WO2008109945A1 - Analyse d'acide ribonucléique - Google Patents

Analyse d'acide ribonucléique Download PDF

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Publication number
WO2008109945A1
WO2008109945A1 PCT/AU2008/000341 AU2008000341W WO2008109945A1 WO 2008109945 A1 WO2008109945 A1 WO 2008109945A1 AU 2008000341 W AU2008000341 W AU 2008000341W WO 2008109945 A1 WO2008109945 A1 WO 2008109945A1
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rna
methylation
nucleic acid
sample
dna
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PCT/AU2008/000341
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English (en)
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Douglas Spencer Millar
John R. Melki
Geoffrey W. Grigg
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Human Genetic Signatures Pty Ltd
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Priority claimed from AU2007901277A external-priority patent/AU2007901277A0/en
Application filed by Human Genetic Signatures Pty Ltd filed Critical Human Genetic Signatures Pty Ltd
Publication of WO2008109945A1 publication Critical patent/WO2008109945A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the present invention relates to assays to detect or determine methyiation of ribonucleic acid (RNA).
  • the target DNA is most commonly separated on the basis of size by gel electrophoresis and transferred to a solid support prior to hybridisation with a probe complementary to the target sequence (Southern and Northern blotting).
  • the probe may be a natural nucleic acid or analogue such as INA or locked nucleic acid (LNA), PNA, HNA, ANA and MNA.
  • the probe may be directly labelled (eg. with 32 P) or an indirect detection procedure may be used. Indirect procedures usually rely on incorporation into the probe of a "tag" such as biotin or digoxigenin and the probe is then detected by means such as enzyme-linked substrate conversion or chemiluminescence.
  • a capture probe is coupled to a solid support and the target DNA, in solution, is hybridised with the bound probe. Unbound target DNA is washed away and the bound DNA is detected using a second probe that hybridises to the target sequences. Detection may use direct or indirect methods as outlined above.
  • the "branched DNA” signal detection system is an example that uses the sandwich hybridization principle.
  • a rapidly growing area that uses nucleic acid hybridisation for direct detection of nucleic acid sequences is that of DNA micro-arrays (Young RA Biomedical discovery with DNA arrays. Cell 102: 9-15 (2000); Watson, New tools. A new breed of high tech detectives. Science 289:850-854 (2000)).
  • individual nucleic acid species that may range from oligonucleotides to longer sequences such as cDNA clones, were fixed to a solid support in a grid pattern.
  • a tagged or labelled nucleic acid population was then hybridised with the array and the level of hybridisation with each spot in the array is quantified.
  • radioactively or fluorescently-labelled nucleic acids eg. cDNAs
  • PCR polymerase chain reaction
  • oligonucleotides generally 15 to 30 nucleotides in length on complementary DNA strands and at either end of the DNA region to be amplified, were used to prime DNA synthesis on denatured single- stranded DNA. Successive cycles of denaturation, primer hybridisation and DNA strand synthesis using thermostable DNA polymerases allows exponential amplification of the sequences between the primers.
  • RNA sequences can be amplified by first copying using reverse transcriptase to produce a cDNA copy.
  • Amplified DNA fragments can be detected by a variety of means including gel electrophoresis, hybridisation with labelled probes, use of tagged primers that allow subsequent identification (eg. by an enzyme linked assay), use of fluorescently-tagged primers that give rise to a signal upon hybridisation with the target DNA (eg. Beacon and TaqMan systems).
  • ligase chain reaction Barany F Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc. Natl. Acad. Sci. USA 88:189-193 (1991 )).
  • the method of choice to detect methylation changes in DNA were dependent on PCR amplification of such sequences after bisulphite modification of DNA.
  • cytosines were converted to uracils (and hence amplified as thymines during PCR) while methylated cytosines were non-reactive and remain as cytosines (Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL and Paul CL.
  • a genomic sequencing protocol which yields a positive display of 5-methyl cytosine residues in individual DNA strands.
  • Primers may be chosen to amplify non-selectively a region of the genome of interest to determine its methylation status, or may be designed to selectively amplify sequences in which particular cytosines were methylated (Herman JG, Graff JR, Myohanen S, Nelkin BD and Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. PNAS 93:9821-9826 (1996)).
  • Alternative methods for detection of cytosine methylation of DNA include digestion with restriction enzymes whose cutting is blocked by site-specific DNA methylation, followed by Southern blotting and hybridisation probing for the region of interest.
  • PNA peptide nucleic acids
  • INA intercalating nucleic acid
  • RNA can be methylated and have now developed assays to detect or measure methylation of RNA.
  • the present invention provides an assay for RNA methylation comprising: treating RNA with an agent that modifies unmethylated cytosine; and measuring or detecting the presence or absence of methylated RNA.
  • the present invention provides use of an agent which modifies unmethylated cytosine in an assay to estimate, detect or measure RNA methylation.
  • the present invention provides an assay for RNA methylation comprising:
  • the present invention provides a method for detecting methylation of a target RNA in a sample comprising:
  • the present invention provides a method for estimating extent of methylation of a target RNA in a sample comprising:
  • the capture ligand or the detector ligand can be in any suitable form including oligonucleotide, nucleotide analogue, PNA or intercalating nucleic acid (INA).
  • INA intercalating nucleic acid
  • the present invention provides a method for detecting a methylated non CpG-containing RNA comprising:
  • the present invention provides a method for estimating extent of methylation of a target RNA in a sample, the method comprising: (a) treating a sample containing RNA with bisulphite to modify unmethylated cytosine to uracil;
  • the RNA is from a microorganism, a prokaryote, an eukaryote, cell, cells or a cell population.
  • the RNA is from a eukaryote. In a preferred form, the RNA is mRNA.
  • the assay according to the present invention is suitable to identify one or more of RNA splicing and generation of alternate splice forms, microRNA regulation and function, RNA/DNA interaction, gene expression, interaction of RNA with proteins, siRNA regulation and activity, processing of immature RNA species, RNA stability, RNA secondary structure, accessibility of various proteins that interact with RNA, RNA degradation, regulation of tissue specific RNA expression, DNA methylation, viral latency and subsequent re-expression, viral pathogenicity, cellular immune response to viruses, directing of DNA methylation, or demethylation, secondary structure of the RNA, RNA with enzymic activity, or translation involvement such as directing post translational modifications.
  • RNA can be obtained by any method suitable for isolating RNA from microorganisms, cells or cell population or other tissue or biological source. Such methods are well known in the art; see, for example, Sambrook et al, "Molecular Cloning, A Laboratory Manual” second ed., CSH Press, Cold Spring Harbor, 1989. Examples include but not limited to oligo-dT coated magnetic beads or resins. RNA binding resins specific examples include the following RNeasyTM and OligotexTM (Qiagen), StrataPrepTM total (Stratagene), NucleobondTM (Clontech), RNAgentsTM and PolyATractTM systems (Promega) etc. RNA may also be isolated using density gradient centrifugation techniques.
  • the RNA is treated with an agent capable of modifying unmethylated cytosine but not methylated cytosine.
  • the agent is preferably selected from bisulphite, acetate or citrate.
  • the agent is a bisulphite or acetate reagent. More preferably, the agent is sodium bisulphite, a reagent which in the presence of water, modifies cytosine into uracil.
  • RNA sequence now comprising of only 3 bases, will have less natural self-complementarity than its natural 4 base counterpart.
  • the modified RNA is then available to interact with specific complementary probes without encumbrance.
  • the amount of the target (modified) RNA present can be measured by any suitable means.
  • specific probes directed to the target RNA can be derived from part or all of the corresponding transcription unit of interest.
  • the probes can be derived from any other entity which exhibits base-sequence specificity such an appropriate antibody or antibody fragment or single domain antibody, an oligonucleotide, or a peptide nucleic acid (PNA), locked nucleic acid (LNA) or intercalating nucleic acid (INA) probes of appropriate sequence.
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • INA intercalating nucleic acid
  • the probes of the invention can be designed to be "substantially" complementary to the RNA to be tested.
  • the probes are PNA, LNA, oligonucleotide or INA in nature they would contain A (adenine), T (thymine), or C (cytosine) bases only because the modified RNA contains substantially no unmodified C residues if the RNA was unmethylated. If the RNA species of interest contained a methylated site then the probes would consist of A (adenine), T (thymine), C (cytosine) and G (guanine) bases to pair with the methyl-cytosine containing RNA molecules.
  • the probes can be any suitable ligand such as oligonucleotide probes or PNA, LNA or INA probes.
  • a poly-T DNA or a poly-T PNA or an LNA probe or poly T INA probe can be used which will bind to total treated RNA, all of which have a poly A "tail", from a cell and allow measurement of total gene expression in cells, cell population or tissue.
  • specific probes directed to an RNA of interest can be used to allow the measurement of specific gene expression in a given cell or tissue.
  • cytosine with uracil, or its bisulphite adduct, in order to destabilise random secondary structure formation in the RNA also would significantly reduce the strength of binding of a specific oligo-, PNA, LNA, or INA probe with the modified RNA.
  • An INA molecule when appropriately designed with an intercalating group restricted to terminal locations has enhanced binding characteristics to RNA of complementary sequence structure, To further compensate for this, in place of adenine bases in the probes it is preferred to substitute 2,6-diaminopurine (AP) which forms three hydrogen bonds with thymines (versus the two which adenine can form) in any complementary RNA strand and thus strengthen the binding between probe and RNA.
  • AP 2,6-diaminopurine
  • the intercalating groups are preferably placed at or close to the termini of the INA to enhance binding. Surprisingly, internal placement of intercalating groups may adversely affect hybridization of RNA to complementary DNA and can destabilize rather than stabilize the hybrid structure. Methods for constructing INA probes are described below.
  • RNA can be carried out using INA primers capable of binding to complementary sequences of RNA.
  • the amplification would typically be carried out using reverse transcriptase PCR based methods.
  • hybridizing under high stringency conditions can be synonymous with “stringent hybridization conditions”, a term which is well known in the art; see, for example, Sambrook, "Molecular Cloning, A Laboratory Manual” second ed., CSH Press, Cold Spring Harbor, 1989; “Nucleic Acid Hybridisation, A Practical Approach", Hames and Higgins eds., IRL Press, Oxford, 1985.
  • PNA or oligonucleotide probes may be prepared using any suitable method known to the art. Typically, the PNA probes were prepared according to methods outlined in US 6110676 (Coull et al 2000).
  • INA probes can be prepared by any suitable method.
  • the INA probes are prepared as disclosed on PCT/DK02/00876.
  • RNA from small amounts using INA primers prior to hybridization assays using suitable probes.
  • the present invention is suitable for use in current array technologies such as chips or in randomly addressable high density optical arrays so that large numbers of genes can be assayed rapidly. In this form, the activity of tens of thousands of gene derived mRNA can be measured or assayed in the one test.
  • the invention is also adaptable to assays directed to small numbers of mRNAs using bead technology, for example. Modified RNA species can be spotted or applied to suitable substrates in the form of an array and the array can be measured by various probes.
  • the present invention makes use of the fact that PNA molecules have no net elbctrical charge while RNA molecules, because of their phosphate backbone, are highly negatively charged.
  • Detection of bound PNA probes can utilize a simple molecule such as a positively charged fluorochrome, multiple molecules of which will bind specifically to nucleic acid in proportion to its length and can be directly detected. Many such suitable fluorochromes are known.
  • the detection system can also be an enzyme carrying a positively charged region that will selectively bind to the nucleic acid and that can be detected using an enzymatic assay, or a positively charged radioactive molecule that binds selectively to the captured nucleic acid. It will be appreciated that nanocrystals could also be used.
  • microspheres to which are attached sequence specific probes together with a number of fluorochrome molecules, can be utilized.
  • the microspheres can be attached directly to the probes targeting a particular RNA species, or via secondary non-specific component part of the RNA such as its polyadenine tail.
  • the attachment of the microsphere signal detection system could be via a poly T sequence as an INA, PNA, LNA or oligonucleotide entity.
  • microspheres carrying fluorochrome markers come in a variety of colours or spectra, it is possible in a single experiment to measure the amount of each of several different RNA species present in a single cell sample. Moreover, single microspheres, so labelled, can be readily visualised and counted, so small differences in expression between different RNA species can be determined with considerable accuracy.
  • detecting ligands binding to target modified RNA such as labelling with a suitable radioactive compound or an enzyme coupled with a colour reaction
  • Other methods for detecting ligands binding to target modified RNA such as labelling with a suitable radioactive compound or an enzyme capable of reacting with a substrate to formed a colored product, could also be used for particular applications either attached directly to the capture RNA or the probe or the substrate.
  • INA or PNA or other oligonucleotide probes as one of the ligands in this procedure can have advantages over the use of oligonucleotide probes.
  • INA or PNA binding reaches equilibrium faster and exhibits greater sequence specificity.
  • PNA molecules are uncharged and can bind the target modified RNA molecules with a higher binding coefficient than conventional oligonucleotide probes.
  • INA probes enhance binding between A- and T- and A-U bases. As a consequence of RNA treatment, there are fewer G-C base interactions and a corresponding increase in the number of A-T plus A-U base interactions.
  • the assay can provide a true and accurate measure of the amount of methylated RNA in a sample.
  • the assay is not confounded by potential bias inherent in methods that rely for signal amplification on processes such as PCR, where the enzymes commonly used in such procedures can introduce systematic bias through differential rates of amplification of different sequences.
  • the present invention is suitable for detection of disease states, differentiation states of stem cells and derivative cell populations, detection or measurement of effects of medication on gene expression or cellular function, and any other situation where an accurate indication of gene expression is useful.
  • two detector ligands can be used where one ligand is capable of binding to a region of RNA that contains one or more methylated cytosines and the other ligand capable of binding to a corresponding region of nucleic acid that before treatment (step (a)) contained no methylated cytosines.
  • a sample may contain many copies of a target RNA, the copies may have different amounts of methylation. Accordingly, the ratio of binding of the two ligands will be proportional to the degree of methylation of that RNA target in the sample.
  • the two ligands can be added together in the one test or can be added in separate duplicate tests. Each ligand can contain a unique marker which can be detected concurrently or separately in the one test or have the same marker and detected individually in separate tests.
  • the capture ligand is preferably selected from oligonucleotide, INA probe, PNA probe, LNA probe, HNA probe, ANA probe, MNA probe, CNA probe, oligonucleotide, modified oligonucleotide, single stranded DNA, RNA, aptamer, antibody, protein, peptide, a combination thereof, or chimeric versions thereof.
  • the capture ligand is an INA probe, PNA probe or an oligonucleotide probe. Even more preferably, the capture ligand is an INA probe.
  • the support can be any suitable support such as a plastic materials, fluorescent beads, magnetic beads, shaped particles, plates, microtiter plates, synthetic or natural membranes, latex beads, polystyrene, column supports, glass beads or slides, nanotubes, fibres or other organic or inorganic supports.
  • the support is a magnetic bead, a fluorescent bead, a shaped particle or a microtiter plate with one or more wells.
  • the solid substrate is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an assay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing the molecule to the insoluble carrier.
  • step (b) comprises a plurality of capture ligands arrayed on a solid support.
  • the array may contain multiple copies of the same ligand so as to capture the same target nucleic acid on the array or may contain a plurality of different ligands targeted to different nucleic acid so as to capture a plurality of target nucleic acid molecules on the array.
  • the array typically contains from about 10 to 200,000 capture ligands. It will be appreciated, however, that the array can have any number of capture ligands.
  • capture oligonucleotide probes, INA probes, or capture PNA probes can be placed on an array and used to capture bisulphite-treated nucleic acid to measure methylated states of nucleic acid.
  • Array technology is well known and has been used to detect the presence of genes or nucleotide sequences in untreated samples. The present invention, however, can extend the usefulness of array technology to provide valuable information on methylation states of many different sources of nucleic acid.
  • the sample can be any biological sample such as stem cells, blood, urine, faeces, semen, cerebrospinal fluid, cells or tissue such as brain, colon, urogenital, lung, renal, hematopoietic, breast, thymus, testis, ovary, or uterus, environmental samples, microorganisms including bacteria, virus, fungi, protozoan, viroid and the like.
  • stem cells include populations of cells containing true progenitor cells. This also applies to germ cell populations and also includes stem cells that fuse with somatic cells to form hybrid cells capable of adopting a particular phenotype.
  • nucleic acid modifying agent In order to assist in the reaction of the nucleic acid modifying agent, optional additives such as urea, methoxyamine and mixtures thereof can be added.
  • Step (b) is typically used to capture a RNA of interest which will be analysed for methylation in subsequent steps of the method.
  • step (b) allows the capture and concentration of the RNA of interest.
  • step (b) comprises a plurality of capture ligands arrayed on a solid support.
  • the array may contain multiple copies of the same ligand so as to capture the same target nucleic acid on the array for subsequent testing.
  • the array may contain a plurality of different capture ligands targeted to different RNA molecules so as to capture many different target RNA samples on the array for subsequent testing.
  • the capture ligands are bound to wells of a microtiter plate or a microarray so that multiple assays may be carried out.
  • the ligand has a detectable label attached thereto.
  • the presence of bound label being indicative of the extent of binding of the ligand.
  • Suitable labels include, but not limited to, chemiluminescence, fluorescence, radioactivity, enzyme, hapten, and dendrimer.
  • the detector ligands used in the present invention for detecting RNA in a sample, after bisulphite modification, can specifically distinguish between untreated RNA, methylated, and unmethylated RNA.
  • the INA probes may be prepared using any suitable method known to the art.
  • the probes are prepared in accordance with the teaching of WO 03/051901 , WO 03/052132, WO 03/052133 and WO 03/052134 (Human Genetic Signatures, Australia).
  • the methods according to the present invention relating to methylation states of target RNA can use any suitable RNA sample, in purified or unpurified form, as the starting material, provided it contains, or is suspected of containing, the specific nucleic acid sequence containing the target region.
  • unamplified samples are used in the methods according to the present invention.
  • INA mixtures or specific INA molecules can be used in an amplification enrichment step prior to capture by the detector ligand. Single or large numbers of INAs could be used for specific or random amplification of bisulphite-treated nucleic acid.
  • RNA molecules of interest may be selected or concentrated prior to using the methods according to the present invention.
  • An enrichment or selection step includes, bit not limited to, physical methods including sonication and shearing, enzymatic digestion, enzymatic treatment, restriction digestion, nuclease treatment, concentration, antibody capture, chemical methods including acidic or base digestion and combinations thereof.
  • an antibody directed to 5-methyl cytosine may be used to capture RNA rich in 5-methyl cytosines or highly methylated.
  • the RNA can be derived from mRNA which has undergone cleavage by any suitable physical or enzymatic means in order to break it up into more manageably sized nucleic acid.
  • RNA-containing specimen used for detection of methylated regions may be from any source and may be extracted by.a variety of techniques such as that described by Maniatis, et al (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982).
  • a detectable label may be chemiluminescent, fluorescent, or radioactive or contain a second label or marker in the form of a microsphere, or nanocrystal.
  • the fluorescent or radioactive microsphere or nanocrystal may be covalently bound to the capture or detector ligand.
  • the specificity of hybridization to target RNA is used to discriminate between methylated cytosines and unmethylated cytosines.
  • INA probes as one of the ligands can have a significant advantage over the use of oligonucleotide or PNA probes.
  • INA binding reaches equilibrium faster and exhibits greater sequence specificity and, as INAs carry one or more intercalating groups, they bind the target RNA molecules with a higher binding coefficient than other ligands such as oligonucleotides or PNAs.
  • the binding characteristics can be modified by choosing different numbers of intercalating groups to add to the INA.
  • the methods can give a true and accurate measure of the amount of a target RNA in a sample.
  • the methods are not confounded by potential bias inherent in prior art methods that rely for signal amplification on processes such as PCR, where the enzymes commonly used in such procedures can introduce systematic bias through differential rates of amplification of different sequences.
  • Multi Photon Detection is a proprietary system for the detection of ultra low amounts of selected radioisotopes. It is 1000 fold more sensitive than existing methods. It has a sensitivity of 1000 atoms of iodine 125, with quantitation of zeptomole amounts of biomaterials. It requires less than 1 picoCurie of isotope which is 100 times less activity than in a glass of water.
  • a family of MPD instruments already exists for measuring radioactivity in a sample.
  • MPD uses coincident multichannel detection of photons coupled with computer controlled electronics to selectively count only those photons that are compatible with an operator-selected radioisotope. As many different isotopes can be used, this is a multicolor system.
  • the MPD imager system is at least 100 fold more sensitive than a phosphor imager.
  • Such instrumentation would be particularly suitable in the detection part of the present invention where ligands or supports are made radioactive. Beads containing capture or detector ligands bound thereto can be processed or measured by cell sorters which measure fluorescence. Examples or suitable instruments include flow cytometers and modified versions thereof.
  • the methods according to the present invention are particularly suitable for scaling up and automation for processing many samples.
  • PCR may be used to selectively amplify RNA that has been captured with a ligand directed to methylated or unmethylated RNA.
  • the present invention relates to use of an agent that modifies unmethylated cytosine but not methylated cytosine and one or more ligands capable of distinguishing between methylated and unmethylated cytosine of RNA in methods for assaying methylation of target RNA.
  • the present invention provides a kit for analysing RNA which has been treated with an agent that modifies unmethylated cytosine, the kit comprising at least one ligand capable of distinguishing between methylated and unmethylated cytosine of RNA.
  • the kit contains one or more ligands immobilized to a solid support.
  • the kit may also contain primers for amplifying treated RNA.
  • Figure 1 shows ' results of PCR analysis of the Calcitonin gene using bisulphite treated RNA (SEQ ID NO: 76).
  • Figure 2 shows results of methylation of the TP53 gene (SEQ ID NO: 77).
  • nucleic acid covers the naturally occurring nucleic acids, DNA and RNA.
  • nucleic acid analogues covers derivatives of the naturally occurring nucleic acids, DNA and RNA, as well as synthetic analogues of naturally occurring nucleic acids. Synthetic analogues comprise one or more nucleotide analogues.
  • nucleotide analogue includes all nucleotide analogues capable of being incorporated into a nucleic acid backbone and capable of specific base-pairing (see below), essentially like naturally occurring nucleotides.
  • Nucleic acids or nucleic acid analogues useful for the present invention may comprise a number of different nucleotides with different backbone monomer units.
  • single strands of nucleic acids or nucleic acid analogues are capable of hybridising with an substantially complementary single stranded nucleic acid and/or nucleic acid analogue to form a double stranded nucleic acid or nucleic acid analogue. More preferably such a double stranded analogue is capable of forming a double helix.
  • the double helix is formed due to hydrogen bonding, more preferably, the double helix is a double helix selected from the group consisting of double helices of A form, B form, Z form and intermediates thereof.
  • nucleic acids and nucleic acid analogues useful for the present invention include, but is not limited to DNA, RNA, LNA, PNA, MNA, ANA, HNA and mixtures thereof and hybrids thereof, as well as phosphorous atom modifications thereof, such as but not limited to phosphorothioates, methyl phospholates, phosphoramidites, phosphorodithiates, phosphoroselenoates, phosphotriesters and phosphoboranoates.
  • non-phosphorous containing compounds may be used for linking to nucleotides such as but not limited to methyliminomethyl, formacetate, thioformacetate and linking groups comprising amides.
  • nucleic acids and nucleic acid analogues may comprise one or more intercalator pseudonucleotides.
  • mixture is meant to cover a nucleic acid or nucleic acid analogue strand comprising different kinds of nucleotides or nucleotide analogues.
  • hybrid is meant to cover nucleic acids or nucleic acid analogues comprising one strand which comprises nucleotide or nucleotide analogue with one or more kinds of backbone and another strand which comprises nucleotide or nucleotide analogue with different kinds of backbone.
  • HNA is meant nucleic acids as for example described by Van Aetschot et al.
  • MNA nucleic acids as described by Hossain et al, 1998.
  • ANA refers to nucleic acids described by Allert et al, 1999.
  • LNA may be any LNA molecule as described in WO 99/14226 (Exiqon), preferably, LNA is selected from the molecules depicted in the abstract of WO 99/14226. More preferably, LNA is a nucleic acid as described in Singh et al, 1998, Koshkin et al, 1998 or Obika et al., 1997.
  • PNA refers to peptide nucleic acids as for example described by Nielsen et al, 1991.
  • nucleotide designates the building blocks of nucleic acids or nucleic acid analogues and the term nucleotide covers naturally occurring nucleotides and derivatives thereof as well as nucleotides capable of performing essentially the same functions as naturally occurring nucleotides and derivatives thereof.
  • Naturally occurring nucleotides comprise deoxyribonucleotides comprising one of the four main nucleobases adenine (A), thymine (T), guanine (G) or cytosine (C), and ribonucleotides comprising on of the four nucleobases adenine (A), uracil (U), guanine (G) or cytosine (C).
  • other less common naturally occurring bases which can exist in some nucleic acid molecules include 5-methyl cytosine (met-C) and 6-methyl adenine (met-A).
  • Nucleotide analogues may be any nucleotide like molecule that is capable of being incorporated into a nucleic acid backbone and capable of specific base-pairing.
  • Non-naturally occurring nucleotides includes, but is not limited to the nucleotides comprised within DNA, RNA, PNA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2'-NH)- TNA, (3'-NH)-TNA, ⁇ -L-Ribo-LNA, ⁇ -L-Xylo-LNA, ⁇ -D-Xylo-LNA, ⁇ -D-Ribo-LNA, [3.2.1]- LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, ⁇ -Bicyclo-DNA, Tricyclo- DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, ⁇ -D- Ribopyranosyl-NA, ⁇ -L-Lyxopyranosyl-NA, 2'-R-RNA, ⁇
  • nucleotides and nucleotide analogues The function of nucleotides and nucleotide analogues is to be able to interact specifically with complementary nucleotides via hydrogen bonding of the nucleobases of the complementary nucleotides as well as to be able to be incorporated into a nucleic acid or nucleic acid analogue.
  • Naturally occurring nucleotide, as well as some nucleotide analogues are capable of being enzymatically incorporated into a nucleic acid or nucleic acid analogue, for example by RNA or DNA polymerases.
  • nucleotides or nucleotide analogues may also be chemically incorporated into a nucleic acid or nucleic acid analogue.
  • nucleic acids or nucleic acid analogues may be prepared by coupling two smaller nucleic acids or nucleic acid analogues to another, for example this may be done enzymatically by ligases or it may be done chemically.
  • Nucleotides or nucleotide analogues comprise a backbone monomer unit and a nucleobase.
  • the nucleobase may be a naturally occurring nucleobase or a derivative thereof or an analogue thereof capable of performing essentially the same function.
  • the function of a nucleobase is to be capable of associating specifically with one or more other nucleobases via hydrogen bonds.
  • base-pairing The specific interaction of one nucleobase with another nucleobase is generally termed "base-pairing”.
  • Complementary nucleotides are nucleotides that comprise nucleobases that are capable of base-pairing.
  • nucleotide comprising A is complementary to a nucleotide comprising either T or U
  • nucleotide comprising G is complementary to a nucleotide comprising C.
  • Nucleotides may further be derivatised to comprise an appended molecular entity.
  • the nucleotides can be derivatised on the nucleobases or on the backbone monomer unit. Preferred sites of derivatisation on the bases include the 8-position of adenine, the 5-position of uracil, the 5- or 6-position of cytosine, and the 7-position of guanine.
  • the heterocyclic modifications can be grouped into three structural classes: Enhanced base stacking, additional hydrogen bonding, and the combination of these classes. Modifications that enhance base stacking by expanding the ⁇ -electron cloud of the planar systems are represented by conjugated, lipophilic modifications in the 5- position of pyrimidines and the 7-position of 7-deaza-purines.
  • Substitutions in the 5- position of pyrimidines modifications include propynes, hexynes, thiazoles and simply a methyl group; and substituents in the 7-position of 7-deaza purines include iodo, propynyl, and cyano groups. It is also possible to modify the 5-position of cytosine from propynes to five-membered heterocycles and to tricyclic fused systems, which emanate from the 4- and 5-position (cytosine clamps).
  • a second type of heterocycle modification is represented by the 2-amino-adenine where the additional amino group provides another hydrogen bond in the A-T or U base pair, analogous to the three hydrogen bonds in a G-C base pair.
  • Heterocycle modifications providing a combination of effects are represented by 2-amino-7-deaza-7-modified adenine and the tricyclic cytosine analog having an ethoxyamino functional group of heteroduplexes. Furthermore, N2- modified 2-amino adenine modified oligonucleotides are among common! modifications.
  • Preferred sites of derivatisation on ribose or deoxyribose moieties are modifications of non-connecting carbon positions C-2' and C-4', modifications of connecting carbons C- 1', C-3' and C-5', replacement of sugar oxygen, 0-4', anhydro sugar modifications (conformational restricted), cyclosugar modifications (conformational restricted), ribofuranosyl ring size change, connection sites - sugar to sugar, (C-3' to C-57 C-2' to C-5'), hetero-atom ring - modified sugars and. combinations of above modifications.
  • other sites may be derivatised, as long as the overall base pairing specificity of a nucleic acid or nucleic acid analogue is not disrupted.
  • the backbone monomer unit comprises a phosphate group
  • the phosphates of some backbone * monomer units may be derivatised.
  • Oligonucleotide or oligonucleotide analogue as used herein are molecules essentially consisting of a sequence of nucleotides and/or nucleotide analogues and/or intercalator pseudonucleotides.
  • oligonucleotide or oligonucleotide analogue comprises 5 to 100 individual nucleotides.
  • Oligonucleotide or oligonucleotide analogues may comprise DNA, RNA, LNA, 2'-O-methyl RNA, PNA, ANA, HNA and mixtures thereof, as well as any other nucleotide and/or nucleotide analogue and/or intercalator pseudonucleotide.
  • RNA covers all RNA found in prokaryotes and eukaryotes and includes messenger RNA (mRNA), immature mRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering (siRNA) and microRNA (miRNA) from any source such as cells, genomic RNA from viruses or other microorganisms, transcribed RNA from DNA, RNA copy of corresponding DNA, and the like.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • siRNA short interfering
  • miRNA microRNA
  • nucleic acids, nucleic acid analogues, oligonucleotides or oligonucleotides analogues are considered to be corresponding when they are capable of hybridising.
  • corresponding nucleic acids, nucleic acid analogues, oligonucleotides or oligonucleotides analogues are capable of hybridising under low stringency conditions, more preferably corresponding nucleic acids, nucleic acid analogues, oligonucleotides or oligonucleotides analogues are capable of hybridising under medium stringency conditions, more preferably corresponding nucleic acids, nucleic acid analogues, oligonucleotides or oligonucleotides analogues are capable of hybridising under high stringency conditions.
  • High stringency conditions as used herein shall denote stringency as normally applied in connection with Southern blotting and hybridisation as described e.g. by
  • Medium stringency conditions shall denote hybridisation in a buffer containing 1 mM EDTA, 1OmM Na 2 HPO 4 H 2 O, 140 mM NaCI, at pH 7.0. Preferably, around 1.5 ⁇ M of each nucleic acid or nucleic acid analogue strand is provided.
  • medium stringency may denote hybridisation in a buffer containing 50 mM KCI, 10 mM TRIS-HCI (pH 9,0), 0.1% Triton X-100, 2 mM MgCI2 .
  • Low stringency conditions denote hybridisation in a buffer constituting 1 M NaCI, 1O mM Na 3 PO 4 at pH 7,0.
  • nucleic acids, nucleic acid analogues, oligonucleotides or oligonucleotides, nucleic acid analogues, oligonucleotides or oligonucleotides substantially complementary to each other over a given sequence such as more than 70% complementary, for example more than 75% complementary, such as more than 80% complementary, for example more than 85% complementary, such as more than 90% complementary, for example more than 92% complementary, such as more than 94% complementary, for example more than 95% complementary, such as more than 96% complementary, for example more than 97% complementary.
  • the given sequence is at least 10 nucleotides long, such as at least 15 nucleotides, for example at least 20 nucleotides, such as at least 25 nucleotides, for example at least 30 nucleotides, such as between 10 and 500 nucleotides, for example between 10 and 100 nucleotides long, such as between 10 and 50 nucleotides long. More preferably corresponding oligonucleotides or oligonucleotides analogues are substantially complementary over their entire length.
  • cross-hybridisation covers unintended hybridisation between at least two nucleic acids or nucleic acid analogues.
  • cross-hybridization may be used to describe the hybridisation of for example a nucleic acid probe or nucleic acid analogue probe sequence to other nucleic acid sequences or nucleic acid analogue sequences than its intended target sequence.
  • cross-hybridization occurs between a probe and one or more corresponding non-target sequences, even though these have a lower degree of complementarity than the probe and its corresponding target sequence. This unwanted effect could be due to a large excess of probe over target and/or fast annealing kinetics.
  • Cross-hybridization also occurs by hydrogen bonding between few nucleobase pairs, e.g. between primers in a PCR reaction, resulting in primer dimer formation and/ or formation of unspecific PCR products.
  • Nucleic acids comprising one or more nucleotide analogues with high affinity for nucleotide analogues of the same type tend to form dimer or higher order complexes based on base pairing.
  • Probes comprising nucleotide analogues such as, but not limited to, LNA, 2'-O-methyl RNA and PNA generally have a high affinity for hybridising to other oligonucleotide analogues comprising backbone monomer units of the same type. Hence even though individual probe molecules only have a low degree of complementarity they tend to hybridize. Self-hybridisation
  • self-hybridisation covers the process wherein a nucleic acid or nucleic acid analogue molecule anneals to itself by folding back on itself, generating a secondary structure like for example a hairpin structure. In most applications it is of importance to avoid self-hybridization.
  • the generation of secondary structures may inhibit hybridisation with desired nucleic acid target sequences. This is undesired in most assays for example when the nucleic acid or nucleic acid analogue is used as primer in PCR reactions or as fluorophore/ quencher labelled probe for exonuclease assays. In both assays, self-hybridisation will inhibit hybridization to the target nucleic acid and additionally the degree of fluorophore quenching in the exonuclease assay is lowered.
  • Nucleic acids comprising one or more nucleotide analogues with high affinity for nucleotide analogues of the same type tend to self-hybridize.
  • Probes comprising nucleotide analogues such as, but not limited to, LNA, 2'-O-methyl RNA and PNA generally have a high affinity for self-hybridising. Hence even though individual probe molecules only have a low degree of self-complementary they tend to self-hybridize.
  • Melting of nucleic acids refer to the separation of the two strands of a double- stranded nucleic acid molecule.
  • the melting temperature (T m ) denotes the temperature in degrees Celsius at which 50% helical (hybridized) versus coil (unhybridized) forms are present.
  • a high melting temperature is indicative of a stable complex and accordingly of a high affinity between the individual strands.
  • a low melting temperature is indicative of a relatively low affinity between the individual strands. Accordingly, usually strong hydrogen bonding between the two. strands results in a high melting temperature.
  • intercalation of an intercalator between nucleobases of a double stranded nucleic acid may also stabilise double stranded nucleic acids and accordingly result in a higher melting temperature.
  • the melting temperature is dependent on the physical/chemical state of the surroundings. For example the melting temperature is dependent on salt concentration and pH. The melting temperature may be determined by a number of assays, for example it may be determined by using the UV spectrum to determine the formation and breakdown (melting) of hybridisation.
  • INAs Intercalating Nucleic Acids
  • INAs are a unique class of DNA binding molecules.
  • INAs are comprised of nucleotides and/or nucleotide analogues and intercalating pseudonucleotide (IPN) monomers.
  • IPN intercalating pseudonucleotide
  • INAs have a very high affinity for complementary DNA with stabilisations of up to 10 degrees for internally placed IPNs and up to 11 degrees for end position IPNs.
  • the INA itself is a selective molecule that prefers to hybridise with DNA over complementary RNA. It has been shown that INAs bind about 25 times less efficiently to RNA than oligonucleotide primers.
  • INAs are the first truly selective DNA binding agents.
  • INAs have a higher specificity and affinity for complementary DNA that other natural DNA molecules.
  • IPNs stabilise DNA best in AT-rich surroundings which make them especially useful in the field of epigenomics research.
  • the IPNs are typically placed as bulge or end insertions in to ' the INA molecule.
  • the IPN is essentially a planar (hetero) polyaromatic compound that is capable of co-stacking with nucleobases in a nucleic acid duplex.
  • the INA molecule has also been shown to be resistant to exonuclease attack. This makes these molecules especially useful as primers for amplification using enzymes such as phi29. As phi29 has inherent exonuclease activity, primers used as templates for amplification must be specially modified at their 3' terminus to prevent enzyme degradation. INA molecules, however, can be added without further modification.
  • INAs can be used in conventional PCR amplification reactions and behave as conventional primers. INAs, however, in certain circumstances have a higher specificity for DNA over RNA templates making them ideal for the use in situations where template is limiting and sensitivity of the reaction is critical. INAs stabilise DNA best in AT-rich surroundings which make them especially useful for amplification of bisulphite treated DNA sequences. This is due to the fact that after bisulphite conversion, all the cytosine residues are converted to uracil and subsequently thymine after PCR or other amplification. Bisulphite treated DNA is therefore very T rich. Increasing the number of IPN molecules in the INA results in increased stabilization of the INA/DNA duplex. The more IPNs in the INA, the greater the melting temperature of the DNA/INA duplex.
  • the present applicant has previously developed a class of intercalator pseudonucleotides which, when incorporated into an oligonuceotide or oligonuceotide analogue, form an intercalating nucleic acid (INA) (WO 03/051901 , WO 03/052132, WO 03/052133 and WO 03/052134) which has novel and useful properties as a supplement to, or replacement of, oligonucleotides.
  • INA intercalating nucleic acid
  • the intercalator pseudonucleotide is preferably selected from phosphoramidites of 1-(4,4'-dimethoxytriphenylmethyloxy)-3-pyrenemethyloxy-2-propanol.
  • the intercalator pseudonucleotide is selected from the phosphoramidite of (S)-1-(4,4'- dimethoxytriphenylmethyloxy)-3-pyrenemethyloxy-2-propanol or the phosphoramidite of (R)-1-(4,4'-dimethoxytriphenylmethyloxy)-3-pyrenemethyloxy-2-propanol.
  • the oligonucleotide or oligonucleotide analogue can be selected from DNA, RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), MNA, altritol nucleic acid (ANA), hexitol nucleic acid (HNA), intercalating nucleic acid (INA), cyclohexanyl nucleic acid (CNA) and mixtures thereof and hybrids thereof, as well as phosphorous atom modifications thereof, such as but not limited to phosphorothioates, methyl phospholates, phosphoramidites, phosphorodithiates, phosphoroselenoates, phosphotriesters and phosphoboranoates.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • MNA altritol nucleic acid
  • ANA altritol nucleic acid
  • HNA hexitol nucleic acid
  • INA intercalating nucleic acid
  • CNA cyclo
  • Non-naturally occurring nucleotides include, but not limited to the nucleotides comprised within DNA, RNA, PNA, INA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2'-NH)-TNA, (3'-NH)-TNA, ⁇ -L-Ribo-LNA, ⁇ -L-Xylo-LNA, ⁇ -D-Xylo-LNA, ⁇ -D-Ribo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi- Bicyclo-DNA, ⁇ -Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, ⁇ -D-Ribopyranosyl-NA, ⁇ -L-Lyxopyranosyl-NA, 2'-R-RNA,
  • non-phosphorous containing compounds may be used for linking to nucleotides such as but not limited to methyliminomethyl, formacetate, thioformacetate and linking groups comprising amides.
  • nucleic acids and nucleic acid analogues may comprise one or more intercalator pseudonucleotides.
  • PNA Peptide nucleic acid
  • Peptide nucleic acids are non-naturally occurring polyamides which can hybridize to nucleic acids (DNA and RNA) with sequence specificity. (See U.S. Pat. No. 5,539,082 and Egholm et al., Nature (1993) 365, 566-568). PNAs are candidates as alternatives/substitutes to nucleic acid probes in probe-based hybridization assays because they exhibit several desirable properties. PNAs are achiral polymers which hybridize to nucleic acids to form hybrids which are more thermodynamically stable than a corresponding nucleic acid/nucleic acid complex. Being non-naturally occurring molecules, they are not known to be substrates for the enzymes which are known to degrade peptides or nucleic acids.
  • PNAs should be stable in biological samples, as well as, have a long shelf-life. Unlike nucleic acid hybridization which is very dependent on ionic strength, the hybridization of a PNA with a nucleic acid is fairly independent of ionic strength and is favoured at low ionic strength under conditions which strongly disfavour the hybridization of nucleic acid to nucleic acid. The effect of ionic strength on the stability and conformation of PNA complexes has been extensively investigated. Sequence discrimination is more efficient for PNA recognizing DNA or RNA than for DNA recognizing DNA. However, the advantages in single base change, indel, or polymorphism discrimination with PNA probes, as compared with DNA probes, in a hybridization assay appears to be somewhat sequence dependent. As an additional advantage, PNAs hybridize to nucleic acid in both a parallel and antiparallel orientation, though the antiparallel orientation is preferred.
  • PNAs are synthesized by adaptation of standard peptide synthesis procedures in a format which is now commercially available.
  • Labelled and unlabelled PNA oligomers can be purchased (See: PerSeptive Biosystems Promotional Literature: BioConcepts, Publication No. NL612, Practical PNA, Review and Practical PNA, Vol. 1, Iss. 2) or prepared using the commercially available products. There are indeed many differences between PNA probes and standard nucleic acid probes.
  • nucleic acids are biological materials that play a central role in the life of living species as agents of genetic transmission and expression. Their in vivo properties are fairly well understood. PNA, however, is a recently developed totally artificial molecule, conceived in the minds of chemists and made using synthetic organic chemistry. It has no known biological function.
  • PNA also differs dramatically from nucleic acid. Although both can employ common nucleobases (A, C, G, T, and U), the backbones of these molecules are structurally diverse. The backbones of RNA and DNA are composed of repeating phosphodiester ribose and 2-deoxyribose units. In contrast, the backbones of PNA are composed on N-(2-aminoethyl)glycine units. Additionally, in PNA the nucleobases are connected to the backbone by an additional methylene carbonyl unit.
  • PNA is not an acid and contains no charged acidic groups such as those present in DNA and RNA. Because they lack formal charge, PNAs are generally more hydrophobic than their equivalent nucleic acid molecules. The hydrophobic character of PNA allows for the possibility of non-specific (hydrophobic/hydrophobic interactions) interactions not observed with nucleic acids. . Furthermore, PNA is achiral, providing it with the capability of adopting structural conformations the equivalent of which do not exist in the RNA/DNA realm.
  • PNA binds to its complementary nucleic acid more rapidly than nucleic acid probes bind to the same target sequence. This behaviour is believed to be, at least partially, due to the fact that PNA lacks charge on its backbone. Additionally, recent publications demonstrate that the incorporation of positively charged groups into PNAs will improve the kinetics of hybridization (See: Iyer et al. J. Biol. Chem. (1995) 270, 14712-14717). Because it lacks charge on the backbone, the stability of the PNA/nucleic acid complex is higher than that of an analogous DNA/DNA or RNA/DNA complex. In certain situations, PNA will form highly stable triple helical complexes or form small loops through a process called "strand displacement". No equivalent strand displacement processes or structures are known in the DNA/RNA world.
  • PNAs hybridize to nucleic acids with sequence specificity
  • PNA probes are not the equivalent of nucleic acid probes.
  • both the exact target sequence and a closely related sequence e.g. a non-target sequence having a single point mutation (single base pair mismatch) will often exhibit detectable interaction with a labelled nucleic acid or labelled PNA probe (See: Nielsen et al. Anti-Cancer Drug Design at p. 56-57 and Weiler et al. at p. 2798, second full paragraph). Any hybridization to a closely related non-target sequence will result in the generation of undesired background signal.
  • the sample was then incubated overnight at 55 0 C.
  • the samples can be cycled in a thermal cycler as follows: incubate for about 4 hours or overnight as follows: Step 1 , 55 0 C / 2 hr cycled in PCR machine; Step 2, 95 0 C / 2 min.
  • Step 1 can be performed at any temperature from about 37 0 C to about 9O 0 C and can vary in length from 5 minutes to 8 hours.
  • Step 2 can be performed at any temperature from about 70 0 C to about 99 0 C and can vary in length from about 1 second to 60 minutes, or longer.
  • additives are optional and can be used to improve the yield of DNA obtained by co- precitpitating with the target DNA especially when the DNA is present at low concentrations.
  • the use of additives as carrier for more efficient precipitation of nucleic acids is generally desired when the amount of nucleic acid is ⁇ 0.5 ⁇ g.
  • An isopropanol cleanup treatment was performed as follows: 800 ⁇ l of water were added to the sample, mixed and then 1 ml isopropanol was added.
  • the water or buffer reduces the concentration of the bisulphite salt in the reaction vessel to a level at which the salt will not precipitate along with the target nucleic acid of interest.
  • the dilution is generally about 1/4 to 1/1000 so long as the salt concentration is diluted below a desired range, as disclosed herein.
  • the sample was mixed again and left at 4 0 C for a minimum of 5 minutes.
  • the sample was spun in a microfuge for 10-15 minutes and the pellet was washed 2x with 70% ETOH, vortexing each time. This washing treatment removes any residual salts that precipitated with the nucleic acids.
  • RNA sample was allowed to dry and then resuspended in a suitable volume of T/E (10 mM Tris/0.1 mM EDTA) pH 7.0-12.5 such as 50 ⁇ l. Buffer at pH 10.5 has been found to be particularly effective.
  • the sample was incubated at 37 0 C to 95 0 C for 1 min to 96 hr, as needed to suspend the nucleic acids. Detection of methylation of RNA
  • RNA methylation there is methylation of RNA and this methylation can be analysed according to the present invention, it will be appreciated that methods used for the detection of methylation in DNA would be suitable for use in the present invention for analysis of RNA methylation. Examples of methods that could be used to detect RNA methylation include, but not limited, to the following:
  • MSP Met ⁇ ylation Specific PCR
  • SNuPE Single Nucleotide Primer Extension
  • Macro- and Microarray approaches including bead based arrays Antibody based methods for methylC enrichment MethylLight
  • Ligation mediated methods such as the ligase chain reaction (LCR)
  • LCR ligase chain reaction
  • Mass Spectrometry including Matrix-Assisted Laser Desorption Ionisation-Time of Flight Reverse-transcriptase PCR approaches
  • Isothermal amplification based methods including Nucleic Acid Sequence Based Amplification, Transcription-based Amplification System,
  • Hybridisation such as Northern, Southern blotting, In-situ Hybridisation Fluorescent In situ Hybridisation
  • Agents suitable for the present invention include bisulphite reagents such as acetate, sodium bisulphite, sodium metabisulphite, guanidinium hydrogen sulphite as described in WO 2005054502.
  • bisulphite reagents such as acetate, sodium bisulphite, sodium metabisulphite, guanidinium hydrogen sulphite as described in WO 2005054502.
  • the detection of a methylated cytosine in RNA can be carried out wherein guanidinium hydrogen sulphite is used for the preparation of a solution containing guanidinium ions and sulphite ions and subsequent modification of the RNA.
  • a un-methylated cytosine is converted to uracil and a methylated cytosine is not converted.
  • the present invention covers the use of any suitable agents that act in a similar manner similar to known bisulphite reagents found to act on unmethylated cytosines in DNA having 5-methyl-cytosine bases.
  • a bisulphite reaction shall mean a reaction for the conversion of a cytosine base, in particular cytosine bases, in a nucleic acid to an uracil base, or bases, preferably in the presence of bisulphite ions whereby preferably 5-methyl-cytosine bases are not significantly converted.
  • This reaction for the detection of methylated cytosines in DNA is described in detail by Frommer et al and Grigg and Clark.
  • the bisulphite reaction contains a deamination step and a desulfonation step which can be conducted separately or simultaneously.
  • 5-methyl-cytosine bases are not significantly converted shall only take the fact into account that it cannot be excluded that a small percentage of 5- methyl-cytosine bases is converted to uracil although it is intended to convert only and exclusively the (non-methylated) cytosine bases in RNA.
  • cytosines present in a ribonucleotide (ribo-cytosines).
  • RNA can be extracted from cells by any suitable means.
  • An example of extraction is set out below:
  • the upper aqueous phase was removed into a clean tube ensuring the pipette tip stayed away from the interface and 1 ⁇ l of 20mg/ml glycogen added and the samples vortexed.
  • RNA was then spun in a microfuge for 10 seconds the residual ethanol removed and the pellet immediately resuspended in 25 ⁇ l of RNase free water. NB if the pellet dries out then it is very difficult to resuspend the RNA and the 260/280 ratio will be less than 1.6. XIII. The OD 2 60/280/3 1 0 was then recorded and the RNA stored at -7O 0 C until required.
  • RNA sample was resuspended in 20 ⁇ l of nuclease free water after extraction from the desired cells or tissue. The sample was heated at 60-10O 0 C for 2-3 minutes to resolve secondary structure and immediately used in the bisulphite reaction.
  • RNA Two ⁇ g of RNA is resuspended in a total of 20 ⁇ l RNase free water. The sample was then incubated at 65 0 C for 2 minutes to remove secondary structure. After the incubation, 208 ⁇ l 2 M Sodium Metabisulphite pH 5.0 (7.6 g in 20 ml water or 10 mM Tris/1 mM EDTA with 416 ⁇ l 10 N NaOH; BDH AnalaR #10356.4D; freshly made) was added in succession. RNase inhibitors can also be added at this point such as RNaseOUT (invitrogen cat # 10777-019) according to the manufacturers instructions. The sample was overlaid with 200 ⁇ l of mineral oil.
  • the overlaying of mineral oil prevents evaporation and oxidation of the reagents but is not essential.
  • the sample was then incubated overnight at 55 0 C. This incubation can be performed at any temperature from about 37 0 C to about 9O 0 C and can vary in length from 5 minutes to 16 hours.
  • the oil was removed, and 1 ⁇ l glycogen (20 mg/ml) was added especially if the RNA concentration was low.
  • This additive is optional and can be used to improve the yield of RNA obtained by co- precitpitating with the target RNA especially when the RNA is present at low concentrations.
  • the use of additives as carrier for more efficient precipitation of nucleic acids is generally desired, when the amount nucleic acid is ⁇ 0.5 ⁇ g.
  • An isopropanol cleanup treatment was performed as follows: 800 ⁇ l of RNase free water was added to the sample, mixed and then 1 ml isopropanol was added.
  • the water or buffer reduces the concentration of the bisulphite salt in the reaction vessel to a level at which the salt will not precipitate along with the target nucleic acid of interest.
  • the dilution is generally about 1/4 to 1/1000 so long as the salt concentration is diluted below a desired range, as disclosed herein.
  • the sample was mixed again and left at 4 0 C for a minimum of 5 minutes but can be up to 60 minutes.
  • the sample was spun in a microfuge for 10-15 minutes and the pellet was washed 2x with 80% ETOH. This washing treatment removes any residual salts that precipitated with the nucleic acids.
  • the pellet was allowed to dry briefly to remove residual ethanol but ensuring that the pellet did not dry out totally as this can reduce the final RNA yield and then resuspended in a suitable volume of T/E (10 mM Tris/0.1 mM EDTA) pH 7.0-12.5 such as 50 ⁇ l.
  • RNase inhibitors can also be added at this point such as RNaseOUT (invitrogen cat # 10777-019) according to the manufacturers instructions. Buffer at pH 10,5 has been found to be particularly effective.
  • the sample was incubated at 37 0 C to 95 0 C for 1 min to 96 hr, as needed to suspend the nucleic acids.
  • Tubes were removed from the PCR machine and cooled on ice for 2 minutes then spun briefly to collect contents. Samples were then incubated @ 42 0 C for 2 minutes.
  • the tubes were heated at 72 0 C for 7 minutes then stored @ -7O 0 C until required.
  • PCR amplification was performed on 1 ⁇ l of bisulphite treated RNA, PCR amplifications were performed in 25 ⁇ l reaction mixtures containing 1 ⁇ l of bisulphite- treated genomic DNA, using the Promega PCR master mix, 6 ng/ ⁇ l of each of the primers. One ⁇ l of 1 st round amplification was transferred to the second round amplification reaction mixtures. Samples of PCR products were amplified in a ThermoHybaid PX2 thermal cycler under the conditions described in Clarke et a/.
  • Agarose gels (2%) were prepared in 1% TAE containing 1 drop ethidium bromide (CLP #5450) per 50 ml of agarose.
  • Five ⁇ l of the PCR derived product was mixed with 1 ⁇ l of 5X agarose loading buffer and electrophoresed at 125 mA in X1 TAE using a submarine horizontal electrophoresis tank. Markers were the low 100-1000 bp type. Gels were visualised under UV irradiation using the Kodak UVIdoc EDAS 290 system.
  • the INA used for attachment to the magnetic beads can be modified in a number of ways.
  • the INA contained either a 5' or 3' amino group for the covalent attachment of the INA to the beads using a hetero-bifunctional linker such as EDC.
  • the INA can also be modified with 5' groups such as biotin which can then be passively attached to magnetic beads modified with avidin or steptavidin groups.
  • the beads were mixed then magnetised and the supernatant discarded.
  • the beads were washed x2 in 100 ⁇ l of PBS per wash and finally resuspended in 90 ⁇ l of 50 mM MES buffer pH 4.5 or another buffer as determined by the manufactures' specifications.
  • One ⁇ l of 250 ⁇ M INA concentration dependant on the specific activity of the selected INA as determined by oligo hybridisation experiments is added to the sample and the tube vortexed and left at room temperature for 10-20 minutes.
  • the samples were then magnetised, the supernatant discarded and the beads may be blocked by the addition of 100 ⁇ l either 0.25 M NaOH or 0.5 M Tris pH 8.0 for 10 minutes.
  • the beads were then washed x2 with PBS solution and finally resuspended in 100 ⁇ l PBS solution.
  • the buffers may also contain either cationic/anionic or zwittergents at known concentration or other additives such as Heparin and poly amino acids.
  • RNA 1-5 ⁇ l was then added to the above solution and the tubes vortexed and then incubated at 55 0 C or another temperature depending on the melting temperature of the chosen INA/RNA hybrid for 20-60 minutes.
  • the samples were magnetised and the supernatant discarded and the beads washed x2 with 0.1XSSC/0.1%SDS at the hybridisation temperature from earlier step for 5 minutes per wash, magnetising the samples between washes.
  • An INA or oligo molecule can be either 3' or 5' labelled with a molecule such as an amine group, thiol group or biotin.
  • the labelled molecule can also have a second label such as P 32 or I 125 incorporated at the opposite end of the molecule to the first label.
  • This dual labelled detector molecule can now be covalently coupled to a carboxylate or modified latex bead of known size using a hetero-bifunctional linker such as EDC.
  • the unbound molecules can then be removed by washing leaving a bead coated with large numbers of specific detector/signal amplification molecules.
  • An INA or oligo molecule can be either 3' or 5' labelled with a molecule such as . an amine group, thiol group or biotin.
  • the labelled molecule can also have a second label such as Cy-3, Cy-5, FAM, HEX, TET, TAMRA or any other suitable fluorescent molecule incorporated at the opposite end of the molecule to the first label.
  • This dual labelled detector molecule can now be covalently coupled to a carboxylate or modified latex bead of known size using a hetero-bifunctional linker such as EDC.
  • the unbound molecules can then be removed by washing, leaving a bead coated with large numbers of specific detector/signal amplification molecules.
  • RNA sample of interest can then be hybridised with the RNA sample of interest to produce signal amplification.
  • An INA or oligo molecule can be either 3' or 5' labelled with a molecule such as an amine group or a thiol group.
  • the labelled molecule can also have a second label such as biotin or other molecules such as horse-radish peroxidase or alkaline phosphatase conjugated on via a hetero-bifunctional linker at the opposite end of the molecule to the first label.
  • This dual labelled detector molecule can now be covalently coupled to a carboxylate or modified latex bead of known size using a hetero-bifunctional linker such as EDC.
  • the unbound molecules can then be removed by washing leaving a bead coated with large numbers of specific detector/signal amplification molecules.
  • These beads can then be hybridised with the nucleic acid sample of interest to produce signal amplification.
  • Signal amplification can then be achieved by binding of a molecule such as
  • Streptavidin or an enzymatic reaction involving a colorimetric substrate.
  • the initial hybridization event preferably involves the use of magnetic beads coated with a INA complimentary to the RNA of interest.
  • a second hybridisation event can involve any of the detection methods mentioned above.
  • This hybridisation reaction can be done with either a second INA complimentary to the nucleic acid of interest or an oligo or modified oligo complementary to the RNA of interest.
  • Dendrimers are branched tree-like molecules that can be chemically synthesised in a controlled manner so that multiple layers can be generated that were labelled with specific molecules. They were synthesised stepwise from the centre to the periphery or visa-versa.
  • One of the most important parameters governing dendrimer structure and its generation is the number of branches generated at each step; this determines the number of repetitive steps required to build the desired molecule.
  • Dendrimers can be synthesised that contain radioactive labels such as I 125 or P 32 or fluorescent labels such as Cy-3, Cy-5, FAM, HEX, TET, TAMRA or any other suitable fluorescent molecule to enhance signal amplification.
  • radioactive labels such as I 125 or P 32 or fluorescent labels such as Cy-3, Cy-5, FAM, HEX, TET, TAMRA or any other suitable fluorescent molecule to enhance signal amplification.
  • dendrimers can be synthesised to contain carboxylate groups or any other reactive group that could be used to attach a modified PNA or DNA molecule.
  • Detection system using arrays Treated RNA can be applied to any suitable substrate to form arrays such as microarrays that can be screened for activity of genes or expression units of interest. Persons skilled in the art would be familiar with the appropriate technology for making suitable arrays.
  • TERT-F1-1 ATTATYGYGAGGTGTTGYYGTTGGTT 21
  • TERT-R1-5 CAAACCCTATAAATATCRTCCAAAC 31
  • TERT-R1-13 CAAAAAAATCTTATAAATATTAATAC 40
  • RASS-F2-6 GAAAGTTTTTGGTGGTGGATGATTT 63
  • RASS-R1-8 CAAAATACRTAAAAAATTATATAATTCAAAC 64
  • R-FLC-1 R1 ACATACTRTTTCCCATATCAATCAAA 68
  • RNA samples 2-5 ⁇ g were reverse transcribed with Superscript III reverse transcriptase (Invitrogen) as follows or a 1-step RT-PCR performed as outlined in the patent using standard premixes (see below) in the second round amplification: 11 ⁇ l converted RNA template
  • PCR reactions were prepared as follows. Promega 2x master mix 12.5 ⁇ l
  • Figure 1 shows the sequence generated from the Calcitonin gene after bisulphite conversion of the RNA and ' PCR amplification. As can be seen all cytosine residues (vertical highlights) were converted to thymine upon amplification. The solid line represents the exon/intron splice site between exohs 5 and 6 indicating the product was indeed amplified from mRNA and not contaminating DNA sequences.
  • Table 3 shows the methylation status of exons 1-3 of the calcitonin gene showing that no methylation was detected at any site in the 3 exons of the Calcitonin gene analysed. The degree of methylation at each site was - 0%.
  • CL1 U87MG cell line
  • CL2 T98G cell line
  • CL3 M059K cell line
  • CL4 TT cell line.
  • Figure 2 shows the sequence generated from the TP53 gene after bisulphite conversion of the RNA and PCR amplification. The methylated RNA bases in the TP53 gene are indicated by the vertical arrows.
  • Table 4 shows the methylation status of the TP53 gene.
  • CL2 T98G cell line
  • CL3 M059K cell line
  • CL4 TT cell line.
  • the degree of methylation at each site is as follows - 0%, + 1-25%, ++ 26-50%, +++ 51-75%, ++++ 76-100%.
  • the sequence context of methylation at each positive site is displayed above the box. As can be seen from the results, metyaltion was detected in a number of exons of this gene.
  • Table 5 shows detailed methylation profiling of the RASSF1 gene.
  • CL2 T98G cell line
  • CL3 M059K cell line
  • CL4 TT cell line.
  • the degree of methylation at each site is as follows - 0%, + 1-25%, ++ 26-50%, +++ 51-75%, ++++ 76-100%.
  • the sequence context of methylation at each positive site is displayed above the box.
  • Table 6 shows detailed methylation profiling of the PTGS2 gene.
  • CL1 U87MG cell line
  • CL2 T98G cell line.
  • the degree of methylation at each site is as follows - 0%, + 1-25%, ++ 26-50%, +++ 51-75%, ++++ 76-100%.
  • the sequence context of methylation at each positive site is displayed above the box. As can be seen from the results, metyaltion was detected in a number of exons Qf this gene.
  • Table 7 shows the detailed methylation status of the GSTP1 gene.
  • Table 8 shows the detailed methylation status of the gene TERT gene.
  • CL2 T98G cell line
  • CL3 M059K cell line
  • CL4 TT cell line.
  • the degree of methylation at each site is as follows - 0%, + 1-25%, ++ 26-50%, +++ 51-75%, ++++ 76- 100%.
  • the sequence context of methylation at each positive site is displayed above the box. As can be seen from the results, metyaltion was detected in a number of exons of this gene.
  • Table 9 shows the methylation profiles of the plant Arabidopsis strain C24 with wild type strain Columbia.
  • the arrows represent methylated sites within exon 4 adjacent to the splice site of the Ababidopsis Columbia ecotype that are not present in the C24 strain.
  • the bold line show the splice site between exon 3 and 4. The experiment was repeated on two occasions to demonstrate the reproducibility of the- method.
  • RNA methylation was also observed around splice sites indicating a possible role for RNA methylation in controlling alternate splicing.
  • Table 3. Methylation status of exons 1-6 of the Calcitonin gene from Figure 1.
  • Table 9 shows the RNA methylation observed at the splice site between exon 3 and exon 4 in the FLC mRNA.
  • the splice site is between sites 1 and 2.
  • the degree of methylation at each site is as follows - 0%, + 1-25%, ++ 26-50%, +++ 51-75%, ++++ 76- 100%.
  • the sequence context of methylation at each positive site is displayed above the box.As can be seen ecotype C24 is not methylated at the splice site whereas wild type Columbia shows heavy methylation at the splice junction.
  • the results were repeated using a fresh extract of RNA that was subsequently amplified using all samples to demonstrate the reproducibility of the method.
  • RNA methylation may be involved in numerous processes such as RNA splicing and the generation of splice variants, processing of immature RNA species, RNA protein interactions such as RNA methyltransferases, ribosomes, spliceomes and RNA interference, RNA regulation, RNA expression, RNA degradation, RNA directed DNA methylation, microRNA regulation and function, RNA/DNA interactions, siRNA regulation and activity, RNA stability such as short lived RNAs vs long lived RNAs, RNA secondary structure, regulation of tissue specific RNA expression, changing the secondary structure of the RNA may be involved in RNAs with enzymic activity such as Xist, important in translation such as directing post translational modifications, viral latency and subsequent re-expression, viral pathogenicity and host interactions and RNA function. Now that the present inventors have detected methylation of RNA, detection of the presence, amount or absence of methylation of RNA maybe a useful tool for biological analysis.
  • RNA protein interactions such as RNA methyltrans

Abstract

La présente invention concerne des analyses pour la méthylation de l'ARN consistant à traiter de l'ARN avec un agent qui modifie la cytosine non méthylée, et à mesurer ou détecter la présence ou l'absence d'ARN méthylé. De préférence, l'agent est du bisulfite.
PCT/AU2008/000341 2007-03-12 2008-03-12 Analyse d'acide ribonucléique WO2008109945A1 (fr)

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Cited By (9)

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WO2009070843A1 (fr) * 2007-12-05 2009-06-11 Human Genetic Signatures Pty Ltd Traitement de l'arn avec du bisulfite
EP2126120A1 (fr) * 2007-03-16 2009-12-02 Human Genetic Signatures Pty Ltd Dosage de l'expression genetique
US7833942B2 (en) 2004-12-03 2010-11-16 Human Genetic Signatures Pty. Ltd. Methods for simplifying microbial nucleic acids by chemical modification of cytosines
US7846693B2 (en) 2003-09-04 2010-12-07 Human Genetic Signatures Pty. Ltd. Nucleic acid detection assay
US8685675B2 (en) 2007-11-27 2014-04-01 Human Genetic Signatures Pty. Ltd. Enzymes for amplification and copying bisulphite modified nucleic acids
WO2014136870A1 (fr) * 2013-03-08 2014-09-12 国立大学法人熊本大学 Procédé de détection simple pour la modification d'arn et procédé de détection du diabète de type ii à l'aide dudit procédé de détection
JP2016123338A (ja) * 2014-12-26 2016-07-11 いであ株式会社 マイクロｒｎａにおけるメチル化修飾部位を計測する方法
US9732375B2 (en) 2011-09-07 2017-08-15 Human Genetic Signatures Pty. Ltd. Molecular detection assay using direct treatment with a bisulphite reagent
CN114250267A (zh) * 2021-12-13 2022-03-29 南京诺唯赞生物科技股份有限公司 一种构建含修饰位点的rna的测序文库的方法

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WO2004096825A1 (fr) * 2003-05-02 2004-11-11 Human Genetic Signatures Pty Ltd Traitement d'acide nucleique
US20050266458A1 (en) * 2004-04-30 2005-12-01 Applera Corporation Methods and kits for methylation detection
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US5786146A (en) * 1996-06-03 1998-07-28 The Johns Hopkins University School Of Medicine Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
US20060014144A1 (en) * 2001-12-18 2006-01-19 Christensen Ulf B Pseudonucleotide comprising an intercalator
US20070042365A1 (en) * 2003-01-24 2007-02-22 Millar Douglas S Assay for detecting methylation changes in nucleic acids using an intercalating nucleic acid
US20060058518A1 (en) * 2003-01-29 2006-03-16 Frank Bergmann Method for bisulfite treatment
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7846693B2 (en) 2003-09-04 2010-12-07 Human Genetic Signatures Pty. Ltd. Nucleic acid detection assay
US7833942B2 (en) 2004-12-03 2010-11-16 Human Genetic Signatures Pty. Ltd. Methods for simplifying microbial nucleic acids by chemical modification of cytosines
EP2126120A4 (fr) * 2007-03-16 2011-03-30 Human Genetic Signatures Pty Dosage de l'expression génétique
EP2126120A1 (fr) * 2007-03-16 2009-12-02 Human Genetic Signatures Pty Ltd Dosage de l'expression genetique
US8685675B2 (en) 2007-11-27 2014-04-01 Human Genetic Signatures Pty. Ltd. Enzymes for amplification and copying bisulphite modified nucleic acids
AU2008331435B2 (en) * 2007-12-05 2013-01-10 Human Genetic Signatures Pty Ltd Bisulphite treatment of RNA
WO2009070843A1 (fr) * 2007-12-05 2009-06-11 Human Genetic Signatures Pty Ltd Traitement de l'arn avec du bisulfite
US9732375B2 (en) 2011-09-07 2017-08-15 Human Genetic Signatures Pty. Ltd. Molecular detection assay using direct treatment with a bisulphite reagent
WO2014136870A1 (fr) * 2013-03-08 2014-09-12 国立大学法人熊本大学 Procédé de détection simple pour la modification d'arn et procédé de détection du diabète de type ii à l'aide dudit procédé de détection
JPWO2014136870A1 (ja) * 2013-03-08 2017-02-16 国立大学法人 熊本大学 Ｒｎａ修飾の簡易検出法、及び該検出法を用いた２型糖尿病の検査方法
US10526654B2 (en) 2013-03-08 2020-01-07 National University Corporation Kumamoto University Simple detection method for RNA modification, and method for detecting type-II diabetes using said detection method
JP2016123338A (ja) * 2014-12-26 2016-07-11 いであ株式会社 マイクロｒｎａにおけるメチル化修飾部位を計測する方法
CN114250267A (zh) * 2021-12-13 2022-03-29 南京诺唯赞生物科技股份有限公司 一种构建含修饰位点的rna的测序文库的方法

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