associated with osteoporosis, nerve injury or malformation, and suppression of the innate immune system, particularly in terms of antiviral activity. In certain embodiments, the methods of the application can be used to detect polynucleotide modifications that are found to be associated with development and regulation in plants, particularly under different environmental conditions, including, for example, disease and insect interactions, and for crop selection and improvement.

[0090] Additional disclosure and claimable subject matter

[0091] 1. A method for detecting at least one modified nucleotide base in a target polynucleotide sequence of a sample, the method comprising: a) optionally, isolating the target nucleotide sequence from the sample; b) binding an affinity binding element to the at least one modified nucleotide base, wherein the affinity binding element comprises an oligonucleotide primer sequence, and a first portion of the oligonucleotide primer sequence hybridizes to the target polynucleotide sequence at a position proximal to the at least one modified nucleotide base; and c) detecting the at least one modified nucleotide base.

[0092] 2. The method of embodiment 1, wherein the target nucleotide sequence is a target RNA sequence.

[0093] 3. The method of embodiment 1, wherein the target nucleotide sequence is a target DNA sequence.

[0094] 4. The method of embodiment 2, further comprising after step b): initiating reverse transcription using the oligonucleotide primer sequence to reverse transcribe the target polynucleotide sequence to generate a cDNA molecule; and optionally, synthesizing a DNA molecule complementary to the cDNA molecule.

[0095] 5. The method of embodiment 1, wherein the affinity binding element further comprises an aptamer, a peptide, or a protein.

[0096] 6. The method of embodiment 5, wherein the protein is an antibody.

[0097] 7. The method of embodiment 6, wherein the antibody is an anti-N6-methyladenosine (m6A), an anti-inosine (I), an anti-pseudouridine (Ψ), an anti-1-methyladenosine (mlA), an anti-2-methyladenosine (m2A), an anti-N6,N6-dimethyladenosine (m6,6A), an anti-8-methyladenosine (m8A), an anti-5-methylcytosine, an anti-5-hydroxymethylcytosine, an anti-5-formylcytosine, or an anti-5-carboxylcytosine.

[0098] 8. The method of embodiment 5, wherein the protein is a YT521-B homology (YTH) domain family protein.

[0099] 9. The method of embodiment 8, wherein the YTH domain family protein is YTHDF1, YTHDF2, or YTHDF3.

[0100] 10. The method of embodiment 1, wherein the oligonucleotide primer sequence is about 6 to about 100 nucleotides in length.

[0101] 11. The method of embodiment 1, wherein the hybridization reaction conditions comprise a high annealing temperature or low ionic conditions.

[0102] 12. The method of embodiment 11, wherein the hybridization occurs through the concerted action of the affinity binding element binding to the at least one modified nucleotide base and the transient hybridization of the oligonucleotide primer sequence to the target polynucleotide sequence.

[0103] 13. The method of embodiment 1, wherein the oligonucleotide primer sequence is random.

[0104] 14. The method of embodiment 1, wherein the oligonucleotide primer sequence hybridizes to the target polynucleotide sequence at a position that is about 0 to about 200 nucleotides upstream or downstream from the modified nucleotide base.

[0105] 15. The method of embodiment 1, wherein the target polynucleotide sequence comprises two or more modified bases.

[0106] 16. The method of embodiment 1, wherein the affinity binding element is specific for at least two, three, four, five, or more different modified nucleotide bases.

[0107] 17. The method of embodiment 1, further comprising detecting the modified nucleotide base in two or more different biological samples, or further comprising detecting at least two modified nucleotide bases in a single biological sample.

[0108] 18. The method of embodiment 1, wherein the target polynucleotide sequence is viral, bacterial, archaeal, eukaryotic, cell-derived, tissue-derived, present on the surface of a cell, derived from the cytoplasm of a fixed cell, derived from the nucleus of a fixed cell, derived from a single cell, derived from a bodily fluid, or any combination thereof.

[0109] 19. The method of embodiment 1, wherein the modified nucleotide base is methylated adenosine, methylated adenine, methylated cytosine, acetylated cytidine, pseudouridine, inosine, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), or 5-carboxylcytosine (5-caC).

[0110] 20. The method of embodiment 4, further comprising amplifying the cDNA molecule using at least one DNA polymerase, at least one dNTP, and at least one buffer having a pH suitable for the polymerase activity of the at least one DNA polymerase.

[0111] 21. The method of embodiment 4, comprising amplifying part or all of the cDNA molecule using a single primer or a pair of oligonucleotide primers.

[0112] 22. The method of embodiment 1, wherein the detecting comprises real-time PCR (qPCR), digital PCR (dPCR), digital PCR performed in droplets (dPCRd), next generation sequencing, mass spectrometry, protein arrays, flow cytometry, fluorescence imaging, or any combination thereof.

[0113] 23. The method of embodiment 1, wherein the second portion of the oligonucleotide primer sequence is double-stranded and does not hybridize to the target polynucleotide.

[0114] 24. A method for detecting at least one modified nucleotide base in a target polynucleotide sequence of a sample, the method comprising: a) optionally, isolating the target polynucleotide sequence from the sample; b) binding a first molecule comprising an affinity binding element and an oligonucleotide sequence to the at least one modified nucleotide base, and binding a second molecule comprising an oligonucleotide homologous to the target polynucleotide sequence at a position proximal to the at least one modified nucleotide; c) directly linking the first molecule to the second molecule, or linking the first molecule to the second molecule using an oligonucleotide linker; or hybridizing the first molecule to the second molecule and extending the first molecule and the second molecule, thereby generating a complement of a region of the second molecule; and d) generating a PCR-detectable reporter molecule using the linked first molecule and second molecule or the complement of the region of the second molecule; e) detecting the PCR-detectable reporter molecule.

[0115] 25. The method of embodiment 24, wherein the affinity binding element is an aptamer, a peptide, or a protein.

[0116] 26. The method of embodiment 25, wherein the protein is an antibody.

[0117] 27. The method of embodiment 26, wherein the antibody is an anti-N6-methyladenosine (m6A), an anti-inosine (I), an anti-pseudouridine (Ψ), an anti-1-methyladenosine (mlA), an anti-2-methyladenosine (m2A), an anti-N6,N6-dimethyladenosine (m6,6A), an anti-8-methyladenosine (m8A), an anti-5-methylcytosine, an anti-5-hydroxymethylcytosine, an anti-5-formylcytosine, or an anti-5-carboxylcytosine.

[0118] 28. The method of embodiment 25, wherein the protein is a YT521-B homology (YTH) domain family protein.

[0119] 29. The method of embodiment 28, wherein the YTH domain family protein is YTHDF1, YTHDF2, or YTHDF3.

[0120] 30. The method of embodiment 24, wherein the oligonucleotide homologous to the target polynucleotide sequence is about 10 to about 200 nucleotides in length.

[0121] 31. The method of embodiment 24, wherein the oligonucleotide sequence of the affinity binding element is about 10 to about 200 nucleotides in length.

[0122] 32. The method of embodiment 24, wherein the oligonucleotide linker is about 6 to about 50 nucleotides in length.

[0123] 33. The method of embodiment 24, wherein the oligonucleotide homologous to the target polynucleotide hybridizes to the target polynucleotide sequence at a position that is about 0 to about 200 nucleotides upstream or downstream from the modified nucleotide base.

[0124] 34. The method of embodiment 24, wherein the target polynucleotide sequence comprises two or more modified bases.

[0125] 35. The method of embodiment 24, further comprising detecting the modified nucleotide base in two or more different biological samples, or further comprising detecting at least two modified nucleotide bases in a single sample.

[0126] 36. The method of embodiment 24, wherein the target polynucleotide sequence is viral, bacterial, archaeal, eukaryotic, cell-derived, tissue-derived, cell surface-presented, cytoplasmic from a fixed cell, nuclear from a fixed cell, single cell-derived, body fluid-derived, or any combination thereof.

[0127] 37. The method of embodiment 24, wherein the modified nucleotide base is methylated adenosine, methylated adenine, methylated cytosine, acetylated cytidine, pseudouridine, inosine, 5-hmC, 5-fC, or 5-caC.

[0128] 38. The method of embodiment 24, further comprising amplifying the PCR detectable reporter molecule using at least one DNA polymerase, at least one dNTP, and at least one buffer having a pH suitable for polymerase activity of the at least one DNA polymerase.

[0129] 39. The method of embodiment 24, further comprising detecting the PCR detectable reporter molecule using at least one fluorescent oligonucleotide probe.

[0130] 40. The method of embodiment 39, wherein each fluorescent probe has a different fluorescent signal.

[0131] 41. The method of embodiment 39, comprising using a single primer or a pair of oligonucleotide primers to amplify part or all of the PCR detectable reporter molecule.

[0132] 42. The method of embodiment 24, wherein the detecting comprises qPCR, dPCR, or dPCRd.

[0133] 43. The method of embodiment 24, wherein part of the first molecule is double stranded and does not hybridize to the second molecule, part of the first molecule is double stranded and does not hybridize to the oligonucleotide linker, part of the second molecule is double stranded and does not hybridize to the oligonucleotide linker, and / or part of the second molecule is double stranded and does not hybridize to the target polynucleotide.

[0134] 44. The method of embodiment 24, further comprising mixing the target polynucleotide sequence from the sample with the second molecule at a temperature of about 60 °C to about 70 °C for about 3 minutes to about 15 minutes.

[0135] 45. The method of embodiment 44, further comprising cooling the mixed second molecule with the target polynucleotide sequence from the sample to about 30 °C to about 45 °C.

[0136] 46. The method of embodiment 45, further comprising cooling the mixed second molecule with the target polynucleotide sequence from the sample to about 37 °C and adding the first molecule to the mixed second molecule with the target polynucleotide sequence from the sample.

[0137] 47. The method of embodiment 24, wherein step b) is performed at a temperature of about 4 °C to about 45 °C for about 15 minutes to about 12 hours, followed by cooling at a temperature of about 1 °C to about 10 °C.

[0138] 48. The method of embodiment 47, wherein step b) is performed at a temperature of about 37 °C for about 1 hour, followed by cooling at a temperature of about 4 °C.

[0139] 49. The method of embodiment 24, further comprising mixing a DNA ligase with the oligonucleotide linker and with the target polynucleotide sequence from the sample and the first and second molecules and incubating at a temperature of about 30 °C to about 45 °C for about 5 minutes to about 60 minutes, followed by incubating the target polynucleotide sequence from the sample, the first and second molecules, the DNA ligase, and the oligonucleotide linker at a temperature of about 1 °C to about 10 °C for up to 10 minutes prior to the ligation step of step c).

[0140] 50. The method of embodiment 49, further comprising mixing a DNA ligase with an oligonucleotide linker and with the target polynucleotide sequence from the sample and the first and second molecules and incubating the DNA ligase with the oligonucleotide linker at a temperature of about 37 °C for about 10 minutes, followed by incubating the DNA ligase with the oligonucleotide linker at a temperature of about 4 °C for up to 10 minutes.

[0141] All patents, patent applications, provisional applications, and publications cited or described in this document are hereby incorporated by reference in their entirety for all purposes, to the extent that they are not inconsistent with the explicit teachings of this specification.

[0142] Example 1 - Quantification of RNA containing M6A modifications

[0143] Two different RNA oligos were used as samples, each containing a single N 6 - methyladenosine base (e.g. m6A RNA). RNA template sequence 1 is 90 bases long and RNA template sequence 2 is 80 bases long. The same RNA oligos without m6A modification were used as negative controls (N.C. RNA).

[0144] The samples and negative control RNA oligos were diluted separately to form a concentration series ranging from 0.03 nM to 100 nM RNA. To measure background levels, a no template control sample (NTC) was used without RNA.

[0145] Detection probes were prepared as follows. For each RNA template, a different probe A oligo was designed based on the RNA sequence. Each probe A consists of a DNA oligo 68 bases long, with the last 20 bases at the 3’ end homologous to a stretch of sequence on the RNA template 15 bases away from the m6A modification. Figure 2 The 5’ end of the probe A oligo was phosphorylated. Probe B consists of an anti-m6A antibody coupled to the 5’ end of a DNA oligo 45 bases long. The third DNA oligo is a 20 base linker homologous to the 10 bases at the 5’ end of probe A and the 10 bases at the 3’ end of the probe B oligo.

[0146] Five microliters of RNA sample or control was mixed with 2.5 μL Probe A, incubated at 65 °C for 5 minutes, cooled to 37 °C, and then mixed with 2.5 μL Probe B. The final binding reaction consisted of 100 pM Probe A, 100 pM Probe B, and 1 nM ligation adaptor in ligation buffer. The binding reaction was incubated at 37 °C for an additional hour, followed by a treatment at 4 °C. Next, 90 μL of ligation mix consisting of T4 DNA ligase and oligonucleotide adaptor was added to each binding reaction and incubated at 37 °C for 10 minutes, followed by an incubation at 4 °C for up to 10 minutes. Finally, the resulting ligation templates were quantified using a qPCR system and the resulting Cq values were fit using a 5-PL regression model.

[0147] It is to be understood that the embodiments and implementations described herein are merely for illustrative purposes and that various modifications or changes in light thereof will be obvious to those skilled in the art and they are intended to be included within the spirit and purview of the application and scope of the appended claims. Moreover, any element or limitations of any application or implementation disclosed herein can be combined with any and / or all other elements or limitations (alone or in any combination), or any other application or implementation, and all such combinations are contemplated to be within the scope of the application and intended to be an aspect of the application, but not limited thereto.

Claims

1. A method for detecting at least one modified nucleotide base in a target polynucleotide sequence of a sample, the method comprising: a) Optionally, isolate the target polynucleotide sequence from the sample; b) Bind a first molecule containing an affinity-binding element and an oligonucleotide sequence to the at least one modified nucleotide base, and bind a second molecule containing an oligonucleotide homologous to the target polynucleotide sequence to a position close to the at least one modified nucleotide. c) Directly link the first molecule to the second molecule, or use an oligonucleotide linker to link the first molecule to the second molecule; or The first molecule is hybridized with the second molecule, and the first and second molecules are extended to generate a complementary sequence in a certain region of the second molecule. as well as d) Generate PCR-detectable reporter molecules using complementary sequences of regions of the first and second molecules or the second molecule; e) Detect the PCR-detectable reporter molecules.

2. The method of claim 1, wherein the affinity-binding element is an aptamer, peptide, or protein.

3. The method as described in claim 2, wherein, The protein in question is an antibody.

4. The method of claim 3, wherein the antibody is anti-N6-methyladenosine (m6A), anti-inosine (I), anti-pseudouridine (Ψ), anti-1-methyladenosine (m1A), anti-2-methyladenosine (m2A), anti-N6,N6-dimethyladenosine (m6,6A), anti-8-methyladenosine (m8A), anti-5-methylcytosine, anti-5-hydroxymethylcytosine, anti-5-formylcytosine, or anti-5-carboxycytosine.

5. The method of claim 2, wherein the protein is a YT521-B homology (YTH) domain family protein, for example, the YTH domain family protein is YTHDF1, YTHDF2 or YTHDF3.

6. The method of claim 1, wherein the length of the oligonucleotide homologous to the target polynucleotide sequence is about 10 to about 200 nucleotides; the length of the oligonucleotide sequence of the affinity binding element is about 10 to about 200 nucleotides; and / or the length of the oligonucleotide linker is about 6 to about 50 nucleotides.

7. The method of claim 1, wherein the oligonucleotide homologous to the target polynucleotide hybridizes with the target polynucleotide sequence at a position of about 0 to about 200 nucleotides upstream or downstream of the modified nucleotide base.

8. The method of claim 1, wherein the target polynucleotide sequence comprises two or more modified bases.

9. The method of claim 1, further comprising detecting the modified nucleotide bases in two or more different biological samples, or further comprising detecting at least two modified nucleotide bases in a single biological sample.

10. The method of claim 1, wherein the target polynucleotide sequence is viral, bacterial, archaea, eukaryotic, cellular, tissue-derived, present on the cell surface, derived from the cytoplasm of a fixed cell, derived from the nucleus of a fixed cell, derived from a single cell, derived from body fluids, or any combination thereof.

11. The method of claim 1, wherein the modified nucleotide base is methylated adenosine, methylated adenine, methylated cytosine, acetylated cytosine, pseudouridine, inosine, 5-hmC, 5-fC, or 5-caC.

12. The method of claim 1, further comprising using at least one DNA polymerase, at least one dNTP, and at least one buffer having a pH suitable for the polymerase activity of said at least one DNA polymerase to amplify the PCR-detectable reporter molecule.

13. The method of claim 1, further comprising using at least one fluorescent oligonucleotide probe to detect the PCR-detectable reporter molecule.

14. The method of claim 13, wherein each fluorescent probe has a different fluorescence signal.

15. The method of claim 13, wherein the method comprises using a single primer or a pair of oligonucleotide primers to amplify part or all of the PCR-detectable reporter molecule.

16. The method of claim 1, wherein the detection comprises qPCR, dPCR, or dPCRd.

17. The method of claim 1, wherein a portion of the first molecule is double-stranded and does not hybridize with the second molecule, a portion of the first molecule is double-stranded and does not hybridize with the oligonucleotide linker, a portion of the second molecule is double-stranded and does not hybridize with the oligonucleotide linker, and / or a portion of the second molecule is double-stranded and does not hybridize with the target polynucleotide.

18. The method of claim 1, further comprising mixing the target polynucleotide sequence from the sample with the second molecule at a temperature of about 60°C to about 70°C for about 3 minutes to about 15 minutes.

19. The method of claim 18, further comprising cooling the mixed second molecule with the target polynucleotide sequence from the sample to about 30°C to about 45°C.

20. The method of claim 19, further comprising cooling the mixed second molecule and the target polynucleotide sequence from the sample to approximately 37°C, and adding the first molecule to the mixed second molecule and the target polynucleotide sequence from the sample.

21. The method of claim 1, wherein step b) is performed at a temperature of about 4°C to about 45°C for about 15 minutes to about 12 hours, followed by cooling at a temperature of about 1°C to about 10°C.

22. The method of claim 21, wherein step b) is performed at a temperature of about 37°C for about 1 hour, followed by cooling at a temperature of about 4°C.

23. The method of claim 1, further comprising mixing the DNA ligase with the oligonucleotide linker and mixing it with the target polynucleotide sequence from the sample and the first and second molecules, and incubating at a temperature of about 30°C to about 45°C for about 5 minutes to about 60 minutes, and then, prior to the ligation step in step c), incubating the target polynucleotide sequence from the sample, the first and second molecules, the DNA ligase, and the oligonucleotide linker at a temperature of about 1°C to about 10°C for up to 10 minutes.

24. The method of claim 23, further comprising mixing the DNA ligase with the oligonucleotide linker and mixing it with the target polynucleotide sequence from the sample and the first and second molecules, and incubating at a temperature of about 37°C for about 10 minutes, followed by incubating the DNA ligase with the oligonucleotide linker at a temperature of about 4°C for up to 10 minutes.

25. A method for detecting at least one modified nucleotide base in a target polynucleotide sequence of a sample, the method comprising: a) Optionally, isolate the target nucleotide sequence from the sample; b) Binding an affinity-binding element to at least one modified nucleotide base, wherein the affinity-binding element comprises an oligonucleotide primer sequence, and a first portion of the oligonucleotide primer sequence hybridizes with a target polynucleotide sequence at a position close to the at least one modified nucleotide base; c) Detect at least one modified nucleotide base.

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