WO2008109945A1 - Analysis of ribonucleic acid - Google Patents

Abstract

The present invention relates to assays for RNA methylation comprising treating RNA with an agent that modifies unmethylated cytosine and measuring or detecting the presence or absence of methylated RNA. Preferably, the agent is bisulphite.

Description

ANALYSIS OF RIBONUCLEIC ACID

Technical Field

The present invention relates to assays to detect or determine methyiation of ribonucleic acid (RNA).

Background Art

A number of procedures were available for the detection of specific nucleic acid molecules. These procedures typically depend on sequence-dependent hybridisation between the target DNA and nucleic acid probes which may range in length from short oligonucleotides (20 bases or less) to sequences of many kilobases.

For direct detection, 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 ³²P) 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.

Another method for direct detection of nucleic acid that has been used widely is "sandwich" hybridisation. In this method, 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)). In this process, 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. Most commonly, radioactively or fluorescently-labelled nucleic acids (eg. cDNAs) were used for hybridisation, though other detection systems were employed.

The most widely used method for amplification of specific sequences from within a population of nucleic acid sequences is that of polymerase chain reaction (PCR) (Dieffenbach C and Dveksler G eds. PCR Primer: A Laboratory Manual. Cold Spring Harbor Press, Plainview NY). In this amplification method, 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).

As well as PCR, a variety of other techniques have been developed for detection and amplification of specific sequences. One example is the 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, such as were found in the GSTP1 gene promoter region in prostate cancer, were dependent on PCR amplification of such sequences after bisulphite modification of DNA. In bisulphite- treated 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. PNAS 89: 1827-1831 (1992); Clark SJ, Harrison J, Paul CL and Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22: 2990-2997 (1994)). Thus, after bisulphite treatment, DNA containing 5-methyl cytosine bases will be different in sequence from the corresponding unmethylated DNA. The Frommer et al 1992 results are the basis of the bisulphite method for sequencing 5-methyl cytosine residues in DNA. Several years later this assay was used as the basis of a PCR assay for the methylation status of CpG islands in US 5786146. 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. This approach is limited to circumstances where a significant proportion (generally >10%) of the DNA is methylated at the site and where there is sufficient DNA, usually 10 μg, to allow for detection. Digestion with restriction enzymes whose cutting is blocked by site-specific DNA methylation, followed by PCR amplification using primers that flank the restriction enzyme site(s). This method can utilise smaller amounts of DNA but any lack of complete enzyme digestion for reasons other than DNA methylation can lead to false positive signals. Several years ago, peptide nucleic acids (PNA) in which the entire deoxyribose- phosphate backbone has been exchanged with a structurally homomorphous uncharged polyamide backbone composed of N-(2-aminoethyl)glycine units have been developed (Ray A and Norden B. Peptide nucleic acid (PNA): its medical and biotechnical applications and for the future. FASEB J 14: 1041-1060 (2000)). Methods have been developed utilizing PNA ligands for the sensitive and specific detection of DNA which do not require PCR amplification (WO 02/38801 ). ^■ Recently, a new DNA ligand, intercalating nucleic acid (INA), has been developed which has unique and useful properties.

Although there has been much work carried out on methylation of DNA and its consequences to cell function, to date, methylation of RNA in cells has not been observed or recognized.

The present inventors have discovered that RNA can be methylated and have now developed assays to detect or measure methylation of RNA.

Disclosure of Invention

In a first aspect, 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.

In a second aspect, the present invention provides use of an agent which modifies unmethylated cytosine in an assay to estimate, detect or measure RNA methylation. In a third aspect, the present invention provides an assay for RNA methylation comprising:

(a) treating RNA with an agent that that modifies unmethylated cytosine;

(b) reverse transcribing and amplifying the treated RNA using primers capable of binding to complementary sequences of RNA; and (c) measuring the presence or amount of treated and amplified RNA so as to obtain an indication of RNA methylation.

In a fourth aspect, the present invention provides a method for detecting methylation of a target RNA in a sample comprising:

(a) treating a sample containing RNA with an agent that modifies unmethylated cytosine;

(b) providing to the treated sample a detector ligand capable of distinguishing between methylated and unmethylated cytosine of RNA and allowing sufficient time for a detector ligand to bind to a target RNA; and

(c) detecting binding of the detector ligand to the RNA in the sample such binding is indicative of the extent of methylation of the RNA.

In a fifth aspect, the present invention provides a method for estimating extent of methylation of a target RNA in a sample comprising:

(a) treating a sample containing RNA with an agent that modifies unmethylated cytosine; (b) providing a support to which is bound a capture ligand which is capable of recognising a first part of a target RNA sequence;

(c) contacting the support with the treated sample for sufficient time to allow RNA to bind to a capture ligand such that target RNA in the sample binds to the support via the capture ligand; (d) contacting the support with a detector ligand capable of distinguishing between methylated and unmethylated cytosine of RNA such that the detector ligand binds to any target nucleic acid on the support; and (e) detecting binding of the detector ligand to the support such that the degree or amount of binding is indicative of the extent of methylation of the target nucleic acid. The capture ligand or the detector ligand can be in any suitable form including oligonucleotide, nucleotide analogue, PNA or intercalating nucleic acid (INA). In a sixth aspect, the present invention provides a method for detecting a methylated non CpG-containing RNA comprising:

(a) treating a sample containing RNA with bisulphite to modify unmethylated cytosine to uracil in the RNA;

(b) providing to the treated sample a detector ligand capable of distinguishing between methylated and unmethylated cytosine of RNA; and

(c) detecting the methylated RNA based on the presence or absence of binding of the detector ligand.

In a seventh aspect, 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;

(b) providing a solid support to which is bound a capture ligand capable of recognising a first part of a target RNA sequence;

(c) contacting the support with the treated sample suspected of containing the target RNA such that target RNA in the sample binds to the support via the capture ligand;

(d) contacting the support with a detector ligand capable of distinguishing between methylated and unmethylated cytosine of RNA; and

(e) determining the extent of methylation of the RNA bound to the support by measuring the amount of bound detector ligand. In a preferred form, the RNA is from a microorganism, a prokaryote, an eukaryote, cell, cells or a cell population.

In a preferred form, 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 RNeasy™ and Oligotex™ (Qiagen), StrataPrep™ total (Stratagene), Nucleobond™ (Clontech), RNAgents™ and PolyATract™ systems (Promega) etc. RNA may also be isolated using density gradient centrifugation techniques.

Preferably, 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. Preferably, 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.

Sodium bisulphite (NaHSO₃) reacts readily with the 5,6-double bond of cytosine to form a sulfonated cytosine reaction intermediate which is susceptible to deamination, and in the presence of water gives rise to a uracil sulphite. If necessary, the sulphite group can be removed under mild alkaline conditions, resulting in the formation of uracil. Thus, potentially all cytosines will be converted to uracils. As uracil bases can form only two hydrogen bonds with any complementary base rather than the three hydrogen bonds which cytosines can form, the tendency for the RNA to reform complex secondary structures is greatly reduced. Furthermore the converted RNA sequence, now comprising of only 3 bases, will have less natural self-complementarity than its natural 4 base counterpart. Thus 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. For example, specific probes directed to the target RNA can be derived from part or all of the corresponding transcription unit of interest. Alternatively, 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.

The probes of the invention can be designed to be "substantially" complementary to the RNA to be tested. When 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. For example, 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. Alternative, 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.

The replacement of 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.

In the case of INA probes designed to bind to RNA molecules, 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.

Amplifying the 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. With respect to equivalent sequences capable of hybridizing under high stringency conditions or having a high sequence similarity with nucleic acid molecules employed in the invention, "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. Preferably, the INA probes are prepared as disclosed on PCT/DK02/00876.

It is also possible to amplify treated 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.

In one form, 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.

Another suitable detection system is the use of quantum dot bioconjugates (Chan and Nie 1998 Science 282: 2016-2018). Alternatively, 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. In this latter instance, the attachment of the microsphere signal detection system could be via a poly T sequence as an INA, PNA, LNA or oligonucleotide entity.

As 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.

Other methods of detecting ligands binding to target modified RNA, such as labelling with a suitable radioactive compound or an enzyme coupled with a colour reaction, could also be used for particular applications. 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.

Using 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. In particular, 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.

As the invention can use direct detection methods, 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.

In step (b), 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. As 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.

More preferably, 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. Preferably, 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.

In a preferred form, 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. Typically, the array contains from about 10 to 200,000 capture ligands. It will be appreciated, however, that the array can have any number of capture ligands.

In one form, 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.

In a preferred form 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. Stems 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.

In many situations there is no need to amplify the nucleic acid to obtain the required information thus overcoming potential errors and resulting in a faster and more simple assay amenable to automation. Amplification after capture or nucleic acid selection prior to treatment is also possible for the present invention. .

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. Thus, step (b) allows the capture and concentration of the RNA of interest.

In one preferred form, 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. Alternatively, 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. In a preferred form, the capture ligands are bound to wells of a microtiter plate or a microarray so that multiple assays may be carried out. In order to detect binding of the detector ligand to a target nucleic acid, preferably 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. Preferably, 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. In one preferred form, 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. For example, 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.

The 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.

Preferably the specificity of hybridization to target RNA is used to discriminate between methylated cytosines and unmethylated cytosines. . Using 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.

As the present invention can use direct detection methods, 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.

There are a number of detector systems and instruments available for detecting or measuring fluorescence or radioactivity. Improvements and advancement in instrumentation are being made by a number of manufacturers. It will be appreciated that many different measuring instruments can be used for the present invention. For example, 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. They consist of instruments that are configured for 96 well, 384 well and higher. 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.

Notwithstanding the above, the methods described can be used in conjunction with such amplification procedures if it is necessary to amplify limiting amounts of RNA in order provide enough material for detection. In addition, PCR may be used to selectively amplify RNA that has been captured with a ligand directed to methylated or unmethylated RNA.

In a eighth aspect, 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. In an ninth aspect, 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.

Preferably, the kit contains one or more ligands immobilized to a solid support. The kit may also contain primers for amplifying treated RNA.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of the invention.

In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following drawings and examples. Brief Description of the Drawings

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).

Mode(s) for Carrying Out the Invention

DEFINITIONS

Nucleic acids

The term "nucleic acid" covers the naturally occurring nucleic acids, DNA and RNA. The term "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. The term 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.

Hence the terms "nucleic acid" or "nucleic acid analogues" designate any molecule which essentially consists of a plurality of nucleotides and/or nucleotide analogues and/or intercalator pseudonucleotides. Nucleic acids or nucleic acid analogues useful for the present invention may comprise a number of different nucleotides with different backbone monomer units.

Preferably, 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. Preferably, 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.

Hence, 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. In addition 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. In particular nucleic acids and nucleic acid analogues may comprise one or more intercalator pseudonucleotides.

Within this context "mixture" is meant to cover a nucleic acid or nucleic acid analogue strand comprising different kinds of nucleotides or nucleotide analogues. Furthermore, within this context, "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. By HNA is meant nucleic acids as for example described by Van Aetschot et al.,

1995. By MNA is meant 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.

The term "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). In addition to the main or common bases above, 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, α-L-Ribo-LNA, α-L-Xylo-LNA, β-D-Xylo-LNA, α-D-Ribo-LNA, [3.2.1]- LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, α-Bicyclo-DNA, Tricyclo- DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, β-D- Ribopyranosyl-NA, α-L-Lyxopyranosyl-NA, 2'-R-RNA, α-L-RNA or α-D-RNA, β-D-RNA. 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. However, nucleotides or nucleotide analogues may also be chemically incorporated into a nucleic acid or nucleic acid analogue.

Furthermore 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. Thus it is an important feature of a nucleobase that it can only form stable hydrogen bonds with one or a few other nucleobases, but that it can not form stable hydrogen bonds with most other nucleobases usually including itself. The specific interaction of one nucleobase with another nucleobase is generally termed "base-pairing".

The base pairing results in a specific hybridisation between predetermined and complementary nucleotides. Complementary nucleotides are nucleotides that comprise nucleobases that are capable of base-pairing.

Of the common naturally occurring nucleobases, adenine (A) pairs with thymine (T) or uracil (U); and guanine (G) pairs with cytosine (C). Accordingly, a nucleotide comprising A is complementary to a nucleotide comprising either T or U, and a 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 π-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. However, other sites may be derivatised, as long as the overall base pairing specificity of a nucleic acid or nucleic acid analogue is not disrupted. Finally, when 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. Preferably 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

As used herein, 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.

Corresponding nucleic acids Nucleic acids, nucleic acid analogues, oligonucleotides or oligonucleotides analogues are considered to be corresponding when they are capable of hybridising. Preferably 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

Southern E. M., 1975, J. MoI. Biol. 98:503-517. For such purposes it is routine practise to include steps of prehybridization and hybridization. Such steps are normally performed using solutions containing 6x SSPE, 5% Denhardt's, 0.5% SDS, 50% formamide, 100 μg/ml denatured salmon testis DNA (incubation for 18 hrs at 42⁰C), followed by washing with 2x SSC and 0.5% SDS (at room temperature and at 37⁰C), and washing with 0.1x SSC and 0.5% SDS (incubation at 68⁰C for 30 min), as^' described by Sambrook et al., 1989, in "Molecular Cloning/A Laboratory Manual", Cold Spring Harbor).

Medium stringency conditions as used herein shall denote hybridisation in a buffer containing 1 mM EDTA, 1OmM Na₂HPO₄ H₂O, 140 mM NaCI, at pH 7.0. Preferably, around 1.5 μM of each nucleic acid or nucleic acid analogue strand is provided. Alternatively 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 Na₃PO₄ at pH 7,0.

Alternatively, corresponding 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. Preferably 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

The term cross-hybridisation covers unintended hybridisation between at least two nucleic acids or nucleic acid analogues. Hence the term 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.

Often 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

The term 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 temperature

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. Similarly, 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. Furthermore, 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. In addition, 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.

INA / IPN Definition

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. 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. Whereas, conventional oligonucleotides, oligonucleotide analogues and PNAs have an equal affinity for both RNA and DNA. Thus INAs are the first truly selective DNA binding agents. In addition, INAs have a higher specificity and affinity for complementary DNA that other natural DNA molecules.

In addition, 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.

The intercalator pseudonucleotide is preferably selected from phosphoramidites of 1-(4,4'-dimethoxytriphenylmethyloxy)-3-pyrenemethyloxy-2-propanol. Preferably, 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. 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, α-L-Ribo-LNA, α-L-Xylo-LNA, β-D-Xylo-LNA, α-D-Ribo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi- Bicyclo-DNA, α-Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, β-D-Ribopyranosyl-NA, α-L-Lyxopyranosyl-NA, 2'-R-RNA, α- L-RNA or α-D-RNA, β-D-RNA. In addition 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. In particular nucleic acids and nucleic acid analogues may comprise one or more intercalator pseudonucleotides.

When IPNs are placed in an INA molecule for the specific detection of methylated sites, the present inventor has found that it is useful to avoid placing an IPN between potential CpG sites. This is due to the fact that when a CpG site is split using an IPN the specificity of the resulting INA is reduced. Peptide nucleic acid (PNA)

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. Therefore, 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. (For a general review of the preparation of PNA monomers and oligomers please see: Dueholm et al., New J. Chem. (1997), 21 , 19-31 or Hyrup et. al., Bioorganic & Med. Chem. (1996) 4, 5-23). 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. These differences can be conveniently broken down into biological, structural, and physico-chemical differences. As discussed above and below, these biological, structural, and physico-chemical differences may lead to unpredictable results when attempting to use PNA probes in applications where nucleic acids have typically been employed. This non-equivalency of differing compositions is often observed in the chemical arts.

With regard to biological differences, 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.

Structurally, 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.

Despite its name, 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.

The physico/chemical differences between PNA and DNA or RNA are also substantial. 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.

In summary, because PNAs hybridize to nucleic acids with sequence specificity, PNAs are useful candidates for developing probe-based assays. Importantly, PNA probes are not the equivalent of nucleic acid probes. Nonetheless, even under the most stringent conditions 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. Because the sequences are so closely related, point mutations are some of the most difficult of all nucleic acid modifications to detect using a probe-based assay. Numerous diseases, such as sickle cell anemia and cystic fibrosis, are sometimes caused by a single point mutation of genomic nucleic acid. Consequently, any method, kits or compositions which could improve the specificity, sensitivity and reliability of probe-based assays would be useful in the detection, analysis and quantitation of DNA containing samples.

Sodium bisulphite

Methods for treating nucleic acid with sodium bisuphite can be found in a number of references including Frommer et al 1992, Proc Natl Acad Sd 89:1827-1831 ; Grigg and Clark 1994 BioAssays 16:431-436; Shapiro et al 1970, J Amer Chem Soc 92:422 to 423; Wataya and Hayatsu 1972, Biochemistry 11 :3583 - 3588.

Methods have also been developed by the present applicant to improve or enhance success of bisulphite treatment of nucleic acids. An exemplary protocol for effective bisulphite treatment of nucleic acid is set out below. The protocol results in retaining substantially all DNA treated. This method is ^■ also referred to herein as the Human Genetic Signatures (HGS) method. It will be appreciated that the volumes or amounts of sample or reagents can be varied.

Preferred method for bisulphite treatment can be found in US 10/428310 or PCT/AU2004/000549.

To 2 μg of DNA, which can be pre-digested with suitable restriction enzymes if so desired, 2 μl (1/10 volume) of 3 M NaOH (6g in 50 ml water, freshly made) was added in a final volume of 20 μl. This step denatures the double stranded DNA molecules into a single stranded form, since the bisulphite reagent preferably reacts with single stranded molecules. The mixture was incubated at 37⁰C for 15 minutes. Incubation at temperatures above room temperature can be used to improve the efficiency of denaturation.

After the incubation, 208 μl 2 M Sodium Metabisulphite (7.6 g in 20 ml water with 416 ml 10 N NaOH; BDH AnalaR #10356.4D; freshly made) and 12 μl of 10 mM Quinol (0.055 g in 50 ml water, BDH AnalR #103122E; freshly made) were added in succession. Quinol is a reducing agent and helps to reduce oxidation of the reagents. Other reducing agents can also be used, for example, dithiothreitol (DTT), mercaptoethanol, quinone (hydroquinone), or other suitable reducing agents. The sample was overlaid with 200 μl 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 55⁰C. Alternatively the samples can be cycled in a thermal cycler as follows: incubate for about 4 hours or overnight as follows: Step 1 , 55⁰C / 2 hr cycled in PCR machine; Step 2, 95⁰C / 2 min. Step 1 can be performed at any temperature from about 37⁰C to about 9O⁰C and can vary in length from 5 minutes to 8 hours. Step 2 can be performed at any temperature from about 70⁰C to about 99⁰C and can vary in length from about 1 second to 60 minutes, or longer.

After the treatment with Sodium Metabisulphite, the oil was removed, and 1 μl tRNA (20 mg/ml) or 2 μl glycogen were added if the DNA concentration was low. These 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 μg.

An isopropanol cleanup treatment was performed as follows: 800 μl 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 4⁰C 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.

The pellet 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 μl. Buffer at pH 10.5 has been found to be particularly effective. The sample was incubated at 37⁰C to 95⁰C for 1 min to 96 hr, as needed to suspend the nucleic acids. Detection of methylation of RNA

The discovery that 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:

Metήylation Specific PCR (MSP) . Single Nucleotide Primer Extension (SNuPE)

Combination of Bisuulphite and Restriction Analysis (COBRA) Pyrosequencing

Sequencing including dideoxy, sequencing by polymerisation and ligation based methods

Macro- and Microarray approaches including bead based arrays Antibody based methods for methylC enrichment MethylLight

Real-time PCR including TaqMan, Beacon, Scorpion and other probe based approaches

Ligation mediated methods such as the ligase chain reaction (LCR) Molecular inversion probe type approaches 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,

3SR (self sustained sequence replication), Transcription Mediated Amplification, QB replicase, Helicase Chain Reaction Strand Displacement Amplification Invader and other such approaches

Restriction enzyme approaches Whole genome amplification approaches. Linker mediated amplification approaches

Hybridisation such as Northern, Southern blotting, In-situ Hybridisation Fluorescent In Situ Hybridisation

Hybrid-Capture and other such approaches HeavyMethyl Head-loop amplification Aptamers/Dendrimers.

Agent that modify unmethylated cytosines

Agents suitable for the present invention include bisulphite reagents such as acetate, sodium bisulphite, sodium metabisulphite, guanidinium hydrogen sulphite as described in WO 2005054502. In this regard, 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. Thereby, a un-methylated cytosine is converted to uracil and a methylated cytosine is not converted. Thus, 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. According to the invention the term a "bisulphite reaction", "bisulphite treatment" or "bisulphite method" 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.

The statement that 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.

When referring to unmethylated or methylated cytosines, it can be taken to include cytosines present in a ribonucleotide (ribo-cytosines). Cell lines

Table 1. Cell lines used for RNA analysis

RNA extraction

RNA can be extracted from cells by any suitable means. An example of extraction is set out below:

I. 1 ml of Trizol was added directly to the cells (90% confluent) after removal of media.

II. Samples mixed well and left at room temperature for 5 minutes to dissociate nucleoprotein complexes.

III. 0.5 ml removed into a clean RNase free 1.5 ml centrifuge tube.

IV. The samples were then spun @ 12,000Xg for 10 minutes@ 4⁰C to remove high molecular weight DNA and other contaminants. V. The supernatant removed into a clean tube and 100μl of 100% chloroform added and the samples mixed vigerously by hand for 15 seconds then incubated @ room temperature for 2-3 minutes.

VI. The samples were then spun @ 12,000Xg for 10 minutes© 4⁰C to separate the phases.

VII. The upper aqueous phase was removed into a clean tube ensuring the pipette tip stayed away from the interface and 1 μl of 20mg/ml glycogen added and the samples vortexed.

VIII. An equal volume of 100% isopropanol (0.25ml) was added the tubes vortexed then left @ room temp for 10 minutes.

IX. The samples were then.spun @ 12,000Xg for 10 minutes© 4⁰C to pellet the RNA.

X. The supernatant removed and the pellet washed with 0.75 ml of 80% ethanol to removed inhibitors of the cDNA synthesis reaction, vortexed briefly then spun @ 7,500Xg for 5 minutes® 4⁰C to pellet the RNA. Xl. Step X was repeated a further X1.

XII. The pellet was then spun in a microfuge for 10 seconds the residual ethanol removed and the pellet immediately resuspended in 25 μl 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 ₂60/280/3₁0 was then recorded and the RNA stored at -7O⁰C until required.

Preparation of RNA

A RNA sample was resuspended in 20 μl of nuclease free water after extraction from the desired cells or tissue. The sample was heated at 60-10O⁰C for 2-3 minutes to resolve secondary structure and immediately used in the bisulphite reaction.

Bisulphite treatment of RNA

An exemplary protocol demonstrating the effectiveness of the bisulphite treatment of RNA according to the present invention is set out below. The protocol successfully resulted in retaining substantially all RNA treated. This method of the invention is also referred to herein as the Human Genetic Signatures (HGS) method. It will be appreciated that the volumes or amounts of sample or reagents can be varied.

Two μg of RNA is resuspended in a total of 20 μl RNase free water. The sample was then incubated at 65⁰C for 2 minutes to remove secondary structure. After the incubation, 208 μl 2 M Sodium Metabisulphite pH 5.0 (7.6 g in 20 ml water or 10 mM Tris/1 mM EDTA with 416 μl 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 μl 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 55⁰C. This incubation can be performed at any temperature from about 37⁰C to about 9O⁰C and can vary in length from 5 minutes to 16 hours. After the treatment with Sodium Metabisulphite, the oil was removed, and 1 μl 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 μg.

An isopropanol cleanup treatment was performed as follows: 800 μl 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 4⁰C 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 μl. 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 37⁰C to 95⁰C for 1 min to 96 hr, as needed to suspend the nucleic acids. cDNA synthesis

The following reagents were prepared for each cDNA synthesis reaction in thin- wall 0.2 ml RNase free tubes.

RNA 3 ■5 (1 μg)

Random hexamers (10 μM) 1

Deionised water 2 .5

The contents were mixed and spun briefly in a microfuge. The samples were incubated @ 7O⁰C for 3 minutes to denature the RNA. While the RNA was being denatured the following master mix was prepared:

Per rxn

5x first strand buffer 2

DTT (20 mM) 1 50x dNTP mix 1

Total volume 4 μl

Tubes were removed from the PCR machine and cooled on ice for 2 minutes then spun briefly to collect contents. Samples were then incubated @ 42⁰C for 2 minutes.

0.5 μl of Powerscript Reverse Transcriptase was added per reaction (1.75μl) and the master mix mixed well by pipetting.

4.5 μl of the complete master mix was added to each sample and control tube and the samples then incubated @ 42⁰C for 60 minutes then the samples transferred to ice. 40 μl of 1OmM Tris/1mM EDTA pH 7.6 was added to each sample.

The tubes were heated at 72⁰C for 7 minutes then stored @ -7O⁰C until required.

PCR amplification

PCR amplification was performed on 1 μl of bisulphite treated RNA, PCR amplifications were performed in 25 μl reaction mixtures containing 1 μl of bisulphite- treated genomic DNA, using the Promega PCR master mix, 6 ng/μl of each of the primers. One μl 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 μl of the PCR derived product was mixed with 1 μl 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.

Detection system using beads

Coating Magnetic beads

The INA used for attachment to the magnetic beads can be modified in a number of ways. In this example, 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. However, 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.

Ten μl of carboxylate modified Magnabind™ beads (Pierce) or 100 μl of Dynabeads™ Streptavidin (Dynal) were transferred to a clean 1.5 ml tube and 90 μl of PBS solution added.

The beads were mixed then magnetised and the supernatant discarded. The beads were washed x2 in 100 μl of PBS per wash and finally resuspended in 90 μl of 50 mM MES buffer pH 4.5 or another buffer as determined by the manufactures' specifications. One μl of 250 μM 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.

Ten μl of a freshly prepared 10 mg/ml EDC solution (Pierce/Sigma) is then added, the sample vortexed and incubated at either room temperature or 4°C for up to 60 minutes.

The samples were then magnetised, the supernatant discarded and the beads may be blocked by the addition of 100 μl 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 μl PBS solution.

Hybridisation using the magnetic beads Ten μl of INA coated Magnabind™ beads were transferred to a clean tube and

40 μl of either ExpressHyb™ buffer (Clontech) either neat or diluted 1 :1 in distilled water, or Ultrahyb™ buffer (Ambion), either neat or diluted 1 :1, 1 :2 or 1 :4 in distilled water added or an in house hybridisation buffer. The buffers may also contain either cationic/anionic or zwittergents at known concentration or other additives such as Heparin and poly amino acids.

Sample RNA 1-5 μl was then added to the above solution and the tubes vortexed and then incubated at 55⁰C 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.

Preparation of radio-labelled detector spheres

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 P³² or I¹²⁵ 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.

These beads can then be hybridised with the nucleic acid sample of interest to produce signal amplification. Preparation of fluorescent labelled detector spheres

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.

These beads can then be hybridised with the RNA sample of interest to produce signal amplification.

Preparation of enzyme labelled detector spheres

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.

INA oligomer combinations

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, if required, 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 and aptamers

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 I¹²⁵ or P³² or fluorescent labels such as Cy-3, Cy-5, FAM, HEX, TET, TAMRA or any other suitable fluorescent molecule to enhance signal amplification.

Alternatively 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.

EXAMPLES Primers

Table 2. Primers used to detect RNA methylation in a number of genes.

Gene Primer SEQ ID NO:

CaIc-FI -1 GTTGTTATYG I I I I I GATTTAAGTT 1

Calc-F2-1 GTTAGGTGAGTTTYGAGATTT 2 .

CaIc-RI -3 CACTTCRTCCTCACTAAACRTAAC 3

Calc-R2-3 AACCTTCATCTACACATAATTC 4

CaIc-FI -3 GAATTATGTGTAGATGAAGGTT 5

CaIc-RI -4 ACCCCAATTACAATTTAAAAAAAC 6

CaIc-FI -5 TAATGTGGGTTTTAAAGTTTTTGGT 7

CaIc-RI -6 ACTTATTCTACATTTAACAATAATTC 8

PTGS2-F1-1 GTTGTGATGTTYGTTYGYGTTTTGTTGT 9

PTGS2-R1-3 TACACTATATTTAAAATAAATTTCAA 10

PTGS2-R2-3 TTCACAACRTTCCAAAATCCCTTA 11

PTGS2-F1-3 TAATATTTTTTTTTTTYGAAATGT 12

PTGS2-R1-5 AAACTAATACRTAAAATACTAAAC 13

PTGS2-F1-5 TTAAGATAGATTATAAGYGAGGGTT 14

PTGS2-R1-7 ACCCCACAACAAACCRTAAATACTCAA 15

PTGS2-F1-7 GTATGYGATGTGTTTAAATAGGAGT 16

PTGS2-R1-8 CAACAACAATACRATTTTAATACTA 17

PTGS2-F1-8 ATTGTTGGAATATGGAATTATTTAC 18

PTGS2-R1-10 CCRAAACTTTTCTACCAAAAAAAC 19

PTGS2-R2-10 CTACCATAATTTCACCAAAAATAAC 20

TERT-F1-1 ATTATYGYGAGGTGTTGYYGTTGGTT 21

TERT-F2-1 GGGGTTTTAGGGTTGGYGGTTGGTGT 22

TERT-R1-2 TCCAACAACRCRAAACCRAAAACCAAC 23

TERT-R2-2 ACTACRCACRCTAATAATAAAAAC 24

TERT-F1-2 GTATAAYGAAYGTYGTTTTTTTAGGAAT 25

TERT-F2-2 ATTTTTTTGGGGAAGTATGTTAAGT 26

TERT-R1-3 CAACCAATACAAAAACTTAACCAA 27

TERT-F1-3 GGAGATTAYGTTTTAAAAGAATAGGT 28

TERT-R1-4 AACCTAACTTCCCRATACTACCTA 29

TERT-F1-4 GGTTGYGGTYGATTGTGAATATGGAT 30

TERT-R1-5 CAAACCCTATAAATATCRTCCAAAC 31

TERT-F1-5 GTTTTATTTYGAGGGTGAAGGT 32

TERT-R1-6 ACCTATCCTAAAAAATAATATCRTAC 33

TERT-F1-6 GTTATYGTTAGTATTATTAAATTTTAG 34

TERT-R1-7 ACTAATCTCCTACAAATAAACC 35

TERT-R1-8 CACRACRTAATAACACATAAAAC 36

TERT-R1-9 CAAAACAACRTAAAAAAAATAAAAC 37 Gene Primer SEQ ID NO:

TERT-F1-10 GGTGGATGATTTTTTGTTGGTGAT 38

TERT-F1 -11 ^" GTGAA I I I I I I I GTAG AAG AYG AG GTT 39

TERT-R1-13 CAAAAAAATCTTATAAATATTAATAC 40

TERT-F1-14 ATTAGTAAGTTTGGAAGAATT 41

TERT-F2-14 TTGTTATTTTATTTTGAAAGTTAAG 42

TERT-R1-16 TAACTACRACCTCCAAAACAATCAAC 43

TERT-R2-16 CCAAAATAATCTTAAAATCTAAAAAC 44

TP53-F1-1 GAYGGTGATAYG I I I I I TTGGAT 45

TP53-R1-4 CATTATTCAATATCRTCCAAAAACAAC 46

TP53-R2-4 CTAAAAACTTCATCTAAACCTAAATC 47

TP53-F1-4 AGGGTAGTTAYGGTTTTYGTTTGGGT 48

TP53-R1-6 TTCCTTCCACTCRAATAAAATACTA 49

TP53-R2-6 CTATATCRAAAAATATTTCTATC 50

TP53-F1-6 ATATTTTTYGATATAGTGTGGTGGTGTT 51

TP53-R1-8 CTATACRCCRATCTCTCCCAAAACAAAC 52

TP53-F1-8 GAATTTTYGTAAGAAAGGGGAGTT 53

TP53-R1-10 CTAAATCAAACCCTTCTATCTTAAAC 54

TP53-R2-10 AATAAAAAACTATCAATAAAAAAC 55

RASS-F1-1 GGTGYGATTTTTGTGGYGATTTTATTTG 56

RASS-F1-2 GGTTTTTAGAYGTTTAGGTGGTT 57

RASS-R1-3 TAAACTTACAATCTACAAAAAAAC 58

RASS-R2-3 ACAACRATAATAACAAATAAACTTAC 59

RASS-F1-4 GAGGATTYGGATTYGGAGTTTGAGT 60

RASS-R1-5 CTAAACATTATACTCCTTAATCTTCTAC 61

RASS-F1-6 GATGTTGTTAAGTATTTGTATGTGT 62-

RASS-F2-6 GAAAGTTTTTGGTGGTGGATGATTT 63

RASS-R1-8 CAAAATACRTAAAAAATTATATAATTCAAAC 64

RASS-R2-8 ACAATAAAAATACTTCTACAAAATC 65

R-FLC-1 F1 TGAATTGAGAATAAAAGTAGTTGAT 66

R-FLC-1 F2 TTGTAATGGTTTTATTGAGAAAGT 67

R-FLC-1 R1 ACATACTRTTTCCCATATCAATCAAA 68

R-FLC-1 R2 ATAACTCATAATATAAACCATAATTCAA 69

R-FLC-2F1 TTTGATTGATATGGGAAAYAGTATGT 70

R-FLC-2F2 AAGTTTTGAATTATGGTTTATATTATGAGT 71

R-FLC-2R1 CTCAACAAACTTCAACATAAATTCAA 72

R-FLC-2R2 . ACTAACCAAAACCTRATTC 73

R-FLC-3R1 AAAATTATCAAAAATTTATCCAA 74

R-FLC-3R2 CTAATTAAATAATAAAAAAATCACCA 75 RNA bisulphite treatment

RNA was extracted from samples (cell lines CU = U87MG cell line, CL2 = T98G cell line, CL3 = M059K cell line, CL4 = TT cell line, and others) using Trizol according to the manufacturers instructions. Bisulphite treatment of the samples was carried out as outlined above. Arabidopsis RNA was kindly provided by Dr Ming-Bo Wang (CSIRO Plant Industry).

RNA samples 2-5 μg, 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 μl converted RNA template

1 μl random primer (300 ng/μl) 1 μl dNTPs (10 mM)

Samples were heated at 65⁰C for 5 minutes, then placed immediately on ice for at least one minute, after which the following reagants were added: 4 μl 5x First strand buffer

1 μl RNase OUT (40 U/μl)

1 μl DTT (10OmM)

1 μl Superscript III (200 U/μl)

The samples were reverse transcribed using the following conditions: • 25⁰C, 12 minutes

27⁰C, 2 minutes

29⁰C, 2 minutes

31⁰C, 2 minutes

33°C, 2 minutes 35⁰C, 2 minutes

37⁰C, 30 minutes

45⁰C, 15 minutes

5O⁰C, 5 minutes

75⁰C, 5 minutes Two μl of cDNA was then PCR amplified with primer sets specific for each gene of interest. 2μl of the 1^st round material was then amplified with the inner nested primer sets.

PCR reactions were prepared as follows. Promega 2x master mix 12.5 μl

Forward primer 50 ng

Reverse primer 50 ng

Water up to 23 μl x μl

Two μl of reverse transcribed RNA was then added to the mixes and cycling carried out as below.

95⁰C, 1 minute

45⁰C, 2 minutes 3Ox

65⁰C, 2 minutes

RESULTS

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, and CL4 = TT cell line. Figure 2 shows 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, and 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, and 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. CL1 =

U87MG cell line, CL2 = T98G cell line, CL3 = M059K cell line, and 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 8 shows the detailed methylation status of the gene TERT gene. CL2 = T98G cell line, CL3 = M059K cell line, and 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.

In summary RNA methylation was most commonly observed in the context of CAN (where N = G, A, T or C) unlike DNA methylation that is found most commonly as part of CpG dinucleotides. In addition, 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. Methylation pattern obtained from the FLC gene of Arabidopsis. c c c c a t a a a t a a

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.

USES

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.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

Claims:

1. 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.

2. The assay according to claim 1 wherein the agent is selected from bisulphite or acetate.

3. The assay according to claim 2 wherein the agent is sodium bisulphite.

4. The assay according to any one of claims 1 to 3 wherein the RNA is eukaryote RNA.

5. The assay according to claim 4 wherein the RNA is mRNA.

6. The assay according to any one of claims 1 to 5 further comprising: reverse transcribing and amplifying the treated RNA using primers capable of binding to complementary sequences of RNA.

7. The assay according to any one of claims 1 to 6 wherein detection of methylated RNA is 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.

8. A method for detecting methylation of a target RNA in a sample comprising: treating a sample containing RNA with an agent that modifies unmethylated cytosine; providing to the treated sample a detector ligand capable of distinguishing between methylated and unmethylated cytosine of RNA and allowing sufficient time for a detector ligand to bind to a target RNA; and detecting binding of the detector ligand to the RNA in the sample such binding is indicative of the extent of methylation of the RNA.

9. A method for estimating extent of methylation of a target RNA in a sample comprising: treating a sample containing RNA with an agent that modifies unmethylated cytosine; providing a support to which is bound a capture ligand which is capable of recognising a first part of a target RNA sequence; contacting the support with the treated sample for sufficient time to allow RNA to bind to a capture ligand such that target RNA in the sample binds to the support via the capture ligand; contacting the support with a detector ligand capable of distinguishing between methylated and unmethylated cytosine of RNA such that the detector ligand binds to any target nucleic acid on the support; and detecting binding of the detector ligand to the support such that the degree or amount of binding is indicative of the extent of methylation of the target nucleic acid.

10. The assay according to claim 8 or 9 wherein the agent is selected from a bisulphite or acetate.

11. The assay according to claim 10 wherein the agent is sodium bisulphite.

12. A method for detecting a methylated non-CpG containing RNA comprising: treating a sample containing RNA with bisulphite to modify unmethylated cytosine to uracil in the RNA; providing to the treated sample a detector ligand capable of distinguishing between methylated and unmethylated cytosine of RNA; and detecting the methylated RNA based on the presence or absence of binding of the detector ligand.

13. A method for estimating extent of methylation of a target RNA in a sample, the method comprising: treating a sample containing RNA with bisulphite reagent to modify unmethylated cytosine to uracil; providing a solid support to which is bound a capture ligand capable of recognising a first part of a target RNA sequence; contacting the support with the treated^' sample suspected of containing the target RNA such that target RNA in the sample binds to the support via the capture ligand; contacting the support with a detector ligand capable of distinguishing between methylated and unmethylated cytosine in RNA; and determining the extent of methylation of the RNA bound to the support by measuring the amount of bound detector ligand.

14. The assay according to any one of claims 8 to 13 wherein the ligand is an INA.

15. The assay according to any one of claims 8 to 13 wherein the RNA is eukaryote RNA.

16. The assay according to any one of claims 8 to 13 wherein the RNA is messenger RNA (mRNA), immature mRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering (siRNA) or microRNA (miRNA).

17. The assay according to claim 16 wherein the RNA is mRNA.

18. The assay according to any one of claims 8 to 13 wherein the RNA is prokaryotic RNA.

19. The assay according to claim 18 wherein the RNA is from a bacterium or virus.

20. Use of bisuphite in an assay to estimate, detect or measure RNA methylation in an eukaryote or prokaryote.