Detection of a modification to a polynucleotide through proximity assays

Abstract

The subject invention pertains to the detection of modifications to a polynucleotide by binding an affinity binding element to the nucleotide sequence. The affinity binding element can comprise a compound that binds to the modified nucleotide base and a nucleotide sequence that is complementary to the target nucleotide sequence, an oligonucleotide primer that is complementary to the target nucleotide sequence, or a nucleotide sequence complementary to a connector oligonucleotide. The invention provides methods of detecting one or more modifications to a nucleotide sequence. The invention further provides affinity binding elements and, optionally, primer oligonucleotides and / or probe oligonucleotides, and methods of using said affinity binding elements in assays to a detect modified base. The methods of the invention detect a modified nucleotide that are implicated in cancer, psychiatric disorders, or metabolic diseases.

Description

[0001] DETECTION OF A MODIFICATION TO A POLYNUCLEOTIDE THROUGH

[0002] PROXIMITY ASSAYS

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 529,794, filed July 31, 2023, the disclosure of which is hereby incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences.

[0005] BACKGROUND OF THE INVENTION

[0006] The presence of modifying marks on polynucleotides has been implicated in a variety of cellular processes and in human disease, including, for example, cancer, psychiatric disorders, and metabolic disease. Therefore, the detection of nucleic acid modifications is currently of interest in the medical field, in particular in the diagnosing and treating of diseases associated with modifications to polynucleotides.

[0007] Traditional methods of identifying the modifications to polynucleotides involve sodium bisulfite conversion and sequencing or affinity capture of modified polynucleotides and sequencing; however, sequencing requires specialized instrumentation and is time consuming. In addition, existing methods are used to reveal the sites of modifications but cannot be easily used to quantitatively measure the fraction of a polynucleotide that is modified.

[0008] Therefore, there remains a need for methods to efficiently detect modifications to nucleotides with enough specificity to consistently distinguish modified nucleotide bases from unmodified nucleotide bases without the use of sequencing.

[0009] BRIEF SUMMARY OF THE INVENTION

[0010] The invention pertains to the detection of modifications to a polynucleotide, for example modifications to DNA or RNA, including, for example, the methylation of adenosine. The invention provides at least one oligonucleotide and at least one affinity binding element, such as, for example an antibody or aptamer. In certain embodiments, the oligonucleotide is linked to the affinity binding element and serves as an oligonucleotide primer. In certain embodiments, if the modification is an RNA modification, then cDNA is generated using a reverse transcription reaction. The invention further pertains to methods of detecting modifications of a polynucleotide by binding the affinity binding element to the modified polynucleotide base. In certain embodiments, the detection of modifications to a polynucleotide include, for example, using proximity ligation assay (PLA) based immunoaffinity, proximity extension assay (PEA) based immunoaffinity.

[0011] In certain embodiments, the binding of the affinity binding element to the modified polynucleotide and the hybridization of the oligonucleotide to a polynucleotide sequence in proximity to the site of the modified polynucleotide can be used to detect the modified polynucleotide using, for example, nucleic acid amplification, more particularly, by PCR, advantageously by real-time PCR (qPCR), digital PCR (dPCR), or digital PCR performed in droplets (dPCRd). In certain embodiments, multiplex PCR can be advantageously used to detect at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different modifications to a polynucleotide with the use of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different PCR assays. In certain embodiments, multiplex PCR can be advantageously used to detect at least two of the same modifications to a polynucleotide in a single sample or in 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different samples. In certain embodiments, by including a sample barcode into the nucleic acid tail.

[0012] The invention also relates to the oligonucleotide and affinity binding element, as well as to compositions thereof, to biological compositions, to detection kits, and to diagnostic kits.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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.

[0015] FIGs. 1A-1H show the steps of an exemplary immunoaffinity RNA modification assay using the subject affinity binding element and oligonucleotide primer.

[0016] FIG. 2 shows the steps of an exemplary proximity ligation assay (PLA) based immunoaffinity RNA modification assay using the subject affinity binding element and oligonucleotide, using the modification-specific antibody to bind a modified RNA base. The figure further provides an example of a lack of binding by the modification-specific antibody when the target RNA does not contain a modified RNA base.

[0017] FIG. 3 shows the proximity ligation assay (PLA) based immunoaffinity RNA modification assay using the subject affinity binding element and oligonucleotide, using the modification-specific antibody to bind a modified RNA base. The figure further provides an example of quantitative detection of the RNA modification fraction of a specific RNA sequence. FIGs. 4A-4B show the detection of a single N6-methyladenosine base in two different RNA oligonucleotide target sequences.

[0018] DETAILED DISCLOSURE OF THE INVENTION

[0019] 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. To the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”. The transitional terms / phrases (and any grammatical variations thereof) “comprising”, “comprises”, “comprise”, “consisting essentially of’, “consists essentially of’, “consisting” and “consists” can be used interchangeably.

[0020] The phrase “consisting essentially of’ or “consists essentially of’ indicates that the described embodiment encompasses embodiments containing the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the described embodiment.

[0021] The term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. In the context of the lengths of polynucleotides where the terms “about” are used, these polynucleotides contain the stated number of bases or base-pairs with a variation of 0-10% around the value (X ± 10%). In the context of compositions containing amounts of ingredients where the terms “about” or “approximately” are used, these compositions contain the stated amount of the ingredient with a variation (error range) of 0-10% around the stated value (X±10%). In the context of pH, the term “about” or “approximately” is intended to include a value of ±0.2 unit of the stated pH.

[0022] In the present disclosure, ranges are stated in shorthand, so as to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. Values having at least two significant digits within a range are envisioned, for example, a range of 5-10 indicates all the values between 5.0 and 10.0 as well as between 5.00 and 10.00 including the terminal values. The term “antibody” refers to an immunoglobulin or fragmentary form thereof. The term includes, but is not limited to, polyclonal or monoclonal antibodies of the classes IgA, IgD, IgE, IgG, and IgM, derived from human or other mammalian cell lines, including natural or genetically modified forms such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. “Antibody” encompasses composite forms including, but not limited to, fusion proteins containing an immunoglobulin moiety. “Antibody” also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, Fc, single variable domain on a heavy chain (VHH), and other compositions, whether or not they retain antigen-binding function.

[0023] As used herein, the term “positive,” when referring to a result or signal, indicates the presence of an analyte or item that is being detected in a sample. The term “negative,” when referring to a result or signal, indicates the absence of an analyte or item that is being detected in a sample. Positive and negative are typically determined by comparison to at least one control, e.g., a threshold level that is required for a sample to be determined positive, or a negative control (e.g., a known blank). A “control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a test condition, e.g., in the presence of a test compound, and compared to samples from known conditions, e.g., in the absence of the test compound (negative control), or in the presence of a known compound (positive control). A control can also represent an average value gathered from a number of tests or results. One of skill in the art will recognize that controls can be designed for assessment of any number of parameters, and will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are variable in controls, variation in test samples will not be considered as significant.

[0024] As used herein, a “calibration control” is similar to a positive control, in that it includes a known amount of a known analyte. In the case of a real-time PCR assay, the calibration control can be designed to include known amounts of multiple known analytes. The amount of analyte(s) in the calibration control can be set at a minimum cut-off amount, e.g., so that a higher amount will be considered “positive” for the analyte(s), while a lower amount will be considered “negative” for the analyte(s). In some cases, multilevel calibration controls can be used, so that a range of analyte amounts can be more accurately determined. For example, an assay can include calibration controls at known low and high amounts, or known minimal, intermediate, and maximal amounts.

[0025] As used herein, “subject,” “patient,” “individual” and grammatical equivalents thereof are used interchangeably and refer to, except where indicated, mammals, such as humans and non-human primates, as well as rabbits, felines, canines, rats, mice, squirrels, goats, pigs, deer, and other mammalian species. The term “patient” does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical or veterinary supervision. A “patient” can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc.

[0026] 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, a eukaryotic organism, including, for example, fungi, plants, and animals; bacteria; or archaea. The term encompasses bodily fluids from eukaryotic organisms, such as, for example, blood, blood components, saliva, nasal mucous, serum, plasma, cerebrospinal fluid (CSF), urine and other liquid samples of biological origin, solid tissue biopsy, tissue cultures, lysates or supernatant taken from cultured patient cells. In the context of the present disclosure, the biological sample is typically a bodily fluid with detectable amounts of a nucleotide sequence, such as, for example a sequence from a virus or, preferably, from a subject’s genome, e.g., a tissue sample, blood or a blood component (e.g., plasma or serum), saliva, oropharyngeal, nasopharyngeal, or a nasal secretion (mucous). The biological sample can be processed prior to assay, e.g., to remove cells or cellular debris. The term encompasses samples that have been manipulated after their procurement, such as by treatment with reagents, solubilization, sedimentation, or enrichment for certain components.

[0027] As used herein, the terms “nucleic acid”, “oligonucleotide” or “polynucleotide” refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as 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 may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base 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.

[0028] As used herein, the term “isolated nucleic acid” molecule refers to a nucleic acid molecule that is separated from other nucleic acid molecules that are usually associated with the isolated nucleic acid molecule. Thus, an “isolated nucleic acid molecule” includes, without limitation, a nucleic acid molecule that is free of nucleotide sequences that naturally flank one or both ends of the nucleic acid in the genome of the organism from which the isolated nucleic acid is derived (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease digestion). In addition, an isolated nucleic acid molecule can include an engineered nucleic acid molecule such as a recombinant or a synthetic nucleic acid molecule. A nucleic acid molecule existing among hundreds to millions of other nucleic acid molecules within, for example, a nucleic acid library (e.g., a cDNA or genomic library) or a gel (e.g., agarose, or polyacrylamide) containing restriction-digested genomic DNA, is not an “isolated nucleic acid”.

[0029] As used herein, the term “gene” means the segment of DNA 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 and the regulation of the transcription / translation, as well as intervening sequences (introns) between individual coding segments (exons).

[0030] As used herein, the terms “identical” or percent “identity”, in the context of describing two or more polynucleotide or amino acid sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (for example, a nucleotide probe used in the method of this invention has at least 70% sequence identity, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, to a target sequence or complementary sequence thereof), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical”. With regard to polynucleotide sequences, this definition also refers to the complement of a test sequence. The term “hybridizes with” when used with respect to two sequences indicates that the two sequences are sufficiently complementary to each other to allow nucleotide base pairing between the two sequences. Sequences that hybridize with teach other can be perfectly complementary but can also have mismatches to a certain extent. Depending upon the stringency of hybridization, a mismatch of up to about 5% to 20% between the two complementary sequences would allow for hybridization between the two sequences. Typically, high stringency conditions have higher temperature and lower salt concentration and low stringency conditions have lower temperature and higher salt concentration. High stringency conditions for hybridization are preferred.

[0031] Affinity Binding Element and Primer Design and Detection

[0032] In certain embodiments, one or more affinity binding elements that can bind to a modified nucleotide base are provided by the subject invention. In certain embodiments, the modified nucleotide base is a modified DNA base or a modified RNA base. In certain embodiments, the modified nucleotide is a methylated adenosine, methylated adenine, methylated cytosine, acetylated cytidine, a pseudouridine, an inosine, an oxidized product of a 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, for example, RMbase (see Worldwide Website: rna.sysu.edu.cn / rmbase / ), Modomics (see Worldwide Website: genesilico.pl / modomics / ), EWAS Data Hub (see Worldwide Website: hngdc.cncb.ac.cn / ewas / datahub / index) and the National Genomics Data Center (see Worldwide Website: ngdc.cncb.ac.cn).

[0033] In certain embodiments, the affinity binding element can comprise a protein, peptide, or an aptamer that binds to the modified nucleotide base. In certain embodiments, the protein is an antibody, such as, for example, anti-N6-methyladenosine (m6A), anti-Inosine (I), anti- Pseudouridine ( ), anti- 1 -methyladenosine (ml A), anti-2-methyladenosine (m2 A), anti- N6,N6-dimethyladenosine (m6,6A), anti-8-methyladenosine (m8A), anti-5-methylcytosine, anti-5-hydroxymethylcytosine, anti-5-formylcytosine, or anti-5-carboxylcytosine. In certain embodiments, the protein of the affinity binding element can be an RNA-binding protein such as the YT521-B homology (YTH) domain 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 adenosine are available from commercial vendors, such as Synaptic Systems GmbH (Goettingen, GERMANY), Epigentek Group Inc. (Farmingdale, NY, USA), Novus Biologicals LLC (Centenial, CO, USA) or Abeam PLC (Boston, MA, USA).

[0034] 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 upstream or downstream of the target nucleic acid sequence containing the modified base, or portions thereof. Alternatively or additionally, the nucleotide sequence of the affinity binding element can be designed to hybridize to a distinct oligonucleotide molecule, such as, for example, a connector oligonucleotide or an oligonucleotide primer that is complementary to a nucleotide sequence upstream or downstream of the target nucleic acid sequence containing the modified base. In certain embodiments, the connector oligonucleotide can link the nucleotide sequence of the affinity binding element to the oligonucleotide that is complementary to a nucleotide sequence that is upstream or downstream of the target nucleotide sequence containing the 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.

[0035] In certain 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 long. In certain embodiments, the target nucleotide sequence containing the modified base 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 long. In certain embodiments, the oligonucleotide can have an anchor structure, loop structure, foot structure, or any combination thereof such as, for example, a SuperSelective primer type oligonucleotide. In certain embodiments, the nucleotide sequence that is upstream or downstream of the target nucleotide sequence containing the modified base to which the nucleotide sequence of the affinity binding element or the oligonucleotide primer hybridizes (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 long, or longer. In preferred embodiments, the complementary region upstream or downstream of the target nucleotide sequence is about 15 to about 60 base, preferably about 16 to about 50 base, more preferably about 17 to about 40 base, more preferably about 17 to about 35 base, more preferably about 15 to about 30 bases. In certain 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, about 384 or more oligonucleotide primers that are each complementary to a distinct region upstream or downstream of the target nucleotide sequence can be used in a single reaction with an affinity binding element, wherein each oligonucleotide hybridizes to a nucleotide sequence that is in proximity to the site of a modification to a polynucleotide. In certain embodiments, the nucleotide sequence of the oligonucleotide or the nucleotide sequence of the affinity binding element binds immediately adjacent (e.g., 0 nucleotides upstream or downstream) to the site of the modification or 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 from the site of the modification.

[0036] In certain embodiments, the nucleotide sequence of the affinity binding element or the sequence of the oligonucleotide primer can incorporate of sample-specific identifiers (also referenced in the art as indexes, barcodes, zip codes, adapters, etc.) such that when cDNA or proximity sequence is generated different samples can be pooled and detected by using barcode specific primers, by, for example, DNA sequencing.

[0037] In certain embodiments, the oligonucleotide primer or the nucleotide sequence of the affinity binding element can be 100% complementary to a sequence upstream or downstream from the site of the modification or 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 multiple possible alternative nucleotides represented by the IUPAC notation of, for example, 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 include also universal bases, such as, for example, 5- nitroindole.

[0038] In certain embodiments, the oligonucleotide can be complementary to the region upstream or downstream from the site of the modification and can be ligated to a connector oligonucleotide or directly to the nucleotide sequence of the affinity binding element. The ligated oligonucleotides can be used to as the basis for the synthesis of a DNA template, such as, for example, a cDNA molecule and / or a PCR detectable reporter. In certain embodiments, the PCR detectable reporter can be the site at which an oligonucleotide probe can bind. In certain embodiments, the nucleotide sequence of the affinity binding element can prime a reverse transcription reaction to yield single stranded cDNA.

[0039] In certain embodiments, one or more primers can be used to amplify the cDNA resulting from the reverse transcription of the target nucleic acid or DNA template resulting from the ligation of the oligonucleotide primer to the connector oligonucleotide or to the nucleotide sequence of the affinity binding element (sometimes also referred to as target nucleotide sequence) region (or amplicon) of 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 is provided by the subject invention. The primers for the amplification reactions can be designed according to known algorithms or by a skilled artisan. For example, algorithms implemented in commercially available or custom software can be used to design primers for amplifying the target sequences based on the complementarity and stringency of said primers to the target region. Stringency refers to hybridization conditions chosen to optimize binding of polynucleotide sequences with different degrees of complementarity. Stringency is affected by factors such as temperature, salt conditions, the presence of organic solvents in the hybridization mixtures, and the lengths and base compositions of the sequences to be hybridized and the extent of base mismatching, and the combination of parameters is more important than the absolute measure of any one factor.

[0040] Typically, the primers or primer sequences used to amplify DNA can be at least 12 bases, more often 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 typically designed so that all primers participating in a particular reaction have melting temperatures that are within 5°C, and most preferably within 2°C of each other. Primers or primer sequences used to amplify DNA are further designed to avoid priming on themselves or each other. Primer concentration should be sufficient to bind to the amount of target sequences that are amplified so as to provide an accurate assessment of the quantity of amplified sequence. Those of skill in the art will recognize that the amount of concentration of primer will vary according to the binding affinity of the primers as well as the quantity of sequence to be bound. Typical primer concentrations will range from about 0.1 pM to about 1 pM, about 0.2 pM to about 0.8 pM, about 0.3 pM to about 0.7 pM, about 0.4 pM to about 0.6 pM, or about 0.4 pM to about 0.5 pM. In certain embodiments, oligonucleotide probes can be designed to hybridize to a nucleic acid sequence, or portions thereof. In certain embodiments, the complementary nucleotide segment of the 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 long, or longer. In preferred embodiments, the complementary nucleotide segment of the 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 with an oligonucleotide primer complementary to the region upstream or downstream from the site of the modification and affinity binding element or the nucleotide sequence of the affinity binding element. In certain embodiments, multiple oligonucleotide primers complementary to the regions upstream or downstream from the multiple sites of the modification. Furthermore, the probe can be labeled with a fluorescent label (e.g., for use with a quencher label). The concentration of the probes can be optimized to promote the amplification reaction. In certain embodiments, the probes can be 100% complementary to a target sequence or 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 the IUPAC notation of, for example, R, Y, S, W, K, M, B, D, H, V, N, or a gap or “.”) nucleotide.

[0041] A number of formats are available that make use of fluorescent probes. These formats are often based on fluorescence resonance energy transfer (FRET) and include molecular beacon, Scorpion® probes, and TaqMan® probes. FRET is a distance-dependent interaction between a donor and acceptor molecule. The donor and acceptor molecules are fluorophores. If the fluorophores have excitation and emission spectra that overlap, then in close proximity (typically around 10-100 angstroms) the excitation of the donor fluorophore is transferred to the acceptor fluorophore. As a result, the lifetime of the donor molecule is decreased and its fluorescence is quenched, while the fluorescence intensity of the acceptor molecule is enhanced and depolarized. When the excited-state energy of the donor is transferred to a non-fluorophore acceptor, the fluorescence of the donor is quenched without subsequent emission of fluorescence by the acceptor. In this case, the acceptor functions as a quenching reagent.

[0042] One FRET -based format for real-time PCR uses DNA probes known as “molecular beacons” (see, e.g., Tyagi et al., Nat. Biotech. 16:49-53, 1998; U.S. Pat. No. 5,925,517). Molecular beacons have a hairpin structure wherein the quencher dye and reporter dye are in intimate contact with each other at the end of the stem of the hairpin. Upon hybridization with a complementary sequence, the loop of the hairpin structure becomes double stranded and forces the quencher and reporter dye apart, thus generating a fluorescent signal. A related detection method uses hairpin primers as the fluorogenic probe (Nazarenko et al., NucL Acid Res. 25:2516-2521, 1997; U.S. Pat. No. 5,866,336; U.S. Pat. No. 5,958,700). The PCR primers can be designed in such a manner that only when the primer adopts a linear structure, i.e., is incorporated into a PCR product, is a fluorescent signal generated.

[0043] Amplification products can also be detected in solution using a fluorogenic 5' nuclease assay, a TaqMan assay. See Holland etal., Proc. Natl. Acad. Sci. U.S.A. 88: 7276-7280, 1991; U.S. Pat. Nos. 5,538,848, 5,723,591, and 5,876,930. The TaqMan probe is designed to hybridize to a sequence within 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 probe extension and contains a dye that will quench the fluorescence of the 5' fluorophore. During subsequent amplification, the 5' fluorescent label is cleaved off if a polymerase with 5' exonuclease activity is present in the reaction. The excising of the 5' fluorophore results in an increase in fluorescence which can be detected.

[0044] Schematically, said probe can have the following formulae: 5' Fluorophore-probe- Quencher 3' or 5' Quencher-prob e-Fluorophore 3'.

[0045] In addition to the hairpin and 5 '-nuclease PCR assay, other formats have been developed that use the FRET mechanism. For example, single-stranded signal primers have been modified by linkage to two dyes to form a donor / acceptor dye pair in such a way that fluorescence of the first dye is quenched by the second dye. This signal primer contains a restriction site (U.S. Pat. No. 5,846,726) that allows the appropriate restriction enzyme to nick the primer when hybridized to a target. This cleavage separates the two dyes and a change in fluorescence is observed due to a decrease in quenching. Non-nucleotide linking reagents to couple oligonucleotides to ligands have also been described (U.S. Pat. No. 5,696,251).

[0046] 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 enclosed in a silica shell may be easily derivatized for coupling to a biological molecule (Bruchez et al. (1998) Science, 281 : 2013-2016). Similarly, highly fluorescent quantum dots (zinc sulfide-capped cadmium selenide) have been covalently coupled to biomolecules for use in ultrasensitive biological detection (Warren and Nie (1998) Science, 281 : 2016-2018).

[0047] In certain embodiments, multiplex PCR can be used in the subject methods. Multiplex PCR results in the detection of multiple polynucleotide fragments in the same reaction. See, e g., PCR PRIMER, A LABORATORY MANUAL (Dieffenbach, ed. 1995) Cold Spring Harbor Press, pages 157-171. For instance, different oligonucleotide primers complementary to the region upstream or downstream from the site of the modification, different affinity binding elements, or different probes that target a PCR detectable reporter can be added in parallel in the same reaction vessel. Multiplex assays can involve the use of different fluorescent labels to detect the different target sequences that are amplified. In preferred embodiments, a single pair of primers is used to amplify a cDNA sequence in order to degrade the fluorescent probes annealed to a sample nucleic acid sequence.

[0048] In certain embodiments, the probes herein can include any useful label, including fluorescent labels and quencher labels at any useful position in the nucleic acid sequence, such as, for example at the 3'- and / or 5 '-terminus. Exemplary fluorescent labels include a quantum dot or a fluorophore. Examples of fluorescence labels for use in this method include fluorescein, 6-FAM™ (Applied Biosystems, Carlsbad, Calif.), LET™ (Applied Biosystems, Carlsbad, Calif.), VIC™ (Applied Biosystems, Carlsbad, Calif), MAX, HEX™ (Applied Biosystems, Carlsbad, Calif), TYE™ (ThermoFisher Scientific, Waltham, Mass.), TYE665, TYE705, TEX, JOE, Cy™ (Amersham Biosciences, Piscataway, N.J.) dyes (Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7), Texas Red® (Molecular Probes, Inc., Eugene, Oreg.), Texas Red-X, Al exaFluor® (Molecular Probes, Inc., Eugene, Oreg.) dyes (Al exaFluor 350, Al exaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500, AlexaFluor 532, AlexaFluor 546, AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, AlexaFluor 750), DyLight™ (ThermoFisher Scientific, Waltham, Mass.) 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 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 520, ATTO 532, ATTO 550, ATTO 565, ATTO RholOl, 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, ATTO 725, ATTO 740), BODIPY® (Molecular Probes, Inc., Eugene, Oreg.) dyes (BODIPY FL, BODIPY R6G, 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, Calif.) 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, Inc., Eugene, Oreg.), Cascade Yellow, Coumarin, Hydroxycoumarin, Rhodamine Green™-X (Molecular Probes, Inc., Eugene, Oreg.), Rhodamine Red™-X (Molecular Probes, Inc., Eugene, Oreg.), Rhodamine 6G, TMR, ABY™ (Applied Biosystems, Carlsbad, Calif.), TAMRA™ (Applied Biosystems, Carlsbad, Calif.), 5-TAMRA, JUN™ (Applied Biosystems, Carlsbad, Calif.), ROX™ (Applied Biosystems, Carlsbad, Calif.), Oregon Green® (Life Technologies, Grand Island, N.Y.), Oregon Green 500, IRDye® 700 (Li-Cor Biosciences, Lincoln, Nebr.), IRDye 800, WellRED D2, WeHRED D3, WellRED D4, and Lightcycler® 640 (Roche Diagnostics GmbH, Mannheim, Germany). In some embodiments, bright fluorophores with extinction coefficients >50,000 M1cm1and appropriate spectral matching with the fluorescence detection channels can be used.

[0049] In certain embodiments, a fluorescently labeled probe is included in a reaction mixture and a fluorescently labeled reaction product is produced. Fluorophores used as labels to generate a fluorescently labeled probe included in embodiments of methods and compositions of the present invention can be any of numerous fluorophores including, but not limited to, 4- acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid; acridine and derivatives such as acridine and acridine isothiocyanate; 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate, Lucifer Yellow VS; N-(4-anilino-l-naphthyl)maleimide; anthranilamide, Brilliant Yellow; BIODIP Y fluorophores (4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes); coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcoumarin (Coumarin 151); cyanosine; DAPDXYL sulfonyl chloride; 4', 6- diaminidino-2-phenylindole (DAPI); 5',5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansyl chloride); 4-4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); EDANS (5-[(2- aminoethyl)amino]naphthalene-l -sulfonic acid), eosin and derivatives such as eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium such as ethidium bromide; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), hexachlorofluorescenin, 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 derivatives such as EBFP, EBFP2, ECFP, and YFP; IAEDANS (5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-l-sulfonic acid), Malachite Green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerytnin; o-phthaldialdehyde; pyrene and 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); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (Rhodamine 6G), rhodamine isothiocyanate, lissamine rhodamine B sulfonyl chloride, rhodamine B, rhodamine 123, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N-tetramethyl-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid; and terbium chelate derivatives. In certain embodiments, the concentration of the fluorescent probe in the compositions and method of use is about 0.01 pM to about 100 pM, about 0.1 pM to about 100 pM, about 0.1 pM to about 50 pM, about 0.1 pM to about 10 pM, or about 0.11 pM to about 1 pM. In certain embodiments, the concentration of the fluorescent probe is about 0.01 pM, about 0.1 pM, 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.4 pMfor about 0.5 pM.

[0050] Exemplary quencher labels include a fluorophore, a quantum dot, a metal nanoparticle, 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), lowaBlack RQ, QXL™ (AnaSpec, Fremont, CA), QSY 7, QSY 9, QSY 21, QSY 35, IRDye QC, BBQ-650, Atto 540Q, Atto 575Q, Atto 575Q, MGB 3’ CDPI3, and MOBS' CDPI3. In one instance, the term “quencher” refers to a substance which reduces emission from a fluorescent donor when in proximity to the donor. In preferred embodiments, the quencher is 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 the fluorophore is detectably reduced, such as reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more.

[0051] In certain embodiments, each of the probes used in a single reaction can have a distinct fluorophore. In certain embodiments, the quencher of each probe can be identical or distinct. In specific embodiments, the fluorophore used for each probe can be 6-FAM, HEX, Texas Red- X, ATTO 647N, and Cy5.5, while the quenchers are lowaBlack FQ or lowaBlack RQ. In certain embodiments, the methods comprise calculating the fraction of polynucleotide molecules that are modified, by, for example, measuring, for the same sample, the total amount of the specific polynucleotide sequence using qPCR or dPCR for DNA targets or Reverse Transcription qPCR or dPCR for RNA targets, and the amount of modified polynucleotide molecules. The polynucleotide modification fraction is the modified polynucleotide divided by the total polynucleotide amount.

[0052] The invention further provides kits, including at least a single affinity binding element and, optionally, oligonucleotide primers and probes, as discussed above, packaged into suitable packaging material, optionally in combination with instructions for using the kit components, e.g., instructions for performing a method of the invention. In one embodiment, a kit includes an amount of at least a single affinity binding element and, optionally, oligonucleotide probes and primers, and instructions for running the assay on a label or packaging insert. In further embodiments, a kit includes an article of manufacture, for performing the assay. Preferably, said kit comprises at least a single affinity binding element and, optionally, oligonucleotide probes and primers, according to the invention. Said kit comprises more than affinity binding element, e.g. 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 elements, notably when the kit is intended to discriminate polynucleotide sequences with modifications and polynucleotide sequences without modifications, such as, for example, methylated adenosine. Said kit comprises more than one probe or oligonucleotide primer that is complementary to a nucleotide sequence upstream or downstream of the target nucleic acid sequence containing the modified base, e.g. 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, notably when the kit is intended to discriminate polynucleotide sequences with modifications and polynucleotide sequences without modifications, such as, for example, methylated adenosine.

[0053] In the kit according to the invention, the affinity binding elements and / or the oligonucleotides (primers, probes, connectors) can be either kept separately, or partially mixed, or totally mixed.

[0054] Said oligonucleotides or affinity binding elements can be provided under dry form, or solubilized in a suitable solvent, as judged by the skilled person. Suitable solvents include TE, PCR-grade water, and the like.

[0055] In a preferred embodiment, the kit according to the invention can also contain further reagents suitable for a PCR or reverse transcriptase PCR (RT-PCR) step. Such reagents are known to those skilled in the art, and include water, like nuclease- free water, RNase free water, DNAse-free water, PCR-grade water; salts, like magnesium, magnesium chloride, potassium; buffers such as Tris; enzymes, including polymerases, such as Taq, Vent, Pfu (all of them Trade-Marks), activatable polymerase, reverse transcriptase, and the like; nucleotides like deoxynucleotides, dideoxunucleotides, dNTPs, dATP, dTTP, dCTP, dGTP, dUTP; other reagents, like DTT and / or RNase inhibitors; and polynucleotides like polyT, polydT, and other oligonucleotides, e.g., primers.

[0056] In another preferred embodiment, the kit according to the invention comprises PCR controls. Such controls are known in the art, and include qualitative controls, positive controls, negative controls, internal controls, quantitative controls, internal quantitative controls, as well as calibration ranges. The internal control for said PCR step can be a template which is unrelated to the target template in the PCR step. Such controls also may comprise control primers and / or control probes. In certain embodiments, a negative control is included which comprises a polynucleotide sequence associated with the target nucleotide sequence, such as an unmodified portion of the target nucleotide sequence (or amplicon). By way of example, an unmodified portion of the target nucleotide sequence does not contain a methylated adenosine. In certain embodiments, a positive control is the same sequence with the same modification, at the same location, and at a known amount; this can also be used to generate a calibration curve to quantify the modification.

[0057] In a preferred embodiment, the kit according to the invention contains means for extracting and / or purifying nucleic acid from a biological sample, e.g. from blood, serum, plasma, saliva, or nasal secretions. Such means are well known to those skilled in the art.

[0058] In a preferred embodiment, the kit according to the invention contains instructions for the use thereof. Said instructions can advantageously be a leaflet, a card, or the like. Said instructions can also be present under two forms: a detailed one, gathering exhaustive information about the kit and the use thereof, possibly also including literature data; and a quick-guide form or a memo, e.g., in the shape of a card, gathering the essential information needed for the use thereof. Instructions can therefore include instructions for practicing any of the methods of the invention described herein. For example, compositions can be included in a container, pack, or dispenser together with instructions for performing the methylation nucleotide detection assay. Instructions may additionally include storage information, expiration date, or any information required by regulatory agencies such as the Food and Drug Administration or European Medicines Agency for use with a human or animal subject. The instructions may be on “printed matter,” e.g., on paper or cardboard within the kit, on a label affixed to the kit or packaging material, or attached to a vial or tube containing a component of the kit. Instructions may comprise voice or video tape and additionally be included on a computer readable medium, such as a disk (floppy diskette or hard disk), optical CD such as CD- or DVD-ROM / RAM, magnetic tape, flash storage, electrical storage media such as RAM and ROM and hybrids of these such as magnetic / optical storage media.

[0059] The present invention also relates to the field of diagnostics, prognosis and drug / treatment efficiency monitoring.

[0060] Detection of a Modifications to Polynucleotides

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

[0062] In certain embodiments, the methods of detecting modifications to a polynucleotide use the binding of two separate molecules. In certain embodiments, the first molecule can be the affinity binding element containing a nucleotide sequence and the second molecule can be an oligonucleotide that hybridizes to a region of a polynucleotide in proximity to the first binding event of the affinity binding element and on the same polynucleotide molecule.

[0063] In certain embodiments, the first and second molecules can interact to generate a PCR detectable reporter. In certain embodiments, the first and second molecules can hybridize directly, or the first and second molecules can interact indirectly using, for example, at least one intermediary molecule, such as, for example, a connector oligonucleotide, in which the first molecule hybridizes to a first region of the connector oligonucleotide and the second molecule hybridizes to a second region of the connector oligonucleotide, generating a ligation site between the first and second molecules that can then be connected using a ligase enzyme. In certain embodiments, the connector oligonucleotide is about 6 to about 50-bases long, about 10 to about 50-bases long, about 15 to about 25-bases long or, preferably, about 20-bases long. In certain embodiments, the first region of the connector oligonucleotide is about one-half of the connector oligonucleotide, and the second region of the connector oligonucleotide is about one-half of the connector oligonucleotide. In preferred embodiments, the first region of the connector oligonucleotide is homologous to about 10 bases at the 5’ end of the first molecule and 10 bases at the 3’ end of the second molecule. In certain embodiments of the connection step, a ligase is provided. In preferred embodiments, the ligase is a DNA ligase. DNA ligases are enzymes capable of catalyzing the formation of a phosphodiester bond between (the ends of) two polynucleotide strands bound at adjacent sites on a complementary strand. DNA ligases usually 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 DNA ligase. Additional ligases suitable for use in the methods disclosed herein are known in the art and such embodiments are within the purview of the invention.

[0064] In certain embodiments, if the target polynucleotide molecule is double-stranded DNA, the DNA molecule can be denatured by, for example, heating the DNA to a temperature about 70°C to about 99°C, or about 95°C or chemical denaturation. In certain embodiments, sodium hydroxide or a salt can be added to the DNA for chemical denaturation. In certain embodiments, the denaturation of the DNA can be performed before an affinity binding element recognizes and affixes to a modification of interest on a polynucleotide molecule.

[0065] In certain embodiments, the connector oligonucleotide can comprise a nucleic acid sequence. In certain embodiments, the connector oligonucleotide can freely diffuse in the sample or hybridized or otherwise bound to the first or second molecule. In certain embodiments, after the first and second molecules hybridize directly, primer templated extension occurs to make a compliment of both or either the first or the second molecule. In certain embodiments, the PCR detectable reporter can be a nucleic acid sequence that enables binding of primers and, optionally, a probe. Once the nucleic acid sequence is formed comprising the first molecule, second molecule, and, optionally, connector oligonucleotide, it can be used in either enzyme-based nucleic acid amplification to generate a signal for detection and quantification of the modifications to the RNA.

[0066] In certain embodiments, the methods of detecting modifications to polynucleotide use the binding of a single molecule. In certain embodiments, the single molecule can be the affinity binding element containing an oligonucleotide sequence that hybridizes to a region of a target polynucleotide in proximity to the site of the modified base and on the same polynucleotide molecule. In certain embodiments, high temperature does not allow for efficient hybridization of the oligonucleotide to the target polynucleotide. In certain embodiments, the high temperature for inefficient hybridization is at least about 5°C to about 20°C above the melting temperature of the oligonucleotide. In certain embodiments, low ionic conditions do not allow for efficient hybridization of the oligonucleotide to the target polynucleotide. In certain embodiments, the low ionic conditions can be, for example, a concentration of KC1 of about 20 mM to about 50 mM and / or a concentration of MgCh of about 1.5 mM to about 2.5 mM. In certain embodiments, the kinetics of the oligonucleotide sequence binding are driven by proximity once the affinity binding element attaches to the modified a nucleotide base. In certain embodiments, once the oligonucleotide primer hybridizes to complementary sequence on the polynucleotide, a reverse transcription reaction can be initiated. The reverse transcription can synthesize cDNA specific to a region in proximity to the site of the modified base if the polynucleotide is RNA. In certain embodiments, a single DNA primer or pair of primers can be used to amplify the cDNA using enzyme-based nucleic acid amplification. In certain embodiments, this amplification can be sequence specific and allow for detection and quantification of the modified base. In certain embodiments, the hybridization of the oligonucleotide to the target polynucleotide occurs through the cooperativity 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.

[0067] In certain embodiments, the first molecule is double-stranded in some regions that are not homologous to the connector oligonucleotide, and the second molecule is double-stranded in some regions that are not homologous to the target polynucleotide. In certain embodiments, the double-stranded parts can inhibit the formation of a secondary structure and non-specific interactions of the probes with the polynucleotide target. In certain embodiments, the polynucleotide sample and the second molecule can be mixed and heated together, followed by cooling and the addition of the first molecule. In certain embodiments, the pre-heating step can inhibit the formation of a secondary structure and increase the specific interactions of the probes with the polynucleotide target.

[0068] Any detection method or system able to detect a nucleotide can be used in methods according to embodiments of the present invention and such appropriate detection methods and systems are well-known in the art. In certain embodiments, next generation sequencing, mass spectrometry, microarray, nanoarrays, bead / particle binding coupled with flow cytometry, protein arrays, or fluorescent imaging.

[0069] In preferred embodiments, the mixture of the polynucleotide sample or controls, and the second molecule are 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 mins to about 12 hours or about 1 hour, followed by cooling at about 1°C to about 10°C or about 4°C. In certain embodiments, a mixture containing DNA ligase and a connector oligonucleotide in a ligation buffer is added to the target polynucleotide and 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, followed by incubation at about 1°C to about 10°C or about 4°C for up to about 10 minutes. In certain embodiments, the template nucleotide generated from the ligation can be quantified.

[0070] In certain embodiments, fluorescent microscopes, fluorescence scanners, spectrofluorometers and microplate readers, flow cytometers, real-time PCR machines or digital PCR machines can be used to detect fluorescence.

[0071] In certain embodiments, the detection of at least one single-stranded or double stranded nucleic acid, such as, for example, the synthesized cDNA specific to a region in proximity to the site of the modified base or the PCR detectable reporter, is carried out in an enzyme-based nucleic acid amplification method.

[0072] The expression “enzyme-based nucleic acid amplification method” relates to any method wherein enzyme-catalyzed nucleic acid synthesis occurs.

[0073] Such an enzyme-based nucleic acid amplification method can be preferentially selected from the group constituted of LCR, Q-beta replication, NASBA, LLA (Linked Linear Amplification), TMA, 3 SR, Polymerase Chain Reaction (PCR), notably encompassing all PCR based methods known in the art, such as reverse transcriptase PCR (RT-PCR), simplex and multiplex PCR, real time PCR (qPCR), end-point PCR, digital PCR performed in droplets (dPCRd), such as the Droplet Digital™ PCR technology available from Bio-Rad Laboratories, Inc., Hercules, CA; see Worldwide Website: bio-rad.com / en-us / life-science / leaming-center / introduction-to- digital-pcrAvhat-is-droplet-digital-pcr9ID=MDV3 IM4VY#Hindson), digital PCR (dPCR), quantitative or qualitative PCR and combinations thereof. These enzyme-based nucleic acid amplification methods are well known to the man skilled in the art and are notably 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 & Mueller (2005) Clin. Sci. (London) 109:365-379 (real-time Reverse-Transcription PCR). In some embodiments, the enzyme-based nucleic acid amplification method is selected from the group consisting of Polymerase Chain Reaction (PCR) and Reverse-Transcriptase- 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 multiplex PCR, quantitative PCR, or RT-PCR method.

[0074] As intended herein “multiplex” relates to the detection in a single sample or in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different samples of at least two different modified nucleotide bases by using at least two oligonucleotide probes, wherein each one of the said nucleic modified bases is detectable by at least one of said probes. Preferably, the labelling of each probe with a different fluorescent donor makes it possible to detect separately the signal emitted by the distinct probes bound to their target nucleic acid, such as, for example the PCR detectable reporter. In preferred embodiments, at least three oligonucleotide probes are used to detect of at least three different modified bases. In more preferred embodiments, at least four oligonucleotide probes are used to detect of at least four different modified bases. In even more preferred embodiments, at least five oligonucleotide probes are used to detect of at least five different nucleotide bases. Typically, the target polynucleotide sequence contains one or more modified bases that permit the probes to distinguish between the polynucleotide sequences based on the presence or absence of a modified base.

[0075] Exemplary PCR reaction conditions typically comprise either two or three step cycles. Two step cycles have a denaturation step followed by a hybridization / elongation step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step. The polymerase reactions are incubated under conditions in which the primers hybridize to the target sequences and are extended by a polymerase. The amplification reaction cycle conditions are selected so that the primers hybridize specifically to the target sequence and are extended.

[0076] Successful PCR amplification requires high yield, high selectivity, and a controlled reaction rate at each step. Yield, selectivity, and reaction rate generally depend on the temperature, and optimal temperatures depend on the composition and length of the polynucleotide, enzymes and other components in the reaction system. In addition, different temperatures may be optimal for different steps. Optimal reaction conditions may vary, depending on the target sequence and the composition of the primer. Thermal cyclers such as, for example, real-time PCR systems provide the necessary control of reaction conditions to optimize the PCR process for a particular assay. For instance, a real-time PCR system may be programmed by selecting temperatures to be maintained, time durations for each cycle, number of cycles, and the like. In some embodiments, temperature gradients may be programmed so that different sample wells may be maintained at different temperatures, and so on.

[0077] In certain embodiments, the target polynucleotide sequence can be RNA or DNA. RNA or DNA can be artificially synthesized or isolated from natural sources. 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 also be present in any useful amount.

[0078] Reverse transcriptases useful in the present invention can be any polymerase that exhibits reverse transcriptase activity. Preferred enzymes include those that exhibit reduced RNase H activity. Several reverse transcriptases are known in the art and are commercially available (e.g., from Bio-Rad Laboratories, Inc., Hercules, CA; Boehringer Mannheim Corp., Indianapolis, Ind.; Life Technologies, Inc., Rockville, Md.; New England Biolabs, Inc., Beverley, Mass.; Perkin Elmer Corp., Norwalk, Conn.; Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.; Qiagen, Inc., Valencia, Calif.; Stratagene, La Jolla, Calif.). In some embodiments, the reverse transcriptase can be Avian Myeloblastosis Virus reverse transcriptase (AMV-RT), Moloney Murine Leukemia Virus reverse transcriptase (M-MLV- RT), Human Immunovirus reverse transcriptase (HIV-RT), EIAV-RT, RAV2-RT, C. hydrogenoformans DNA Polymerase, rTth DNA polymerase, SUPERSCRIPT I, SUPERSCRIPT II, and mutants, variants and derivatives thereof. It is to be understood that a variety of reverse transcriptases can be used in the present invention, including reverse transcriptases not specifically disclosed above, without departing from the scope or preferred embodiments disclosed herein.

[0079] DNA polymerases useful in the present invention can be any polymerase capable of replicating a DNA molecule. Preferred DNA polymerases are thermostable polymerases and polymerases that have exonuclease activity, which are especially useful in PCR. Thermostable polymerases are isolated from a wide variety of thermophilic bacteria, such as Thermus aquaticus (Taq), Thermus brockianus (Tbr), Thermus flavus (Tfl), Thermus ruber (Tru), Thermus thermophilus (Tth), Thermococcus litoralis (Tli) and other species of the Thermococcus genus, Thermoplasma acidophilum (Tac), Thermotoga neapolitana (Tne), Thermotoga maritima (Tma), and other species of the Thermotoga genus, Pyrococcus furiosus (Pfu), Pyrococcus woesei (Pwo) and other species of the Pyrococcus genus, Bacillus sterothemophilus (Bst), Sulfolobus acidocaldarius (Sac) Sulfolobus solfataricus (Sso), Pyrodictium occultum (Poc), Pyrodictium abyssi (Pab), and Methanobacterium thermoautotrophicum (Mth), and mutants, variants or derivatives thereof.

[0080] Many DNA polymerases are known in the art and are commercially available (e.g., from Bio-Rad Laboratories, Inc., Hercules, CA; Boehringer Mannheim Corp., Indianapolis, Ind.; Life Technologies, Inc., Rockville, Md; New England Biolabs, Inc., Beverley, Mass.; Perkin Elmer Corp., Norwalk, Conn.; Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.; Qiagen, Inc., Valencia, Calif.; Stratagene, La Jolla, Calif.). In some embodiments, the DNA polymerase can be Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT™, DEEPVENT™, and active mutants, variants and derivatives thereof. It is to be understood that a variety of DNA polymerases can be used in the present invention, including DNA polymerases not specifically disclosed above, without departing from the scope or preferred embodiments thereof.

[0081] The reverse transcriptase can be present in any appropriate ratio to the DNA polymerase. In some embodiments, the ratio of reverse transcriptase to DNA polymerase in unit activity is greater than or equal to 3. One of skill in the art will appreciate that other reverse transcriptase to DNA polymerase ratios are useful in the present invention.

[0082] In a preferred embodiment, the reactions according to the invention can also contain further reagents suitable for a PCR step.

[0083] Such reagents are known to those skilled in the art, and include water, like nuclease- free water, RNase free water, DNAse-free water, PCR-grade water; salts, like magnesium, magnesium chloride, potassium; buffers such as Tris; enzymes; nucleotides like deoxynucleotides, dideoxunucleotides, dNTPs, dATP, dTTP, dCTP, dGTP, dUTP and modified nucleotides such as deaza-, locked nucleic acid, and peptide nucleic acid; other reagents, like DTT and / or RNase inhibitors; and polynucleotides like polyT and polydT.

[0084] Certain method and compositions used for amplifying and / or detecting nucleic acids are described in US Pat. No. 9,493,824, US Pat. No. 10,988,762, US Pat. No. 10,053,676, US Pat. No. 6,627,424, US Pat. No. 7,541,170, US Pat. No. 7,666,645, US Pat. No. 7,560,260, US Pat. No. 8,367,376, US Pat. No. 9,145,550, US Pat. No. 9,688,969, US Pat. No. 10,577,593, US Pat. No. 10,301,675, US Pat. No. 8,338,094, and US Pat. No. 9,200,318, which are each entirely incorporated herein by reference. US Pat. No. 9,493,824 describes nucleic acid amplification / detection reaction mixtures and uses thereof; US Pat. No. 10,988,762 describes reverse transcriptases and uses thereof; US Pat. No. 10,053,676 describes polymerase storage compositions and uses thereof; US Pat. No. 6,627,424 describes DNA polymerases and uses thereof; US Pat. No. 7,541,170 describes polymerases and uses thereof; US Pat. No. 7,666,645 describes polymerases and uses thereof; US Pat. No. 7,560,260 describes polymerases, particularly Pfu polymerases, and uses thereof; US Pat. No. 8,367,376 describes polymerases, particularly Pfu polymerases, and uses thereof; US Pat. No. 9,145,550 describes polymerases, particularly Pfu polymerases, and uses thereof; US Pat. No. 9,688,969 describes polymerases, particularly Pfu polymerases, and uses thereof; US Pat. No. 10,577,593 describes polymerases, particularly Pfu polymerases, and uses thereof; US Pat. No. 10,301,675 describes compositions and methods for synthesizing cDNA from an RNA template and replicating the cDNA and kits thereof; US Pat. No. 8,338,094 describes methods for synthesizing cDNA from an RNA template and replicating the cDNA and kits thereof; and US Pat. No. 9,200,318 describes methods for synthesizing cDNA from an RNA template and replicating the cDNA and kits thereof.

[0085] Targets of Polynucleotide Detection

[0086] In certain embodiments, the methods provided by the subject invention can be used to detect one or more modifications to a polynucleotide in a sample (also referred to as “target sequence(s)”, “target nucleic acid sequence(s)”, “target nucleotide sequence(s)”, or “target polynucleotide sequence(s)” herein and which can be used interchangeably). In certain embodiments, modifications can be methylated nucleoside base, such as, for example, 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 distinct modified bases can be detected in a polynucleotide sequence. In preferred embodiments, these one, two, or more modifications are found within a target sequence that is between about 60 and about 5000 nucleotides in length.

[0087] In certain embodiments, the methods of the subject invention can be used to detect modifications of polynucleotides that were found to be associated with various diseases or other ailments in a subject, such as, for example, cancer, psychiatric disorder and metabolic disease, as described in the Modomics database (Worldwide Website: https: / / genesilico.pl / modomics / diseases) or the m6A Modification Database in Disease (see Worldwide Website: m6add.edbc.org, which are each incorporated by reference in their entireties). In certain embodiments, the cancer is caused by abnormal or block differentiation of cancer stem cells, such, as for example, leukemia (e.g., acute myeloid leukemia), Hodgkin 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 psychiatric disorder is, for example, major depression, attend on- 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 to polynucleotides were found to be associated with osteoporosis, nerve injury or malformation, and an inhibited innate immune system, particularly in antiviral activity. In certain embodiments, the methods of the subject invention can be used to detect modifications of polynucleotides that were found to be associated with the development and regulation of plants, particularly under different environmental conditions, including, for example, disease and insect interactions, and for crop selection and improvement.

[0088] Additional Disclosure and Claimable Subject Matter

[0089] 1. A method for the detection of at least one modified nucleotide base in a target polynucleotide sequence from a sample, 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 part of the oligonucleotide primer sequence hybridizes to the target polynucleotide sequence in proximity to the at least one modified nucleotide base; and c) detecting the at least one modified nucleotide base.

[0090] 2. The method of embodiment 1 , wherein the target nucleotide sequence is a target RNA sequence.

[0091] 3. The method of embodiment 1 , wherein the target nucleotide sequence is a target DNA sequence.

[0092] 4. The method of embodiment 2, further comprising, after step b): reverse transcribing the target polynucleotide sequence using the oligonucleotide primer sequence to initiate the reverse transcription, yielding a cDNA molecule; and optionally, synthesizing a DNA molecule complementary to the cDNA molecule. 5. The method of embodiment 1, wherein the affinity binding element further comprises an aptamer, peptide, or protein.

[0093] 6. The method of embodiment 5, wherein the protein is an antibody.

[0094] 7. The method of embodiment 6, wherein the antibody is anti-N6-methyladenosine (m6A), anti -Inosine (I), anti -Pseudouridine ( ), anti- 1 -methyladenosine (ml A), 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-carboxylcytosine.

[0095] 8. The method of embodiment 5, wherein the protein is a YT521-B homology (YTH) domain family protein.

[0096] 9. The method of embodiment 8, wherein the YTH domain family protein is YTHDF1, YTHDF2, or YTHDF3.

[0097] 10. The method of embodiment 1, wherein the oligonucleotide primer sequence is about 6 to about 100 nucleotides in length.

[0098] 11. The method of embodiment 1, wherein the hybridization reaction conditions comprise high annealing temperatures or low ionic conditions.

[0099] 12. The method of embodiment 11, wherein the hybridization occurs through the cooperativity 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.

[0100] 13. The method of embodiment 1, wherein the oligonucleotide primer sequence is random.

[0101] 14. The method of embodiment 1, wherein the oligonucleotide primer sequence hybridizes to the target polynucleotide sequence about 0 to about 200 nucleotides upstream or downstream from the modified nucleotide base.

[0102] 15. The method of embodiment 1, wherein the target polynucleotide sequence contains two or more modified bases.

[0103] 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.

[0104] 17. The method of embodiment 1, further comprising detecting the modified nucleotide base in two or more distinct biological samples or further comprising detecting at least two modified nucleotide bases in a single biological sample. 18. The method of embodiment 1, wherein the target polynucleotide sequence is viral, bacterial, archaeal, eukaryotic, from a cell, from a tissue, present on the surface of a cell, from the cytoplasm of a fixed cell, from a nucleus of a fixed cell, from a single cell, from bodily fluids, or any combination thereof.

[0105] 19. The method of embodiment 1, wherein the modified nucleotide base is a methylated adenosine, a methylated adenine, a methylated cytosine, an acetylated cytidine, a pseudouridine, an inosine, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), or 5- carboxylcytosine (5-caC).

[0106] 20. The method of embodiment 4, further comprising amplifying said cDNA molecule with at least one DNA polymerase, at least one dNTP, and at least one buffer having a pH adapted to the polymerase activity of the at least one DNA polymerase.

[0107] 21. The method of embodiment 4, said method comprising amplification of the cDNA molecule using a single primer or pair of oligonucleotides primers that amplify a portion of the cDNA molecule or the entire cDNA molecule.

[0108] 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, a protein array, flow cytometry, fluorescent imaging, or any combination thereof.

[0109] 23. The method of embodiment 1, wherein a second part of the oligonucleotide primer sequence is double-stranded and does not hybridize to the target polynucleotide.

[0110] 24. A method for the detection of at least one modified nucleotide base in a target polynucleotide sequence from a sample, 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 in proximity to the at least one modified nucleotide; c) ligating the first molecule to the second molecule directly or ligating the first molecule to the second molecule using a connector oligonucleotide; or hybridizing the first molecule to the second molecule and extending the first and second molecule, yielding a complement of a region of the second molecule; and d) generating a PCR detectable reporter molecule using the ligated first molecule and second molecule or the complement of the region of the second molecule; e) detecting the PCR detectable reporter molecule.

[0111] 25. The method of embodiment 24, wherein the affinity binding element is an aptamer, peptide, or protein.

[0112] 26. The method of embodiment 25, wherein the protein is an antibody.

[0113] 27. The method of embodiment 26, wherein the antibody is anti-N6- m ethyladenosine (m6A), anti -Inosine (I), anti -Pseudouridine ( ), anti- 1 -methyladenosine (ml A), anti-2-methyladenosine (m2 A), anti-N6,N6-dimethyladenosine (m6,6A), anti-8- methyladenosine (m8A), anti-5-methylcytosine, anti-5-hydroxymethylcytosine, anti-5- formylcytosine, or anti-5-carboxylcytosine.

[0114] 28. The method of embodiment 25, wherein the protein is a YT521-B homology (YTH) domain family protein.

[0115] 29. The method of embodiment 28, wherein the YTH domain family protein is YTHDF1, YTHDF2, or YTHDF3.

[0116] 30. The method of embodiment 24, wherein the oligonucleotide homologous to the target polynucleotide sequence is about 10 to about 200 nucleotides in length.

[0117] 31. The method of embodiment 24, wherein the oligonucleotide sequence of the affinity binding element is about 10 to about 200 nucleotides in length.

[0118] 32. The method of embodiment 24, wherein the oligonucleotide connector is about 6 to about 50 nucleotides in length.

[0119] 33. The method of embodiment 24, wherein the oligonucleotide homologous to the target polynucleotide hybridizes to the target polynucleotide sequence about 0 to about 200 nucleotides upstream or downstream from the modified nucleotide base.

[0120] 34. The method of embodiment 24, wherein the target polynucleotide sequence contains two or more modified bases.

[0121] 35. The method of embodiment 24, further comprising detecting the modified nucleotide base in two or more distinct biological samples or further comprising detecting at least two modified nucleotide bases in a single biological sample.

[0122] 36. The method of embodiment 24, wherein the target polynucleotide sequence is viral, bacterial, archaeal, eukaryotic, from a cell, from a tissue, present on the surface of a cell, from the cytoplasm of a fixed cell, from a nucleus of a fixed cell, from a single cell, from bodily fluids, or any combination thereof. 37. The method of embodiment 24, wherein the modified nucleotide base is a methylated adenosine, a methylated adenine, a methylated cytosine, an acetylated cytidine, a pseudouridine, an inosine, 5-hmC, 5-fC, or 5-caC.

[0123] 38. The method of embodiment 24, further comprising amplifying said PCR detectable reporter molecule with at least one DNA polymerase, at least one dNTP, and at least one buffer having a pH adapted to the polymerase activity of the at least one DNA polymerase.

[0124] 39. The method of embodiment 24, further comprising detecting said PCR detectable reporter molecule using at least one fluorescent oligonucleotide probe.

[0125] 40. The method of embodiment 39, wherein each of the fluorescent probes have a distinct fluorescent signal.

[0126] 41. The method of embodiment 39, said method comprising amplification of the PCR detectable reporter molecule using a single primer or pair of oligonucleotides primers that amplify a portion of the PCR detectable reporter molecule or the entire PCR detectable reporter molecule.

[0127] 42. The method of embodiment 24, wherein the detecting comprises qPCR, dPCR, or dPCRd.

[0128] 43. The method of embodiment 24, wherein a part of the first molecule is doublestranded and does not hybridize to the second molecule, a part of the first molecule is doublestranded and does not hybridize to the connector oligonucleotide, a part of the second molecule is double-stranded and does not hybridize to the connector oligonucleotide, and / or a part of the second molecule is double-stranded and does not hybridize to the target polynucleotide.

[0129] 44. The method of embodiment 24, further comprising mixing of the target polynucleotide sequence from the sample and the second molecule at a temperature of about 60°C to about 70°C for about 3 minutes to about 15 minutes.

[0130] 45. The method of embodiment 44, further comprising cooling the mixed second molecule and the target polynucleotide sequence from the sample to about 30°C to about 45°C.

[0131] 46. The method of embodiment 45, further comprising cooling the mixed second molecule and the target polynucleotide sequence from the sample to about 37°C, and adding the first molecule to the mixed second molecule and the target polynucleotide sequence from the sample.

[0132] 47. The method of embodiment 24, wherein step b) occurs at a temperature of about 4°C to about 45°C for about 15 mins to about 12 hours followed by cooling at a temperature of about 1°C to about 10°C. 48. The method of embodiment 47, wherein step b) occurs at a temperature of about 37°C for about 1 hour, followed by cooling at a temperature of about 4°C.

[0133] 49. The method of embodiment 24, further comprising mixing a DNA ligase and the connector oligonucleotide and mixing with the target polynucleotide sequence from the sample and first and second molecules and incubating at 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 , first and second molecules, DNA ligase and the connector oligonucleotide at about 1°C to about 10°C for up to 10 minutes before the ligating step of step c).

[0134] 50. The method of embodiment 49, further comprising mixing a DNA ligase and a connector oligonucleotide and mixing with the target polynucleotide sequence from the ample and first and second molecules and incubating at about 37°C for about 10 minutes, followed by incubating the DNA ligase and the connector oligonucleotide at about 4°C for up to 10 minutes.

[0135] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0136] EXAMPLE 1— QUANTITATION OF RNA CONTAINING M6A MODIFICATION

[0137] Two different RNA oligonucleotides, each containing a single N6-methyladenosine base at a specific site (e.g., m6A RNA), were used as samples. RNA template sequence 1 is 90 bases-long, and RNA template sequence 2 is 80 bases-long. The same RNA oligonucleotides without an m6A modification were used as negative controls (N.C. RNA).

[0138] The samples and negative control RNA oligonucleotides were each diluted to form a concentration series at a range of 0.03 nM to 100 nM RNA. To measure background levels, No Template Control samples containing no RNA were used (NTC).

[0139] The detection probes were prepared as follows. For each of the RNA templates, a different Probe A oligonucleotide was designed based on the RNA sequences. Each Probe A consists of a 68 bases-long DNA oligonucleotide, of which the last 20 bases at the 3’ end are homologous to a sequence on the RNA template, located 15 bases apart from the m6A modification (FIG. 2). The 5’ end of the probe A oligonucleotides is phosphorylated. Probe B consists of anti-m6A antibody conjugated to the 5’ end of a 45 bases DNA oligonucleotide. A third DNA oligonucleotide is a 20-bases ligation connector, homologous to 10 bases at the 5’ end of probe A and 10 bases at the 3’ end of probe B oligonucleotide.

[0140] Five microliters of the RNA samples or controls were mixed with 2.5 pL Probe A, incubated at 65 °C for 5 minutes, cooled to 37 °C, and then mixed with 2.5 pL Probe B. The final binding reactions consisted of 100 pM Probe A, 100 pM Probe B, and 1 nM ligation connector, in ligation buffer. The binding reactions were incubated at 37 °C for additional 1 hour, followed by 4 °C. Next, 90 pL of Ligation mixture, consisting of T4 DNA ligase and the connector oligonucleotide, was added to each of the binding reactions, and incubated at 37 °C for 10 minutes, followed by 4 °C for up to 10 minutes. Finally, the ligation template generated was quantified using a qPCR system, and the resulting Cq values were fitted using a 5-PL regression model.

[0141] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and / or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated within the scope of the invention without limitation thereto.

Claims

CLAIMSWe claim:

1. A method for the detection of at least one modified nucleotide base in a target polynucleotide sequence from a sample, 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 in proximity to the at least one modified nucleotide; c) ligating the first molecule to the second molecule directly or ligating the first molecule to the second molecule using a connector oligonucleotide; or hybridizing the first molecule to the second molecule and extending the first and second molecule, yielding a complement of a region of the second molecule; and d) generating a PCR detectable reporter molecule using the ligated first molecule and second molecule or the complement of the region of the second molecule; e) detecting the PCR detectable reporter molecule.

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

3. The method of claim 2, wherein the protein is an antibody.

4. The method of claim 3, wherein the antibody is anti-N6-methyladenosine (m6A), anti -Inosine (I), anti -Pseudouridine ( ), anti- 1 -methyladenosine (ml A), 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-carboxylcytosine.

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

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

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

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

9. The method of claim 1, further comprising detecting the modified nucleotide base in two or more distinct 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, archaeal, eukaryotic, from a cell, from a tissue, present on the surface of a cell, from the cytoplasm of a fixed cell, from a nucleus of a fixed cell, from a single cell, from bodily fluids, or any combination thereof.

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

12. The method of claim 1, further comprising amplifying said PCR detectable reporter molecule with at least one DNA polymerase, at least one dNTP, and at least one buffer having a pH adapted to the polymerase activity of the at least one DNA polymerase.

13. The method of claim 1, further comprising detecting said PCR detectable reporter molecule using at least one fluorescent oligonucleotide probe.

14. The method of claim 13, wherein each of the fluorescent probes have a distinct fluorescent signal.

15. The method of claim 13, said method comprising amplification of the PCR detectable reporter molecule using a single primer or pair of oligonucleotides primers that amplify a portion of the PCR detectable reporter molecule or the entire PCR detectable reporter molecule.

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

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

18. The method of claim 1, further comprising mixing of the target polynucleotide sequence from the sample and 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 and 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 about 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) occurs at a temperature of about 4°C to about 45°C for about 15 mins 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) occurs 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 a DNA ligase and the connector oligonucleotide and mixing with the target polynucleotide sequence from the sample and first and second molecules and incubating at 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 , first and second molecules, DNA ligase and the connector oligonucleotide at about 1°C to about 10°C for up to 10 minutes before the ligating step of step c).

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

25. A method for the detection of at least one modified nucleotide base in a target polynucleotide sequence from a sample, 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 part of the oligonucleotide primer sequence hybridizes to the target polynucleotide sequence in proximity to the at least one modified nucleotide base; c) detecting the at least one modified nucleotide base.

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