WO2008040355A2 - Novel methods for quantification of micrornas and small interfering rnas - Google Patents

Novel methods for quantification of micrornas and small interfering rnas Download PDF

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WO2008040355A2
WO2008040355A2 PCT/DK2007/000429 DK2007000429W WO2008040355A2 WO 2008040355 A2 WO2008040355 A2 WO 2008040355A2 DK 2007000429 W DK2007000429 W DK 2007000429W WO 2008040355 A2 WO2008040355 A2 WO 2008040355A2
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sequence
primer
rna
target
oligonucleotide
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PCT/DK2007/000429
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WO2008040355A3 (en
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Peter Mouritzen
Søren Morgenthaler ECHWALD
Nana Jacobsen
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Exiqon A/S
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to nucleic acids, probes and methods for detection, quantification as well as monitoring the expression of mature microRNAs and small interfering RNAs (siRNAs).
  • the invention furthermore relates to methods for monitoring the expression of other non-coding RNAs, mRNA splice variants, as well as detecting and quantifying RNA editing, allelic variants of single transcripts, mutations, deletions, or duplications of particular exons in transcripts, e.g. alterations associated with human disease, such as cancer.
  • the invention furthermore relates to methods for detection and quantification of a target DNA sequence.
  • the present invention relates to the quantification of target nucleotide sequences in a wide variety of nucleic acid samples anci more specifically to the methods employing the design and use of oligonucleotide probes that are useful for detecting and quanti- fying target nucleotide sequences, especially RNA target sequences, such as mi- croRNA and siRNA target sequences of interest and for detecting differences between nucleic acid samples (e.g., such as samples from a cancer patient and a healthy patient).
  • RNAs such as small nucleolar RNAs, siRNAs, microRNAs and
  • RNAs have been considered as simple molecules that just translate the genetic information into protein. Recently, it has been estimated that although most of the genome is transcribed, almost 97% of the genome does not encode proteins in higher eukaryotes, but putative, non-coding RNAs (Wong et al. 2001 , Genome Research 11 : 1975- 1977).
  • the non-coding RNAs appear to be particularly well suited for regu- latory roles that require highly specific nucleic acid recognition. Therefore, the view of RNA is rapidly changing from the merely informational molecule to comprise a wide variety of structural, informational and catalytic molecules in the cell.
  • miRNAs small non-coding RNA genes
  • the first miRNAs to be discovered were the lin-4 and let-7 that are heterochronic switching genes essential for the normal temporal control of diverse developmental events (Lee et al. 1993, Cell 75:843-854; Reinhart et al. 2000, Nature 403: 901-906) in the roundworm C. elegans.
  • miRNAs have been evolutionar- ily conserved over a wide range of species and exhibit diversity in expression pro- files, suggesting that they occupy a wide variety of regulatory functions and exert significant effects on cell growth and development (Ke et al. 2003, Curr.Opin. Chem. Biol. 7:516-523). Recent work has shown that miRNAs can regulate gene expression at many levels, representing a novel gene regulatory mechanism and supporting the idea that RNA is capable of performing similar regulatory roles as proteins. Under- standing this RNA-based regulation will help us to understand the complexity of the genome in higher eukaryotes as well as understand the complex gene regulatory networks.
  • miRNAs are 21-25 nucleotide (nt) RNAs that are processed from longer endogenous hairpin transcripts (Ambros et al. 2003, RNA 9: 277-279). To date more than 719 mi- croRNAs have been identified in humans, worms, fruit flies and plants according to the miRNA registry database hosted by Sanger Institute, UK, and many miRNAs that correspond to putative genes have also been identified. Some miRNAs have multiple loci in the genome (Reinhart et al. 2002, Genes Dev. 16: 1616-1626) and occasion- ally, several miRNA genes are arranged in tandem clusters (Lagos-Quintana et al. 2001 , Science 294: 853-858).
  • miRNAs are single-stranded RNAs of about 21-25 nt.
  • miRNAs match precisely the genomic regions that can potentially encode precursor RNAs in the form of double-stranded hairpins.
  • miRNAs and their predicted precursor secondary structures are phylogenetically conserved.
  • miRNAs are cleaved by Dicer from the hairpin pre- cursor in the form of duplex, initially with 2 or 3 nt overhangs in the 3' ends, and are termed pre-miRNAs. Cofactors join the pre-miRNP and unwind the pre-miRNAs into single-stranded miRNAs, and pre-miRNP is then transformed to miRNP.
  • miRNAs can recognize regulatory targets while part of the miRNP complex.
  • miRNP RNA-induced silencing complex
  • RISC RNA-induced silencing complex
  • pre-miRNAs The structure of pre-miRNAs is consistent with the observation that 22 nt RNA duplexes with 2 or 3 nt overhangs at the 3' ends are beneficial for reconstitution of the protein complex and might be required for high affinity binding of the short RNA duplex to the protein components (for review, see Ke et al. 2003, Curr.Opin. Chem. Biol. 7:516-523).
  • miRNAs play crucial roles in eukaryotic gene regulation.
  • Other miRNAs are thought to interact with target mRNAs by limited complementary and suppressed translation as well (Lagos-Quintana et al. 2001, Science 294: 853-858; Lee and Ambros 2001 , Science 294: 858-862).
  • SMA spinal muscular atrophy
  • SMA spinal muscular atrophy
  • SMA a paediatric neurodegenerative disease caused by reduced protein levels or loss-of-function mutations of the survival of motor neurons (SMN) gene
  • SMA motor muscular atrophy
  • Two proteins (Gemin3 and Gemin4) that are part of the SMN complex are also com- ponents of miRNPs, whereas it remains to be seen whether miRNA biogenesis or function is dysregulated in SMA and what effect this has on pathogenesis.
  • FXMR fragile X mental retardation
  • FMRP fragile X mental retardation protein
  • RNA from fly embryos, worms or HeLa cells have been size fractionated so that only molecules 25 nucleotides or smaller would be captured (Moss 2002, Curr.Biology 12: R138-R140).
  • Synthetic oligomers have then been ligated directly to the RNA pools using T4 RNA ligase. Then the sequences have been reverse-transcribed, amplified by PCR 1 cloned and sequenced (Moss 2002, Curr.Biology 12: R138-R140).
  • the genome databases have subsequently been queried with the sequences, confirming the origin of the miRNAs from these organisms as well as placing the miRNA genes physically in the context of other genes in the genome.
  • a recent report used cDNA microarrays to monitor the expression of miRNAs during neuronal development with 5 to 10 ⁇ g aliquot of input total RNA as target, but the mature miRNAs had to be separated from the miRNA precursors using micro concentrators prior to microarray hybridizations (Krichevsky et al. 2003, RNA 9: 1274-1281).
  • a PCR approach has also been used to determine the expression levels of mature miRNAs (Grad et al. 2003, MoI. Cell 11 : 1253-1263). This method is useful to clone miRNAs, but highly impractical for routine miRNA expression profiling, since it involves gel isolation of small RNAs and ligation to linker oligonucleotides.
  • Schmittgen et al. (2004, Nucleic Acids Res. 32: e43) describe an alternative method to Northern blot analysis, in which they use real-time PCR assays to quantify the expression of miRNA precursors.
  • the disadvantage of this method is that it only allows quantification of the pre- cursor miRNAs, which does not necessarily reflect the expression levels of mature miRNAs.
  • the biggest challenge in measuring the mature miRNAs as well as siRNAs using real-time quantitative PCR is their small size of the of 21-25 nt.
  • the described method of invention addresses the aforementioned practical problems in detection and quantification of small RNA molecules, miRNAs as well as siRNAs, and aims at ensuring the development of flexible, convenient and inexpensive assays for accurate and specific quantification of miRNA and siRNA transcripts.
  • RNA splicing not only provides functional mRNA, but is also responsible for generating additional diversity. This phenomenon is called alternative splicing, which results in the production of different mRNAs from the same gene.
  • the mRNAs that represent isoforms arising from a single gene can differ by the use of alternative exons or retention of an intron that disrupts two exons. This process often leads to different protein products that may have related or drastically different, even antagonistic, cellular functions.
  • the detection of the detailed structure of the transcriptional output is an important goal for molecular characterization of a cell or tissue. Without the ability to detect and quantify the splice variants present in one tissue, the transcript content or the protein content cannot be described accurately. Molecular medical research shows that many cancers result in altered levels of splice variants, so an accurate method to detect and quantify these transcripts is required. Mutations that produce an aberrant splice form can also be the primary cause of such severe diseases such as spinal muscular dystrophy and cystic fibrosis.
  • RNA-mediated gene regulation is widespread in higher eukaryotes and complex genetic phenomena like RNA interference, co-suppression, transgene silencing, im- printing, methylation, and possibly position-effect variegation and transvection, all involve intersecting pathways based on or connected to RNA signalling (Mattick 2001; EMBO reports 2, 11: 986-991).
  • RNA signalling Mattick 2001; EMBO reports 2, 11: 986-991
  • Recent studies indicate that antisense transcription is a very common phenomenon in the mouse and human genomes (Oka- zaki et al. 2002; Nature 420: 563-573; YeNn et al. 2003, Nature Biotechnol.).
  • antisense modulation of gene expression in eukaryotic cells e.g. human cells appear to be a common regulatory mechanism.
  • the present invention provides a method for quantification of non-coding antisense RNAs, as well as a method for highly accurate mapping of the overlapping regions between sense-anti
  • the present invention solves the current problems faced by conventional approaches to homogeneous assays outlined above by providing a method for the design, synthesis and combined use of novel oligonucleotide tagging probes and detection probes with sufficient sequence specificity and high affinity to short nucleic acid targets, e.g. RNA target sequences- so that they are unlikely to detect a random RNA target molecule and also unlikely to detect pre-mature RNA molecules.
  • tagging probes contain a sequence, anchored to the tagging probes, essential as priming sites for subsequent amplification of the nucleic acids by polymerase chain reaction in real-time quantitative PCR assays.
  • the method of invention utilizes two anchored tagging probes, each designed in combination to detect a complementary target sequence, e.g. a short RNA sequence, where the first tagging probe hybridizes to a first region within a target sequence and the second tagging probe hybridizes to a second region within the same complementary target sequence, e.g. a short RNA target sequence that is adjacent to the first region.
  • one of the tagging probes is 5' phosphorylated enabling covalent coupling of the two contiguous tagging oligonucleotide probes hybridized to the complementary target sequence by a ligase to form a single oligonucleotide sequence.
  • the background in the hybridization to the target RNA sequence in complex nucleic acid samples is eliminated by the use of two tagging probes, where the hybridization of both probes to the complementary target sequence, e.g. short RNA target sequence is required for the covalent joining of the two probes.
  • the method furthermore takes the advantage of substitution of the recognition sequences with high-affinity nucleotide analogues, e.g. LNA, for sensitive and specific hybridization to short target sequences, e.g. miRNAs or siRNAs.
  • the ligation reaction is followed by quantitative real-time PCR of the target sequence, e.g.
  • the detection probe is substituted with duplex- stabilizing, high-affinity nucleotide analogues, e.g. LNA, and preferably oxy-LNA, to allow use of short detection probes in the real-time quantitative PCR assay.
  • duplex- stabilizing, high-affinity nucleotide analogues e.g. LNA, and preferably oxy-LNA
  • the covalent joining of the tagging probes hybridized to the target ribonucleic acid in the nucleic acid sample is carried out using a thermostable ligase, which allows repetitive cycles of denaturation, annealing and ligation at ele- vated temperatures to be carried out in the target sequence tagging reaction, thus generating a plurality of covalently joined template molecules for the subsequent real-time quantitative PCR assay.
  • the annealing temperature is adjusted so as to allow discrimination between highly homologous target ribonucleic acids in complex nucleic acid samples.
  • the annealing temperature is adjusted to allow single mismatch discrimination between highly homologous target sequences.
  • the second tagging probe designated as 2 nd strand tagging probe, is designed so that its recognition sequence is complementary to the reverse transcriptase-extended nucleotide sequence corresponding to the 5'-end of the mature microRNA or siRNA or located 5' to the RNA edited nucleotide, splice junction, single nucleotide polymorphism or point mutation in the ribonucleic acid tar- get sequence
  • the 2 nd strand tagging probe is used as anchored primer to generate the second strand complementary to the primer extension product. The specificity of the reaction is based on the sequential use of the two anchored tagging probes, hybridising to complementary 3'-end and 5'-end regions of the target RNA and complementary DNA sequences, respectively.
  • the recognition se- quence of the RT tagging probe is modified with duplex-stabilizing, high-affinity nucleotide analogues e.g. LNA, and preferably oxy-LNA, to allow use of high-stringency primer annealing conditions.
  • the recognition sequences of both tagging probes are modified with duplex-stabilizing, high-affinity nucleotide analogues e.g.
  • a second strand synthesis is performed as a first step of a real-time PCR to amplify the reverse transcription product produced in the reverse transcription reaction taking place prior to the real time PCR.
  • This first step of the real-time PCR uses a gene specific forward primer with a 3'-recognition nucleotide sequence, which is sequence identical with the target ribonucleic acid sequence, here the mature miRNA.
  • the 5'-sequence of the gene specific forward primer con- tains a universal forward primer sequence. Quantification of the resulting nucleic acids by real-time PCR is performed using a universal forward primer and so called scorpion primer as the reverse primer.
  • the universal forward primer contains sequence identical to the 5'-sequence of the gene specific forward primer and the 3'- region of the scorpion primer contains a sequence stretch complementary to the RNA oligonucleotide adaptor ligated to the target ribonucleic acid sequence.
  • the 5'-region of the scorpion primer contains a detection moiety consisting of a sequence stretch complementary to the target ribonucleic acid sequence.
  • amplification of a mature miRNA results in a short amplicon where the short distance between the scorpion primer and target ribonucleic acid sequence of the ma- ture miRNA facilitates binding of the scorpion primer detection moiety to the target ribonucleic acid sequence. This causes unfolding of the scorpion primer and thereby increased fluorescence.
  • amplification of a precursor miRNA results in a amplicon where the longer distance between the scorpion primer and target ribonucleic acid sequence of the mature miRNA prevents binding of the scorpion primer detection moiety to the target ribonu- cleic acid sequence. Therefore unfolding of the scorpion primer is prevented and fluorescence remains unchanged.
  • Fig. 5 shows a schematic presentation of one method of the invention for quantification of microRNAs by sequence-specific real-time quantitative RT-PCR.comprising the following steps:
  • the 5'-sequence of the Forward primer contain a universal forward primer binding site.
  • FIG. 6 shows a schematic presentation of one method of the invention for quantification of microRNAs by sequence-specific real-time quantitative RT-PCR.comprising the following steps:
  • RNA oligonucleotide adaptor sequence a sequence in the RNA oligonucleotide adaptor sequence.
  • the 5'-sequence of the oligonucleotide RT-primer contain a universal reverse primer binding site.
  • RNA oligonucleotide adaptor sequence as primer binding site for the oligonucleotide RT-primer.
  • the 5'-sequence of the Forward primer contain a universal forward primer binding site.
  • the Forward primer in d) is present in a low concentration to prevent mis-priming whereas the set of universal forward and reverse primers in e) are present in normal concentration.
  • Fig. 7 Bioanalyzer profiles of real-time PCR end-products resulting from use of For- ward primers with different lengths of the sequence which is complementary to the first strand synthesis consisting of reverse transcribed miRNA sequence - here hsa- mir-92 - ligated to RNA adaptor.
  • the used forward primer had 9, 13, or 17 nucleotides of their sequence complementary to the reverse transcription product.
  • the Bioanalyzer profile shows that the priming with the forward primer having only 9 nucleo- tides of complementarity is unspecific with two bands being visible. In contrast, specificity is obtained by increasing the number of complementary nucleotides from 9 to 13 and 17. The increased specificity is shown by the presence of a single band of correct size on the Bioanalyzer profile.
  • dNTP means a mixture of 2'-deoxyadenosine-5'-triphosphate, 2'- deoxycytidine-5'-triphosphate, 2 1 -deoxyguanosine-5'-triphosphate, and 2'- deoxythymidine-5'-triphosphate
  • RT-primer refers to a primer, comprising a recognition sequence, complementary to a sequence in the target deoxyribonucleic and/or ribonucleic acid sequence, e.g. to the 3'-end of the mature microRNA or siRNA, or to an RNA-DNA chimerical moiety, or to a sequence located 3' to a RNA-edited nucleotide, splice junction, single nucleotide polymorphism or point mutation in the target ribonucleic acid sequence, and an anchor sequence essential for subsequent capture or amplification by PCR.
  • the said RT-primer is used as an anchored primer in a reverse transcription reaction to generate a primer extension product, complementary to the target RNA sequence using a reverse transcriptase enzyme.
  • Capture probes or “capture probe” refer to a probe(s), comprising a recognition sequence, complementary to the target sequence, e.g. a short RNA target sequence, and an anchor sequence essential for subsequent capture, reverse tran- scription reaction, or amplification by PCR.
  • the anchor sequence function as priming sites for the RT- or PCR primers in subsequent reverse transcription reaction, realtime PCR, or as tags for capture assays.
  • linker means a thermochemically and photochemi- cally non-active distance-making group that is used to join two or more different nu- cleotide moieties of the types defined above.
  • Linkers are selected on the basis of a variety of characteristics including their hydrophobicity, hydrophilicity, molecular flexibility and length (e.g. see Hermanson et. al., “Immobilized Affinity Ligand Techniques", Academic Press, San Diego, California (1992), p. 137-ff).
  • the length of the linkers is less than or about 400 angstroms, in some applications preferably less than 100 angstroms.
  • the length of the linker may vary, taking into consideration the desired or necessary positioning and spatial orientation of the "ac- tive/functional" part of the group in question in relation to the 5- or 6-membered ring.
  • the linker includes a chemically cleavable group. Examples of such chemically cleavable groups include disulphide groups cleavable under reductive conditions, peptide fragments cleavable by peptidases, etc.
  • a "solid support” may be chosen from a wide range of polymer materials e.g. CPG (controlled pore glass), polypropylene, polystyrene, polycarbonate or polyethylene and is may take a variety of forms such as a tube, a microtiter well plate, a stick, a bead, a particle, a filter etc.
  • the oligonucleotide may be immobilized to the solid support via its 5'- or 3'-end (or via the terminus of a linker attached to the 5'- or 3'-end) by a variety of chemical or photochemical methods usually employed in the immobilization of oligonucleotides or by non-covalent coupling e.g. via binding of a biotinylated oligonucleotide to immobilized streptavidin.
  • U refers to a enzyme unit defined as the amount of enzyme required to convert a given amount reactants to a product using a defined time and temperature.
  • Ligands means something, which binds.
  • Ligands comprise bio- tin and functional groups such as: aromatic groups (such as benzene, pyridine, naph- talene, anthracene, and phenanthrene), heteroaromatic groups (such as thiophene, furan, tetrahydrofuran, pyridine, dioxane, and pyrimidine), carboxylic acids, carboxylic acid esters, carboxylic acid halides, carboxylic acid azides, carboxylic acid hy- drazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines,
  • a cell includes a plurality of cells, including mixtures thereof.
  • a nucleic acid molecule includes a plurality of nucleic acid molecules.
  • Transcriptome refers to the complete collection of transcriptional units of the genome of any species. In addition to protein-coding mRNAs, it also represents non- coding RNAs, such as small nucleolar RNAs, siRNAs, microRNAs and antisense RNAs, which comprise important structural and regulatory roles in the cell.
  • amplicon refers to small, replicating DNA fragments.
  • Sample refers to a sample of cells, or tissue or fluid isolated from an organism or organisms, including but not limited to, for example, skin, plasma, serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs, tumours, and also to samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, recombinant cells and cell components).
  • An “organism” refers to a living entity, including but not limited to, for example, hu- man, mouse, rat, Drosophila, C. elegans, yeast, Arabidopsis thaliana, maize, rice, zebra fish, primates, domestic animals, etc.
  • Tagging probes or “tagging probe” refer to a probe(s), comprising a recognition sequence, complementary to the target sequence, e.g. a short RNA target sequence, and an anchor sequence essential for subsequent capture or amplification by PCR.
  • “Two tagging probes” or a “Pair of tagging probes” refer to two anchored tagging probes, each designed to detect in combination a short complementary target sequence, e.g. a short RNA sequence, where the recognition sequence of the first tagging probe hybridizes to a first region within a target sequence and the recognition sequence of the second tagging probe hybridizes to a second region within the same complementary target sequence, e.g. a short RNA target sequence that is adjacent to the first region.
  • 2 nd strand tagging probe refers to an anchored tagging probe, which recognition sequence is complementary to the reverse transcriptase-extended nucleotide sequence corresponding to the 5'-end of the mature microRNA or siRNA or located 5' to the RNA edited nucleotide, splice junction, single nucleotide polymorphism or point muta- tion in the initial ribonucleic acid target sequence.
  • the 2 nd strand tagging probe is used as anchored primer to generate the second nucleic acid strand, which is complementary to the primer extension product.
  • the specificity of the reaction is based on the sequential use of the two anchored tagging probes with non-overlapping recognition sequences, hybridising to complementary 3'-end and 5'-end regions of the target RNA and complementary DNA sequences, respectively.
  • “Two tagging probes” or a “Pair of tagging probes” refer to two anchored tagging probes, each designed to detect in combination a short complementary target sequence, e.g. a short RNA sequence, where the recognition sequence of the first tagging probe hybridizes to a first region within a target sequence and the recognition sequence of the 2 nd strand tagging probe recognizing a sequence is complementary to the reverse transcriptase-extended nucleotide sequence corresponding to the 5'- end of the mature microRNA or siRNA or located 5 1 to the RNA edited nucleotide, splice junction, single nucleotide polymorphism or point mutation in the initial ribonucleic acid target sequence.
  • the 2 nd strand tagging probe is used as anchored primer to generate the second nucleic acid strand, which is complementary to the primer extension product.
  • the anchor sequences attached to each of the two tagging probes are designed so that they do not cross-hybridize to any target nucleic acid in a given transcriptome or to each other under the hybridization conditions used in the method of invention.
  • the anchor sequences function as priming sites for the PCR primers in subsequent realtime quantitative PCR or as tags for capture assays.
  • the primer is preferably single-stranded for maximum efficiency in amplification by a polymerase or reverse transcriptase, in a suitable buffer ("buffer" includes substituents which are cofactors, or which affect pH, ionic strength, etc.), and at a suitable temperature.
  • buffer includes substituents which are cofactors, or which affect pH, ionic strength, etc.
  • the primer is preferably single-stranded for maximum efficiency in amplification.
  • the amount of primer used in PCR may depend on the experiment. Generally, the two amplification primers should be used in equal concentrations and normal amounts vary from 0.1 ⁇ M to 1 ⁇ M equivalent to 5 - 50 pmol of each primer in a 50 ⁇ l reaction volume.
  • the forward primer is present in a low concentration. Accordingly the concentration ratio between forward primer and the other amplification primer(s) is equal to or less than 0.5, provided that the other amplification primer(s) are present in a normal amount. Preferably the ratio is equal to or less than 0.2 or 0.1 or 0.05 or even less than 0.01.
  • the ratio is 0.2, i.e. the forward primer concentration is 80 nM and the concentration of the other amplification primer is 400 nM.
  • Detection probes or “detection probe” refer to labelled oligonucleotide, which forms a duplex structure with a sequence within the amplified target nucleic acid, e.g. short RNA target sequence, due to complementarity of the probe with a sequence in the target region.
  • the detection probe preferably, does not contain a se- quence complementary to sequence(s) used to prime the polymerase chain reaction. Generally the 3' terminus of the probe will be "blocked” to prohibit incorporation of the probe into a primer extension product.
  • Locking may be achieved by using non- complementary bases or by adding a chemical moiety such as biotin or a phosphate group to the 3 1 hydroxyl of the last nucleotide, which may, depending upon the selected moiety, serve a dual purpose by also acting as a label.
  • a chemical moiety such as biotin or a phosphate group
  • siRNAs refer to_21-25 nt RNAs derived from processing of linear double-stranded RNA.
  • siRNAs assemble in complexes termed RISC (RNA-induced silencing complex) and target homologous RNA sequences for endonucleolytic cleavage. Synthetic siRNAs also recruit RISCs and are capable of cleaving homologous RNA sequences
  • RNA interference refers to a phenomenon where double-stranded RNA homologous to a target mRNA leads to degradation of the targeted mRNA. More broadly defined as degradation of target mRNAs by homologous siRNAs.
  • Recognition sequence refers to a nucleotide sequence that is complementary to a region within the target nucleotide sequence essential for sequence- specific hybridization between the target nucleotide sequence and the recognition sequence.
  • the tagging probes as well as the detection probes of invention contain a target sequence-specific recognition sequence.
  • substituted organic nitroxides or other paramagnetic probes (e.g. Cu 2+ , Mg 2+ ) bound to a biological molecule being detectable by the use of electron spin resonance spectroscopy).
  • paramagnetic probes e.g. Cu 2+ , Mg 2+
  • “Ligation” or “covalent coupling” refers to covalent coupling of two adjacent nucleotide sequences, e.g. the tagging oligonucleotide probe sequences of the invention, to form a single nucleotide sequence.
  • the reaction is catalyzed by the enzyme ligase, which forms a phosphodiester bond between the 5'-end of one nucleotide sequence and the 3'-end of the adjacent nucleotide sequence, e.g. between the two adjacent tagging probes of invention, annealed to their complementary, target nucleic acid sequence.
  • RNA-templated oligonucleotide ligation refers to covalent coupling of two adjacent oligonucleotide probe sequences annealed to a complementary RNA target sequence, to form a single nucleotide sequence.
  • the reaction is catalyzed by the enzyme ligase, which forms a phosphodiester bond between the 5'-end of one nucleo- tide sequence and the 3'-end of the adjacent nucleotide sequence, e.g. between the two adjacent tagging probes of invention.
  • PCR reaction means interchangeable terms used to signify use of a nucleic acid amplification system, which multiplies the target nucleic acids being detected.
  • examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Other methods recently described and known to the person of skill in the art are the nucleic acid sequence based amplification (NASBATM, Cangene, Mississauga, Ontario) and Q Beta Replicase systems.
  • the products formed by said amplification reaction may or may not be monitored in real time or only after the reaction as an end point measurement.
  • nucleic acid refers to primers, probes, oligomer fragments to be detected, oligomer controls and unla- belled blocking oligomers and shall be generic to polydeoxyribonucleotides (contain- ing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), and to any other type of polynucleotide which is an N glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases.
  • oligonucleotide or “nucleic acid” intend a polynucleotide of genomic DNA or RNA, cDNA, semi synthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature; and (3) is not found in nature.
  • a nucleic acid sequence even if internal to a larger oligonucleotide, also may be said to have a 5 1 and 3' ends.
  • the 3' end of one oligonucleo- tide points toward the 5' end of the other; the former may be called the "upstream” oligonucleotide and the latter the "downstream” oligonucleotide.
  • SBC nucleobases Selective Binding Complementary nu- cleobases, i.e. modified nucleobases that can make stable hydrogen bonds to their complementary nucleobases, but are unable to make stable hydrogen bonds to other SBC nucleobases.
  • the SBC nucleobase A' can make-a stable hydrogen bonded pair with its complementary unmodified nucleobase, T.
  • the SBC nucleobase T' can make a stable hydrogen bonded pair with its complementary unmodified nucleobase, A.
  • the SBC nucleobases A' and T' will form an unstable hydrogen bonded pair as compared to the base pairs A'-T and A-T'.
  • a SBC nucleobase of C is designated C and can make a stable hydrogen bonded pair with its complementary unmodified nucleobase G 1 and a SBC nucleobase of G is designated G' and can make a stable hydrogen bonded pair with its complementary unmodified nucleobase C, yet C and G' will form an unstable hydrogen bonded pair as compared to the base pairs C-G and C-G'.
  • a stable hydrogen bonded pair is ob- tained when 2 or more hydrogen bonds are formed e.g. the pair between A' and T, A and T 1 , C and G', and C and G.
  • An unstable hydrogen bonded pair is obtained when 1 or no hydrogen bonds is formed e.g. the pair between A' and T', and C and G'.
  • nucleic acid sequence refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5 1 end of one sequence is paired with the 3' end of the other, is in "antiparallel association.”
  • Bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention include, for example, inosine and 7-deazaguanine. Complementarity may not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases.
  • 5' ⁇ 3' nuclease activity or “5 1 to 3' nuclease activity” refers to that activity of a template-specific nucleic acid polymerase including either a 5' ⁇ 3' ex- onuclease activity traditionally associated with some DNA polymerases whereby nucleotides are removed from the 5 1 end of an oligonucleotide in a sequential manner, (i.e., E. coli DNA polymerase I has this activity whereas the Klenow fragment does not), or a 5' ⁇ 3' endonuclease activity wherein cleavage occurs more than one nucleotide from the 5 1 end, or both.
  • nucleobase covers the naturally occurring nucleobases adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) as well as non-naturally occurring nucleobases such as xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7- deazaxanthine, 7-deazaguanine, N 4 ,N 4 -ethanocytosin, N 6 ,N 6 -ethano-2,6- diaminopurine, 5-methylcytosine, 5-(C 3 -C 6 )-alkynyl-cytosine, 5-fluorouracil, 5- bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanine, inosine and the "non-naturally occurring" nucleobases described in Benner et al., U
  • Universal base refers to a naturally-occurring or desirably a non-naturally occurring compound or moiety that can pair with at least one and preferably all natural bases (e.g., adenine, guanine, cytosine, uracil, and/or thymine), and that has a Tm differential of 15, 12, 10, 8, 6, 4, or 2oC or less as described herein.
  • natural bases e.g., adenine, guanine, cytosine, uracil, and/or thymine
  • LNA monomers also referred to as "oxy-LNA” are LNA monomers which include bicyclic compounds as disclosed in PCT Publication WO 03/020739 wherein the bridge between R 4 and R 2 as shown in formula (I) below together designate - CH 2 -O- or -CH 2 -CH 2 -O-.
  • LNA modified oligonucleotide or "LNA substituted oligonucleotide” is meant a oligonucleotide comprising at least one LNA monomer of formula (I), described infra, having the below described illustrative examples of modifications:
  • P designates the radical position for an internucleoside linkage to a succeeding monomer, or,a 5'-terminal group, such internucleoside linkage or 5'-terminal group optionally including the substituent R 5 .
  • One of the substituents R 2 , R 2* , R 3 , and R 3* is a group P * which designates an internucleoside linkage to a preceding monomer, or a 273'-terminal group.
  • Each of the substituents R r ,.R 2 , R 2* , R 3 , R 4* , R 5 , R 5* , R 6 and R 6* , R 7 , and R 7* which are present and not involved in P, P * or the biradical(s), is independently selected from hydrogen, optionally substituted C 1-12 -alkyl, optionally substituted C 2- i 2 -alkenyl, optionally substituted C 2- i 2 -alkynyl, hydroxy, C ⁇ -alkoxy, C 2- i 2 -alkenyloxy, carboxy, C 1-12 -alkoxycarbonyl, d. ⁇ -alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, aryl- carbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and carbamoyl, mono- and di(C 1-6 -alky
  • Exemplary 5 1 , 3 1 , and/or 2' terminal groups include -H, -OH, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., phenyl or benzyl), alkyl (e.g., methyl or ethyl), alkoxy (e.g., methoxy), acyl (e.g.
  • acetyl or benzoyl aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, ary- loxycarbonyl, aralkoxycarbonyl, acylamino, aroylamino, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, arylthio, het- eroarylthio, aralkylthio, heteroaralkylthio, amidino, amino, carbamoyl, sulfamoyl, al- kene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monometh
  • references herein to a nucleic acid unit, nucleic acid residue, LNA monomer, or similar term are inclusive of both individual nucleoside units and nucleotide units and nucleoside units and nucleotide units within an oligonucleotide.
  • a “modified base” or other similar terms refer to a composition (e.g., a non-naturally occurring nucleobase or nucleosidic base), which can pair with a natural base (e.g., adenine, guanine, cytosine, uracil, and/or thymine) and/or can pair with a non- naturally occurring nucleobase or nucleosidic base.
  • a natural base e.g., adenine, guanine, cytosine, uracil, and/or thymine
  • the modified base provides a T m differential of 15, 12, 10, 8, 6, 4, or 2 0 C or less as described herein.
  • Exemplary modified bases are described in EP 1 072 679 and WO 97/12896.
  • Dual-labelled probe refers to an oligonucleotide with two attached labels. In one aspect, one label is attached to the 5' end of the probe molecule, whereas the other label is attached to the 3' end of the molecule. A particular aspect of the invention contain a fluorescent molecule attached to one end and a molecule which is able to quench this fluorophore by Fluorescence Resonance Energy Transfer (FRET) attached to the other end.
  • FRET Fluorescence Resonance Energy Transfer
  • 5' nuclease assay probes and some Molecular Beacons are examples of Dual labelled probes.
  • “5' nuclease assay probe” refers to a dual labelled probe which may be hydrolyzed by the 5'-3" exonuclease activity of a DNA polymerase.
  • Molecular Beacon refers to a single or dual labelled probe which is not likely to be affected by the 5'-3' exonuclease activity of a DNA polymerase. Special modifications to the probe, polymerase or assay conditions have been made to avoid separation of the labels or constituent nucleotides by the 5'-3" exonuclease activity of a DNA polymerase. The detection principle thus rely on a detectable difference in label elicited signal upon binding of the molecular beacon to its target sequence.
  • the oligonucleotide probe forms an intramolecular hairpin structure at the chosen assay temperature mediated by complementary sequences at the 5'- and the 3'-end of the oligonucleotide.
  • High affinity nucleotide analogue refers to a non-naturally occurring nucleotide analogue that increases the "binding affinity" of an oligonucleotide probe to its complementary recognition sequence when substituted with at least one such high-affinity nucleotide analogue.
  • a probe with an increased "binding affinity" for a recognition sequence compared to a probe which comprises the same sequence but does not comprise a stabilizing nucleotide refers to a probe for which the association constant (K a ) of the probe recognition segment is higher than the association constant of the complementary strands of a double-stranded molecule.
  • the association constant of the probe recognition segment is higher than the dissociation constant (K d ) of the complementary strand of the recognition sequence in the target sequence in a double stranded molecule.
  • Monomers are referred to as being "complementary” if they contain nucleobases that can form hydrogen bonds according to Watson-Crick base-pairing rules (e.g. G with C, A with T or A with U) or other hydrogen bonding motifs such as for example dia- minopurine with T, 5-methyl C with G, 2-thiothymidine with A, inosine with C, pseu- doisocytosine with G, etc.
  • Watson-Crick base-pairing rules e.g. G with C, A with T or A with U
  • other hydrogen bonding motifs such as for example dia- minopurine with T, 5-methyl C with G, 2-thiothymidine with A, inosine with C, pseu- doisocytosine with G, etc.
  • target nucleic acid or “target ribonucleic acid” refers to any relevant nucleic acid of a single specific sequence, e. g., a biological nucleic acid, e. g., derived from a patient, an animal (a human or non-human animal), a plant, a bacteria, a fungi, an archae, a cell, a tissue, an organism, etc.
  • a biological nucleic acid e. g., derived from a patient, an animal (a human or non-human animal), a plant, a bacteria, a fungi, an archae, a cell, a tissue, an organism, etc.
  • the method optionally further comprises selecting the bacteria, archae, plant, non-human animal, cell, fungi, or non-human organism based upon detection of the target nucleic acid.
  • the target nucleic acid is derived from a patient, e.g., a human patient.
  • the invention optionally further includes selecting a treatment, diagnosing a disease, or diagnosing a genetic predisposition to a disease, based upon detection of the target nucleic acid.
  • “Target sequence” refers to a specific nucleic acid sequence within any target nucleic acid.
  • stringent conditions is the “stringency” which occurs within a .range from about T m -5° C. (5° C. below the melting temperature (T m ) of the probe) to about 20° C. to 25° C. below T m .
  • the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences.
  • Hybridization techniques are generally described in Nucleic Acid Hybridization, A Practical Approach, Ed. Hames, B. D. and Higgins, S. J., IRL Press, 1985; Gall and Pardue, Proc. Natl. Acad. ScL, USA 63: 378-383, 1969; and John, et al. Nature 223: 582-587, 1969.
  • the present invention also provides a kit for the isolation, purification, amplification, detection, identification, quantification, or capture of natural or synthetic nucleic acids, where the kit comprises a reaction body and one or more LNA modified oligonucleotides (oligomer) as defined herein.
  • the LNA modified oligonucleotides are pref- erably immobilised onto said reactions-body.. - -
  • the reaction body is preferably a solid support material, e.g. selected from borosilicate glass, soda-lime glass, polystyrene, polycarbonate, polypropylene, polyethylene, polyethyleneglycol terephthalate, polyvinylace- tate, polyvinylpyrrolidinone, polymethylmethacrylate and polyvinylchloride, preferably polystyrene and polycarbonate.
  • a solid support material e.g. selected from borosilicate glass, soda-lime glass, polystyrene, polycarbonate, polypropylene, polyethylene, polyethyleneglycol terephthalate, polyvinylace- tate, polyvinylpyrrolidinone, polymethylmethacrylate and polyvinylchloride, preferably polystyrene and polycarbonate.
  • the reaction body may be in the form of a specimen tube, a vial, a slide, a sheet, a film, a bead, a pellet, a disc, a plate, a ring, a rod, a net, a filter, a tray, a microtitre plate, a stick, or a multi-bladed stick.
  • a written instruction sheet stating the optimal conditions for use of the kit typically accompanies the kits.
  • the present invention relates to the use of an oligonucleotide for the isolation, purification, amplification, detection, identification, quantification, or capture of microRNA or small interfering RNAs characterized in that the oligonucleotide contains a number of nucleoside analogues. More particular the present invention provides methods for detection and quantification of microRNA or small interfering RNAs having a high sensitivity and good selectivity. According to the invention the quantification of microRNA and small interfering RNAs is detectable at levels of from 10 fmol to 10 amol RNA target or less (10 zmol) in the sample corresponding to RNA target concentration of from 100 pM to 10 fM or less (1O aM).
  • the detection of binding is either direct by a measurable change in the properties of one or more of the labels following binding to the target (e.g. a molecular beacon type assay with or without stem structure) or indirect by a subsequent reaction follow- ing binding, e.g. cleavage by the 5' nuclease activity of the DNA polymerase in 5' nuclease assays.
  • the detection probe is yet another novel component of the present invention. It comprises a short oligonucleotide moiety which sequence has been selected to enable specific detection of the short amplified DNA molecules corresponding to the target sequence in the core segment and the anchored sequences used as annealing sites for the PCR primers.
  • novel, short detection probes designed to detect target sequences are enabled by the discovery that very short 8 - 12-mer LNA-DNA chimeric, mix-mer probes are compatible with real-time PCR based assays.
  • modified or nucleobase analogues, nucleosidic bases or nucleotides are incorporated in the tagging probes as well as the detection probe, possibly together with minor groove binders and other modifications, that all aim to stabilize the duplex formed between the probes and the target molecule so that the shortest possible probe sequences can be used to hybridized and detect the target molecules.
  • the modi- fications are incorporation of LNA residues to reduce the length of the detection probe to 8 or 9 or 10 or 11 or 12 to 14 nucleotides while maintaining sufficient stability of the formed duplex to be detectable under standard real-time PCR assay conditions.
  • the target recognition sequences in one or both tagging probes for the ligation reaction or the recognition sequence in the RT tagging probe or the recognition sequences in both the RT tagging probe and the 2 nd strand tagging probe for the RT-PCR reaction are substituted with LNA monomers at every second, every third or every fourth nucleotide position with at least one DNA nucleotide at the 3'-ends of both probes, respectively, allowing highly specific and sensitive hybridization even at elevated temperatures due to the in- creased duplex stability of LNA modified oligonucleotide probes to their complementary target molecules, particularly target RNA molecules.
  • microRNA molecules occur both as longer (over 70 nucleotides) pricursor and precursor molecules as well as in the active form of mature miRNAs (17-25 nu- cleotides).
  • One challenge in the detection of microRNA molecules is to detect the mature form of the molecule only, which is a 17-25 bp long single strand RNA molecule.
  • the mature miRNA functions as a primer, i.e. the miRNA is hybridized to a template and extended by an enzyme capa- ble of RNA-primed DNA-directed DNA synthesis.
  • the detection relies on the occurence of this extension and furthermore the occurence of extension relies on having an -OH termination at the 3'end of the miRNA available at the expected distance from the annealing site to the template, which is used to ensure detection of processed mature miRNA molecules only.
  • the principle of using the target (in this case miRNAs) as a primer in the detection reaction can be applied to other detection formats using other targets (both DNA and RNA).
  • RNA molecules such as microRNA molecules are very short and do not accommodate placement of primers for both reverse transcriptase, PCR am- plification and optionally a labelled detection probe for amplification and detection by PCR.
  • One solution for accommodating this is, according to the present invention, to append additional sequence to the microRNA, preferably by a method that enables the design of mature-specific assays.
  • such sequence(s) may be appended by means of providing (by sequence specific hybridisation) a template for a polymerase-reaction to the microRNA, and providing a polymerase (e.g. a Klenow polymerase) and nucleotides to allow extension, leading to the appending to the mature microRNA of a sequence similar in part to that of the provided template.
  • a polymerase e.g. a Klenow polymerase
  • Such appended sequences may accommodate in part primers for reverse transcriptase, for PCR amplification or for a labelled detection probe, alone or in combination with the nucleic acid sequence of the microRNA.
  • Another means of appending additional sequence may be that of a ligation reaction.
  • an adaptor nucleic acid sequence may be attached to either the 3'-end, the 5'- end or both ends of the microRNA molecule by means of a ligation reaction.
  • a ligation reaction may be assisted by providing a "bridging" nucleic acid sequence comprising a nucleotide sequence specific for a terminal part of a mature target RNA sequence and a nucleotide sequence specific for terminal part of said adapter molecule such that the mature RNA target and said adaptor molecule are place in close vicinity to each other upon sequence specific hybridisation.
  • sequence appended by ligation may accommodate in part primers for reverse transcrip- tase, for PCR amplification or for a labelled detection probe, alone or in combination with the nucleic acid sequence of the microRNA.
  • Yet another means of appending additional sequence to a target small RNA molecule may be that of a template-independent polymerase reaction.
  • a sample of small target RNA molecules are subjected to a polymerase re- action, providing a polyA tail to all microRNAs present in the sample. This could for example be performed by using a polyA polymerase.
  • a sample of small target RNA molecules are subjected to a terminal transferase enzyme reaction, capable of providing an A, C, G or T polynucleotide tail to all microR- NAs present in the sample when respective dATP, dCTP, dGTP or dTTPs are added.
  • Such a microRNA sample provided with a nucleotide tail of similar nucleotides may be converted to cDNA by using a primer comprising the complementary similar nucleotides in a reverse transcriptase reaction, hence providing a cDNA sample of mi- croRNAs with an appended polynucleotide tail of similar nucleotides.
  • the RT-primer may also be specific for a specific mi- croRNA or a group or family of microRNAs.
  • Such a cDNA sample may subsequently serve a template for a PCR amplification reaction using primers specific for specific microRNA sequences, encompassed within the mature microRNA sequence or partly overlapping the sequence appended by means of a template independent polymerase reaction.
  • a broad aspect of the invention thus relates to a method for quantitative determination of a short-length RNA (which can be any of the small RNA types described herein), which has a length of at most 100 nucleotides, comprising a) preparing, from a sample comprising said short-length RNA, a template polynucleotide which consists of 1) a single stranded target sequence consisting of the se- quence of said short-length RNA, its correponding DNA sequence or a nucleotide sequence complementary to the sequence of said short-length RNA and 2) a 5' and/or a 3' adjacent nucleotide sequence, b) using said template polynucleotide in a reverse transcription or a nucleotide po- lymerization to obtain a strand of cDNA, and c) performing a quantitative real-time PCR (qPCR) including as template(s) said cDNA and optionally the template polynucleotide.
  • qPCR quantitative real-
  • This aspect of the invention reflects the underlying concept of the invention, namely that specific detection of short-length RNA can be accomplished by ensuring a rela- tively high degree of specificity in all of steps a to c and that the specificity in each step adds to the general specificity of the method.
  • One main characteristic is the provision of the template polynucleotide in step a, where said template includes appended sequences which can serve as "handles" for primers in the subsequent steps, thus providing space for all primers necessary and for the detection probes used.
  • these "handles" can be both specific and non-specific for the short-length RNA one desires to quantify - in the case of specific sequences, these are appended in a reaction that preferentially or specifically will add the sequences to the short-length RNA but not to sequences which include the short-length RNA.
  • RNA sequence can correspond to a DNA sequence if the complementary sequence to the DNA sequence can be transcribed to the RNA sequence in question.
  • cDNA in this context means a DNA fragment which is obtained by means of either reverse transcription of the template polynucleotide or by means of nucleotide polymerization (such a DNA polymerization) based on the template nucleotide.
  • RNA having lengths of at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, and at most 25 nucleotide residues can conveniently be determined by means of the present methods and kits, but even shorter RNAs such as those having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 nucleotide residues.
  • the short-length RNAs have lengths between 16 and 25 nucleotide residues.
  • oligonucleotides corresponds to or is complementary to a sequence in the 5' or 3' adjacent nucleotide sequence - an embodiment which, especially if both primers relate to the adjacent sequences, benefits from the existence in steps a and b of sequence specific (for the short-length RNA or a sequence derived therefrom) appending of the 5' and/or 3' sequences and/or that step b has utilised an approach specific for the short-length RNA;
  • oligonucleotides corresponds to or is complementary to a contiguous sequence in the template polynucleo- - tide constituted by part of the single stranded target sequence and part of the adjacent 5' or 3' nucleotide sequence - an embodiment, where a relatively high degree of specificity is present in step c due to the specific recognition of part of the short- length RNA (or a sequence derived therefrom) and where it may be advantageous that the 5' or 3 1 nucleotide sequence has been appended based on a sequence spe- cific approach and/or that step b has utilised an approach specific for the short-length RNA; and
  • oligonucleotides wherein one corresponds to a first nucleotide sequence in the single stranded target sequence and the other is complementary to a second nucleotide sequence in the single stranded target sequence - an embodiment, where a high degree of specificity is present in step c due to the specific recognition of the short-length RNA (or a sequence derived therefrom).
  • Said primers used for the qPCR may each independently include a detectable label.
  • the reaction in step (b) utilises a reverse transcription primer or a DNA poymerization primer which corresponds to or is complementary to the sin- gle stranded target sequence or which corresponds to or is complementary to a contiguous sequence in the template polynucleotide constituted by part of the single stranded target sequence and part of the adjacent 5' or 3' nucleotide sequence.
  • the reverse transcription primer or nucleotide polymerization primer is specific for at least one short-length RNA; this reflects the fact that a number of short- length RNAs falls in certain families having a high degree of sequence identity.
  • the appended 5' and/or a 3' adjacent nucleotide sequence is in some embodiments a polynucleotide consisting of identical nucleotides (an effect which can be attained by utilising terminal transferase enzymes for appending the sequence or, alternatively by utilising a polymerase which adds identical nucleotide residues).
  • the single stranded target sequence and the 5' and/or a 3' adjacent nucleotide sequence(s) may be covalently joined but also non-covalently joined - the important issue is whether the template sequence can be subjected to reverse tran- scription or nucleotide polemerization in step b.
  • the 5' and/or a 3' adjacent nucleotide sequence in some embodiments include(s) a detectable label, thus facilitating subsequent detection.
  • the 5' and/or 3' adjacent nucleotide sequence is joined to the single stranded target sequence by an enzymatic reaction, but also non-enzymatic reactions are envisaged.
  • Useful enzymes for adding identical nucleotides include, using the IUBMB Enzyme Nomenclature are provided in the following:
  • Transferases EC 2.7.7.19 (polynucleotide adenylyltransferase), EC 2.7.7.52 (RNA uridylyltransferase), and EC 2.7.7.31 (DNA nucleotidylexotransferase).
  • Ligases EC 6.5.1.1 (DNA ligase (ATP)), EC 6.5.1.2 (DNA ligase (NAD+)), and EC 6.5.1.3 (RNA ligase (ATP)).
  • the 5' and/or 3 1 adjacent nucleotide sequence does not occur naturally in the organism from where the sample RNA is derived. This is believed to reduce the risk of detecting irrelevant sequences in the sample. It is preferred that the 5 1 and/or 3' adjacent nucleotide sequence is non-mammalian.
  • step (a) comprises preparation of the template polynucleotide by ligation of the 5' and/or 3 1 adjacent nucleotide sequence to the short-length RNA 1 or step (a) comprises preparation of the template polynucleotide by joining the 5' and/or 3' adjacent nucleotide sequence to the short-length RNA in a terminal transferase reaction, preferably in a poly-A transferase reaction.
  • the ligation can be both sequence specific (e.g. overhang ligation) and blunt-end ligation, but it is preferred to utilise overhang ligation.
  • the method involves annealing, to the short-length RNA, an oligonucleotide in part complementary to the ligase-reactive end of the 5' or 3' adjacent nucleotide sequence and in part complementary to the ligase-reative end of the short-length RNA molecule so as to position the ligase-reactive end of the 5' or 3' adjacent nucleotide sequence directly adjacent to the ligase-reative end of the small RNA molecule to allow overhang ligation.
  • RNA in the sample can be rendered useful for the subsequent steps (which then, on the other hand, should be highly specific). This enables creation of e.g. a non-specific cDNA library which can later be used for the more specific steps in b and c.
  • ligation or the terminal transferase reaction is only performed at the 3' end of the target sequence, but ligation to the 5 1 end of the target sequence can be performed by phosphorylating the 5' end of the target sequence prior to the ligation reaction.
  • it is preferred to block one of the termini since ligases require 3'-hydroxyl and 5'-phosphate in the molecules to be ligated, this is a fairly easy task for the skilled person).
  • the 5' adjacent nucleotide sequence is blocked at its 5' terminus and the 3' adjacent nucleotide sequence is blocked at its 3 1 terminus prior to ligation, and since these two nucleotide sequences are normally added in separate steps, it is avoided that they self-ligate.
  • step (a) The 5' and/or 3' adjacent nucleotide sequence(s) is/are preferentially or exclusively joined to a defined processing state of said short-length RNA in step (a).
  • the means for appending the adjacent nucleotide sequence utilises a sequence coupling step which depends on the presence of a free 3' or 5' end in the short-length RNA (whereby discrimination is introduced over e.g. a pre-mature RNA that includes the same sequence but not in its relevant terminus).
  • the defined processing state of said RNA is the mature state.
  • step (a) may comprise a step of nucleotide polymerization to attach the adjacent nucleotide sequences.
  • the polymerase used for this purpose can be both a template-independent and a template- dependent polymerase.
  • Typcically employed polymerases are DNA polymerases.
  • polymerization which is template specific
  • the polymerization may also consist in addition of a poly-A, poly-G, poly-T or a poly-C tail to the 3' end of the target sequence.
  • the currently preferred embodiments entail use of template specific approaches.
  • different approaches _ have to be used.
  • the present invention also relates to a kit useful in the quantitative determination of mature short- length RNA having a length of at most 100 nucleotides, said kit comprising - the minimum number of reverse transcription primers and/or nucleotide polymeriza- tion primers and/or primers for qPCR and/or oligonucleotide capture probes and/or helper oligonucleotides and/or oligonucleotide probes, which are used in a method described herein, wherein the reverse transcription primers, nucleotide polymerization primers, primers for qPCR, oligonucleotide capture probes, helper oligonucleotides, and oligonucleotide probes share the features described above; and
  • kits for quantitative determination of the mature short-length RNA using the reverse transcription primers and/or nucleotide polymerization primers and/or primers for qPCR and/or oligonucleotide capture probes and/or helper oligonucleotides and/or oligonucleotide probes. All disclosures relating to the provision of kits apply mutatis mutandis do this kit.
  • the kit may further comprise one or more enzymes and other reagents as described herein.
  • enzymes and other reagents as described herein.
  • the following is provided (the reference primers and probes are optional):
  • Oligonucleotide amount 1 assay 10 assays concentration ._ volume
  • Biotinylated LNA capture probe 0.5 pmol 5 pmol 0.5 ⁇ M 1 ⁇ L miR-specific reverse primer 0.1 pmol 1 pmol 0.1 ⁇ M 1 ⁇ L miR-specific forward primer 2.025 pmol 20.25 pmol 0.9 ⁇ M 2.25 ⁇ L miR-specific reverse primer 2.025 pmol 20.25 pmol 0.9 ⁇ M 2.25 ⁇ L miR-specific dual- labeled probe 0.3125 pmol 3.125 pmol 0.25 ⁇ M 1.25 ⁇ L
  • RNA control oligonucleotide 0.01 pmol 0.1 pmol 0.01 ⁇ M 1 ⁇ L
  • DNA control oligonucleotide 0.01 pmol 0.1 pmol 0.01 ⁇ M 1 ⁇ L
  • LNA substituted tagging probes and detection probes are preferably chemically synthesized using commercially available methods and equipment as described in the art (Tetrahedron 54: 3607-30, 1998).
  • the solid phase phosphoramidite method can be used to produce short LNA probes (Caruthers, et al., Cold Spring Harbor Symp. Quant. Biol. 47:411-418, 1982, Adams, et al., J. Am. Chem. Soc. 105: 661 (1983).
  • LNA-containing-probes are typically labelled during synthesis.
  • the flexibility of the phosphoramidite synthesis approach furthermore facilitates the easy production of LNAs carrying all commercially available linkers, fluorophores and labelling- molecules available for this standard chemistry.
  • LNA may also be labelled by enzymatic reactions e.g. by kinasing.
  • Detection probes according to the invention can comprise si ⁇ gle labels or a plurality of labels.
  • the plurality of labels comprise a pair of labels which interact with each other either to produce a signal or to produce a change in a signal when hybridization of the detection probe to a target sequence occurs.
  • the detection probe comprises a fluorophore moiety and a quencher moiety, positioned in such a way that the hybridized state of the probe can be distinguished from the unhybridized state of the probe by an increase in the fluo- rescent signal from the nucleotide.
  • the detection probe comprises, in addition to the recognition element, first and second complementary sequences, which specifically hybridize to each other, when the probe is not hybridized to a recognition sequence in a target molecule, bringing the quencher molecule in sufficient proximity to said reporter molecule to quench fluorescence of the reporter molecule. Hybridization of the target molecule distances the quencher from the reporter molecule and results in a signal, which is proportional to the amount of hybridization.
  • polymerization of strands of nucleic acids can be detected using a polymerase with 5' nuclease activity.
  • Fluorophore and quencher molecules are incorporated into the probe in sufficient proximity such that the quencher quenches the signal of the fluorophore molecule when the probe is hybridized to its recognition sequence.
  • Cleavage of the probe by the polymerase with 5' nuclease activity results in separation of the quencher and fluorophore molecule, and the presence in increasing amounts of signal as nucleic acid sequences
  • Suitable samples of target nucleic acid molecules may comprise a wide range of eu- karyotic and prokaryotic cells, including protoplasts; or other biological materials, which may harbour target nucleic acids.
  • the methods are thus applicable to tissue culture animal cells, animal cells (e.g., blood, serum, plasma, reticulocytes, lymphocytes, urine, bone marrow tissue, cerebrospinal fluid or any product prepared from blood or lymph) or any type of tissue biopsy (e.g.
  • a muscle biopsy e.g., a liver biopsy, a kidney biopsy, a bladder biopsy, a bone biopsy, a cartilage biopsy, a skin biopsy, a pancreas biopsy, a biopsy of the intestinal tract, a thymus biopsy, a mammae biopsy, a uterus biopsy, a testicular biopsy, an eye biopsy or a brain biopsy, e.g., homogenized in lysis buffer), archival tissue nucleic acids, plant cells or other cells sensitive to osmotic shock and cells of bacteria, yeasts, viruses, mycoplasmas, protozoa, rickettsia, fungi and other small microbial cells and the like.
  • amplifying reactions are well known to one of ordinary skill in the art and include, but are not limited to PCR, RT-PCR, LCR, in vitro transcription, rolling circle PCR, OLA and the like. Multiple primers can also be used in multiplex PCR for detecting a set of specific target molecules.
  • the tagging probes as well as the detection probes of the invention are modified in order to increase the binding affinity of the probes for the target sequence by at least two-fold compared to probes of the same sequence without the modification, under the same conditions for hybridization or stringent hybridization conditions.
  • the preferred modifications include, but are not limited to, inclusion of nucleobases, nucleosidic bases or nucleotides that have been modified by a chemical moiety or replaced by an analogue to increase the binding affinity.
  • the preferred modifications may also include attachment of duplex-stabilizing agents e.g., such as minor-groove- binders (MGB) or intercalating nucleic acids (INA).
  • MGB minor-groove- binders
  • INA intercalating nucleic acids
  • the preferred modifications may also include addition of non-discriminatory bases e.g., such as 5- nitroindole, which are capable of stabilizing duplex formation regardless of the nu- cleobase at the opposing position on the target strand.
  • multi-probes composed of a non-sugar-phosphate backbone, e.g. such as PNA, that are capable of binding sequence specifically to a target sequence are also considered as a modification. All the different binding affinity-increasing modifications mentioned above will in the following be referred to as "the stabilizing modification(s)", and the tagging probes and the detection probes will in the following also be referred to as "modified oligonucleotide". More preferably the binding affinity of the modified oligonucleotide is at least about 3-fold, 4-fold, 5-fold, or 20-fold higher than the binding of a probe of the same sequence but without the stabilizing modification(s).
  • the stabilizing modification(s) is inclusion of one or more LNA nucleotide analogs.
  • Probes from 6 to 30 nucleotides according to the invention may comprise from 1 to 8 stabilizing nucleotides, such as LNA nucleotides. When at least two LNA nucleotides are included, these may be consecutive or separated by one or more non-LNA nucleotides.
  • LNA nucleotides are alpha and/or xylo LNA nucleotides.
  • the invention also provides a probe library comprising tagging probes and detection probes with stabilizing modifications as defined above.
  • the detection probes are less than about 20 nucleotides in length and more preferably less than 15 nucleotides, and most preferably about 7 or 8 or 9 or 10 or 11 nucleotides.
  • the tagging probes are less than about 40 nucleotides in length and more preferably less than 35 nucleotides, and most preferably about 20 or 30 nucleotides.
  • the tagging probes ligation reaction and the RT tagging probe and the 2 nd strand tagging probe for the RT-PCR reaction are composed of a high-affinity tagging recognition sequence of less than about 15 nucleotides in length and more preferably less than 14 nucleotides, and most preferably between 6 and 13 nucleotides, and furthermore of an anchored sequence as a primer site for PCR primers of less than about 30 nucleotides in length and more preferably less than 25 nucleotides, and most preferably between 15 to 20 nucleotides.
  • the probe libraries contain- ing labelled detection probes may be used in a variety of applications depending on the type of detection element attached to the recognition element.
  • the problems with existing quantification assays for microRNAs, siRNAs, RNA- edited transcripts, alternative splice variants and antisense non-coding RNAs as outlined above are addressed by the use of the probes of the invention in combination with any of the methods of the invention consisting of a set of RNA tagging probes and detection probes or sets of RNA RT tagging probes combined with 2 nd strand tagging probes and detection probes, selected so as to recognize or detect a majority of all discovered and detected miRNAs, RNA-edited transcripts, siRNAs, alternative splice variants and antisense non-coding RNAs in a given cell type from a given or- ganism.
  • the probe library comprises probes that tag and detect mammalian mature miRNAs, e.g., such as mouse, rat, rabbit, monkey, or human miRNAs.
  • mammalian mature miRNAs e.g., such as mouse, rat, rabbit, monkey, or human miRNAs.
  • probes according to the invention can be adapted for use in 5' nuclease assays, molecular beacon assays, FRET assays, and other similar assays.
  • the detection probe comprises two labels capable of interacting with each other to produce a signal or to modify a signal, such that a signal ora change in a signal may be detected when the probe hybridizes to a target sequence.
  • the two labels comprise a quencher and a reporter molecule.
  • the probe comprises a target-specific recognition segment capable of specifically hybridizing to a target molecule comprising the complementary recognition sequence.
  • a particular detection aspect of the invention referred to as a "molecular beacon with a stem region" is when the recognition segment is flanked by first and second complementary hairpin-forming sequences which may anneal to form a hairpin.
  • a reporter label is attached to the end of one complementary sequence and a quenching moiety is attached to the end of the other complementary sequence.
  • the stem formed when the first and second complementary sequences are hybridized i.e., when the probe recognition segment is not hybridized to its target) keeps these two labels in close proximity to each other, causing a signal produced by the reporter to be quenched by fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the target detection probe comprises a reporter and a quencher molecule at opposing ends of the short target recognition sequence, so that these moieties are in sufficient proximity to each other, that the quencher substantially reduces the signal produced by the reporter molecule. This is the case both when the probe is free in solution as well as when it is bound to the target nucleic acid.
  • a particular detection aspect of the invention referred to as a "5' nuclease assay” is when the detection probe may be susceptible to cleavage by the 5' nuclease activity of the DNA polymerase. This reaction may possibly result in separation of the quencher molecule from the reporter molecule and the production of a detectable signal.
  • probes can be used in amplification-based assays to detect and/or quantify the amplification process for a target nucleic acid.
  • the invention also provides a method, system and computer program embedded in a computer readable medium ("a computer program product") for designing tagging probes and detection probes comprising at least one stabilizing nucleobase.
  • the method comprises querying a database of target sequences (e.g., such as the - miRNA registry at http://www.sanger.ac.uk/Software/Rfam/mirna/index.shtml ) and designing probes which: i) have sufficient binding stability to bind their respective target sequence under stringent hybridization conditions, ii) have limited propensity to form duplex structures with itself, and iii) are capable of binding to and detect- ing/quantifying at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% of all the target sequences in the given database of.
  • target sequences e.g., such as the - miRNA registry at http://www.sanger.ac.uk/Software/Rfam/mirna/index.s
  • the invention also provides a method, system and computer program embedded in a computer readable medium ("a computer program product") for designing the se- quence of nucleotides to implement the capture probe.
  • the method consists of the following steps:
  • the melting temperature is designated "Tm”.
  • the initial guess is based on the miRNA sequence to match a list of suitable reverse primers found by using a primer finding software (primer3). Random sequences are generated to fill up not initialized parts of the capture probe. The random generation is guided by the use of di-nucleotide Tm tables to ensure sequences with Tm in the neighborhood of the aimed Tm value.
  • the iterative improvement will be directed by a scoring function based on the aims and conditions and of di-nucleotide Tm tables. Random changes are made to avoid suboptimal iteration.
  • the invention also provides a method, system and computer program embedded in a computer readable medium ("a computer program product") for designing nucleotide sequences to implement into the dual-labeled probe.
  • the dual-labeled probe is used for detection of a particular miRNA or a particular family of miRNA's with maximal specificity.
  • the dual-labeled probe must fulfill the following conditions: a) A requirement of low self-annealing and low self-hybridization.
  • the method consist of the following steps:
  • Dual-labeled probe matches are scored by how well they fulfil the conditions above.
  • the dual-labeled probes are scored by how well they fulfil the conditions above according to the scoring functions. The specificity score and the scores from the conditions are then used to deside the best nucleotide sequence of dual-labeled probe.
  • the quencher is preferably selected from dark quencher as disclosed in EP Application No. 2004078170.0, in particular compounds selected from 1 ,4-bis-(3-hydroxy- propylamino)-anthraquinone, 1-(3-(4,4'-dimethoxy-trityloxy)propylamino)-4-(3- hydroxypropylamino)-anthraquinone, 1-(3-(2- cyanoethoxy(diisopropylamino)phosphinoxy)propylamino)-4-(3-(4,4'-dimethoxy- trityloxy)propylamino)-anthraquinone (#Q1 ), 1 ,5-bis-(3-hydroxy-propylamino)- anthraquinone, 1-(3-hydroxypropylamino)-5-(3-(4,4'-dimethoxy- trityloxy)propylamino)-anthraquino
  • the high affinity and specificity of LNA modified oligonucleotides is exploited in the sequence specific capture and purification of natural or synthetic nucleic acids.
  • the natural or synthetic nucleic acids are contacted with the LNA modified oligonucleotide immobilised on a solid surface. In this case hybridisation and capture occurs simultaneously.
  • the captured nucleic acids may be, for instance, detected, characterised, quantified or amplified directly on the surface by a variety of methods well known in the art or it may be released from the surface, before such characterisation or amplification occurs, by subjecting the immobilised, modified oligonucleotide and captured nucleic acid to dehybridising conditions, such as for example heat or by using buffers of low ionic strength.
  • the LNA modified oligonucleotide carries a ligand covalently attached to either the 5' or 3' end.
  • the LNA modified oligonucleotide is con- tacted with the natural or synthetic nucleic acids in solution whereafter the hybrids formed are captured onto a solid support carrying molecules that can specifically bind the ligand.
  • the method further comprises calculating stability based on the assumption that the recognition sequence comprises at least one stabilizing nucleotide, such as an LNA molecule.
  • the calculated stability is " used to eliminate probes with inadequate stability from the database of virtual candi- date probes prior to the initial query against the database of target sequence to initiate the identification of optimal probe recognition sequences.
  • the method further comprises calculating the capability for a given probe sequence to form a duplex structure with itself based on the assumption that the sequence comprises at least one stabilizing nucleotide, such as an LNA mole- cule.
  • the calculated propensity is used to eliminate probe sequences that are likely to form probe duplexes from the database of virtual candidate probes.
  • the plurality of probes comprises a nucleotide coupled to or stably associated with at least one chemical moiety for increasing the stability of binding of the probe.
  • the kits according to the invention allow a user to quickly and efficiently develop an assay for different miRNA targets, siRNA targets, RNA-edited transcripts, non-coding antisense transcripts or alternative splice variants.
  • the invention features the design of high affinity oligonucleotide probes that have duplex stabilizing properties and methods highly useful for a variety of target nucleic acid detection, amplification, and quantification methods (e.g., monitoring expression of microRNAs or siRNAs by real-time quantitative PCR).
  • Some of these oligonucleotide probes contain novel nucleotides created by combining specialized features of target nucleic acid detection, amplification, and quantification methods (e.g., monitoring expression of microRNAs or siRNAs by real-time quantitative PCR).
  • the invention also provides improved methods for detecting and quantifying nucleic acids in a complex nucleic acid sample.
  • the human miR-15a microRNA tagging probe with the 3'-end recognition sequence was enzymatically 5'-phosphorylated in a 50 ⁇ l_ reaction using 10 U T4 polynucleo- tide kinase (New England Biolabs (NEB) USA), 400 pmol hsa-miR-15a microRNA probe 1 (EQ15848), and 1 ⁇ T4 DNA ligase buffer (NEB 1 USA).
  • the reaction was incubated 30 min at 37°C and heat inactivated 10 min at 7O 0 C.
  • the kinase was removed by adding 50 ⁇ l_ DECP-treated H 2 O and filtering the reaction through an YM-30 Microcon spin column (Millipore, USA) 3 min 14000* g.
  • the concentration of the phosphorylated tagging probe was determined on a NanoDrop ND-1000 (Nano- Drop technologies, USA).
  • the ligation reaction was performed in 20 ⁇ l_ consisting of 120 nM miR-15a RNA template (EQ15885), 120 nM of each microRNA tagging probe (phosphylated EQ15848 (see above) and EQ15849), 10 mM Tris-HCI pH 7.0 (Ambion.USA), 10 mM MgCI 2 (PE Biosystems, USA), 0.05* T4 DNA ligase buffer [2.5 mM TRIS-HCI 1 0.5 mM MgCI 2 , 0.5 mM DTT, 50 ⁇ M ATP, 1.25 ⁇ g/mL BSA, pH 7.5 @ 25°C; (NEB, USA)].
  • the reaction comprised (50 ⁇ l_) 1 x SYBR® Green PCR Master Mix (Applied Biosystems, USA) 200 nM of M13 forward primer (EQ7396), 200 nM M13 reverse primer (EQ7655) and 2.5 ⁇ l_ ligation reaction (described above). Cycling procedure: 10 min 95°C, 50 cycles of 15 sec 95°C, 1 min 45°C, 1 min 6O 0 C, and finally dissociation 20 min from 60 0 C to 95°C in an ABI Prism® 7000 Sequence Detection System.
  • the reaction (50 ⁇ l_) was 1 * QuantiTect Probe PCR master mix (Qiagen, Germany) 200 nM hsa miR-15a M13 forward primer (EQ15887), 200 nM hsa miR-15a M13 reverse primer (EQ15888), 100 nM LNA sequence-specific probe (EQ15866 or EQ15867), 2.5 ⁇ l_ ligation reaction (described above). Cycling procedure: 15 min 95 0 C, 50 cycles of 20 sec 95°C, 1 min 6O 0 C in an ABI Prism® 7000 Sequence Detection System.
  • dUTP means 2 1 -deoxyuridine-5'-triphosphate
  • Example 1 Gene specific first strand synthesis of microRNAs and real-time quantitative PCR detection
  • RT reverse transcription
  • 20 ⁇ L consisting of 0.5 ⁇ g Brain Total RNA template (Ambion, USA) spiked with 100, 10, 1, or 0.1 fmol synthetic hsa-miR-145 (EQ16901) template, respectively.
  • RNA templates and the GSP-RT primer were mixed and heated 2 min at 95 0 C and quenched on ice.
  • the thermocycler DYADTM MJ Research DNA engine, USA
  • Temperature profile was 30 min 16 0 C, 30 min 37 0 C, 5 min 85 0 C and cooled down to 4 0 C, finally.
  • the sample recovered after centrifugation was diluted to five times the originally RT starting volume (100 ⁇ L in total).
  • the real-time PCR reaction (50 ⁇ L) was performed in 1* QuantiTect Probe PCR Master Mix (Qiagen, Germany), 400 nM Universal forward primer (EQ 15809, Table M), 400 nM Universal reverse primer (EQ15810, Table II), 80 nM miR-specific forward primer (EQ 24037, Table II), 200 nM hsa-miR 145-Probe1 (EQ20317, Table II), 5 ⁇ L of the reverse transcription (RT) reaction (described above), and 0.5 U Uracil DNA Glycosylase (Invitrogen, USA).
  • the hsa-miR-145 (ace. no. MIMAT0000437, miRBase, Sanger Institute) RT reactions were subsequently detected using real time PCR as described above, universal PCR primers, miR-specific forward primer, and LNA-modified dual-labelled detection probe for the human miR-145 using a no template reaction as a negative control.
  • the Ct values using 100, 10, 1, and 0.1 fmol hsa-miR 145 template were 9.2, 12.6, 16.2, and 20.4 for the LNA-modified dual-labelled detection probe (EQ20317), respectively (Fig. 2).
  • the two positive control samples with 0.5 ⁇ g Brain total RNA, 10 fmol synthetic miR-145 template gave 23.5 and 12.9, respectively whereas no Ct values were detectable for the negative control experiments (no RNA and no cDNA template).
  • Example 2 Application of Universal Primers for real-time PCR amplification of reverse transcribed miRNA using the adaptor ligation approach. __
  • a series of reactions were made to which were added different amounts of a synthetic miRNA (EQ15886) corresponding to the mature sequence of hsa-miR-16.
  • the different reactions contained hsa-miR-16 in 10-fold dilutions starting with 100 fmol, 10 fmol, 1 fmol, 0.1 fmol, and 0 fmol as well as a reaction containing 10 fmol without a total RNA background and a reaction without either synthetic or total RNA.
  • Reverse transcription was performed in a reaction volume of 20 ⁇ L consisting of the following components: the 10 ⁇ L of the terminated ligation reaction described above, 35 pmol of DNA RT primer (EQ23791), 1* First strand buffer (50 mM Tris-HCI pH 8.3 at 20 0 C, 75 mM KCI, 3 mM MgCI2; Invitrogen, USA), 1.25 mM of each of dNTP (Applied Biosystems, USA, 10 mM DTT (Invitrogen, USA), 20 U SUPERase-ln (Ambion, USA), and 200 U Superscript Il reverse transcriptase (Invitrogen, USA).
  • the reaction was incubated for 1 h at 42 0 C followed by heating for 15 min at 70 0 C to terminate the reaction.
  • the volume of the reaction was adjusted to 100 ⁇ L by adding 80 ⁇ L of DEPC H2O.
  • Example 3 Scorpion primer mediated detection of reverse transcribed miRNAs obtained with the gene specific first strand synthesis approach or with the adaptor ligation approach.
  • RNA adaptor EQ23336
  • RT-primer EQ23791
  • RNA adaptor EQ24076
  • RT-primer EQ24077
  • Real-time PCR should be performed in 50 ⁇ l_ consisting of 1 * QuantiTect Probe PCR Master Mix (Qiagen, Germany), 80 nM GSP F-PRIMER ⁇ 5 ⁇ l_ of the reverse transcription (RT) reaction (described above), and 0.5 U Uracil DNA Glycosylase (Invi- trogen, USA).
  • One of the following primer pairs may be used for detection in a concentration of 400 nM each:
  • the real-time RT-PCR analysis may be performed on an Opticon real-time PCR in- strument (MJ Research, USA) or other real-time PCR instruments that are able to detect the FITC fluorophore.
  • a method for discrimination of mature and precursor forms of miRNAs using a Scor- pion primer based detection format is described.
  • RNA sample containing miRNAs should be reverse transcribed using the adaptor ligation approach (described in example 002) using the RNA adaptor (EQ23336) together with RT-primer (EQ23791) or RNA adaptor (EQ24076) together with RT-primer (EQ24077). Both RNA adaptor/RT-primer sets facilitate PCR with universal primers.
  • the real-time PCR is performed as described in Example 3 and the assays are expected to be able to discriminate between mature and precursor forms of miRNAs based on the difference in distance between the 3'-extension site of the Scorpion primer and the probe-binding site on the extended Scorpion primer. This distance will be relatively long and suboptimal when Scorpion primers detect precursor forms whereas is it will be short and optimal when Scorpion primers detect mature forms of the miRNAs (Fig. 4).
  • Table II The design of the microRNA tagging probes, synthetic transcription templates and detection probes.
  • RNA was ligated with 100 pmol of activated RNA adaptor* (EQ23336) using 20 U of T4 DNA Ligase (New England Biolabs, USA) in a total volume of 10 ⁇ L consisting of 1X T4 DNA Ligase Reaction Buffer (50 mM Tris- HCI pH 8.0 at 25 0 C 1 10 mM MgCI2, 3.3 mM dithiothreitol, 10 ⁇ g/ml BSA, and 8.3 % glycerol). Ligation was performed by incubation for 15 min at 37 0 C followed by heating for 10 min at 65 0 C to terminate the reaction.
  • 1X T4 DNA Ligase Reaction Buffer 50 mM Tris- HCI pH 8.0 at 25 0 C 1 10 mM MgCI2, 3.3 mM dithiothreitol, 10 ⁇ g/ml BSA, and 8.3 % glycerol.
  • the three forward primers EQ20573, EQ23439, and EQ23440 had 9, 13 or 17 nucleotides, respectively, complementary to the reverse transcription product produced above in (2), and the probes were designed not to interfere with the forward primers.
  • the following temperature cycling program was used: 10 min at 37 0 C, 15 min at 95 0 C, 1 min at 50 0 C, and 39 cycles of 20 s at 94 0 C and 1 min at 60 0 C.
  • the real-time RT-PCR analysis was performed on a Opticon real-time PCR instrument (MJ Research, USA) with detection at the 60 0 C step. 4. Results

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Abstract

The invention relates to ribonucleic acids, probes and methods for detection, quantification as well as monitoring the expression of mature microRNAs and small interfering RNAs (siRNAs). The invention furthermore relates to methods for monitoring the expression of other non-coding RNAs, mRNA splice variants, as well as detecting and quantifying RNA editing, allelic variants of single transcripts, mutations, deletions, or duplications of particular exons in transcripts, e.g., alterations associated with human disease such as cancer. The invention furthermore relates to methods for detection, quantification as well as monitoring the expression of deoxy nucleic acids.

Description

NOVEL METHODS FOR QUANTIFICATION OF microRNAS AND SMALL INTERFERING RNAS
The present invention relates to nucleic acids, probes and methods for detection, quantification as well as monitoring the expression of mature microRNAs and small interfering RNAs (siRNAs). The invention furthermore relates to methods for monitoring the expression of other non-coding RNAs, mRNA splice variants, as well as detecting and quantifying RNA editing, allelic variants of single transcripts, mutations, deletions, or duplications of particular exons in transcripts, e.g. alterations associated with human disease, such as cancer. The invention furthermore relates to methods for detection and quantification of a target DNA sequence.
Background of the Invention
The present invention relates to the quantification of target nucleotide sequences in a wide variety of nucleic acid samples anci more specifically to the methods employing the design and use of oligonucleotide probes that are useful for detecting and quanti- fying target nucleotide sequences, especially RNA target sequences, such as mi- croRNA and siRNA target sequences of interest and for detecting differences between nucleic acid samples (e.g., such as samples from a cancer patient and a healthy patient).
MicroRNAs
The expanding inventory of international sequence databases and the concomitant sequencing of nearly 200 genomes representing all three domains of life - bacteria, archea and eukaryota - have been the primary drivers in the process of deconstructing living organisms into comprehensive molecular catalogs of genes, transcripts and proteins. The importance of the genetic variation within a single species has become apparent, extending beyond the completion of genetic blueprints of several important genomes, culminating in the publication of the working draft of the human genome sequence in 2001 (Lander, Linton, Birren et al., 2001 Nature 409: 860-921; Venter, Adams, Myers et al., 2001 Science 291: 1304-1351; Sachidanandam, Weissman, Schmidt et a/., 2001 Nature 409: 928-933). On the other hand, the increasing num- ber of detailed, large-scale molecular analyses of transcription originating from the human and mouse genomes along with the recent identification of several types of non-protein-coding RNAs, such as small nucleolar RNAs, siRNAs, microRNAs and
SUBSTITUTE SHEB" antisense RNAs, indicate that the transcriptomes of higher eukaryotes are much more complex than originally anticipated (Wong et al. 2001 , Genome Research 11: 1975-1977; Kampa et al. 2004, Genome Research 14: 331-342).
As a result of the Central Dogma: 'DNA makes RNA, and RNA makes protein', RNAs have been considered as simple molecules that just translate the genetic information into protein. Recently, it has been estimated that although most of the genome is transcribed, almost 97% of the genome does not encode proteins in higher eukaryotes, but putative, non-coding RNAs (Wong et al. 2001 , Genome Research 11 : 1975- 1977). The non-coding RNAs (ncRNAs) appear to be particularly well suited for regu- latory roles that require highly specific nucleic acid recognition. Therefore, the view of RNA is rapidly changing from the merely informational molecule to comprise a wide variety of structural, informational and catalytic molecules in the cell.
Recently, a large number of small non-coding RNA genes have been identified and designated as microRNAs (miRNAs) (for review, see Ke et al. 2003, Curr.Opin. Chem. Biol. 7:516-523). The first miRNAs to be discovered were the lin-4 and let-7 that are heterochronic switching genes essential for the normal temporal control of diverse developmental events (Lee et al. 1993, Cell 75:843-854; Reinhart et al. 2000, Nature 403: 901-906) in the roundworm C. elegans. miRNAs have been evolutionar- ily conserved over a wide range of species and exhibit diversity in expression pro- files, suggesting that they occupy a wide variety of regulatory functions and exert significant effects on cell growth and development (Ke et al. 2003, Curr.Opin. Chem. Biol. 7:516-523). Recent work has shown that miRNAs can regulate gene expression at many levels, representing a novel gene regulatory mechanism and supporting the idea that RNA is capable of performing similar regulatory roles as proteins. Under- standing this RNA-based regulation will help us to understand the complexity of the genome in higher eukaryotes as well as understand the complex gene regulatory networks.
miRNAs are 21-25 nucleotide (nt) RNAs that are processed from longer endogenous hairpin transcripts (Ambros et al. 2003, RNA 9: 277-279). To date more than 719 mi- croRNAs have been identified in humans, worms, fruit flies and plants according to the miRNA registry database hosted by Sanger Institute, UK, and many miRNAs that correspond to putative genes have also been identified. Some miRNAs have multiple loci in the genome (Reinhart et al. 2002, Genes Dev. 16: 1616-1626) and occasion- ally, several miRNA genes are arranged in tandem clusters (Lagos-Quintana et al. 2001 , Science 294: 853-858). The fact that many of the miRNAs reported to date have been isolated just once suggests that many new miRNAs will be discovered in the future. A recent in-depth transcriptional analysis of the human chromosomes 21 and 22 found that 49 % of the observed transcription was outside of any known annotation, and furthermore, that these novel transcripts were both coding and non- coding RNAs (Kampa et al. 2004, Genome Research 14: 331-342). The identified miRNAs to date represent most likely the tip of the iceberg, and the number of miRNAs might turn out to be very large.
The combined characteristics of microRNAs characterized to date (Ke et al. 2003, Curr.Opin. Chem. Biol. 7:516-523; Lee et al. 1993, Cell 75:843-854; Reinhart et al. 2000, Nature 403: 901-906) can be summarized as:
1. miRNAs are single-stranded RNAs of about 21-25 nt.
2. They are cleaved from a longer endogenous double-stranded hairpin precursor by the enzyme Dicer.
3. miRNAs match precisely the genomic regions that can potentially encode precursor RNAs in the form of double-stranded hairpins.
4. miRNAs and their predicted precursor secondary structures are phylogenetically conserved.
Several lines of evidence suggest that the enzymes Dicer and Argonaute are crucial participants in miRNA biosynthesis, maturation and function (Grishok et al. 2001, Cell 106: 23-24). Mutations in genes required for miRNA biosynthesis lead to genetic developmental defects, which are, at least in part, derived from the role of generating miRNAs. The current view is that miRNAs are cleaved by Dicer from the hairpin pre- cursor in the form of duplex, initially with 2 or 3 nt overhangs in the 3' ends, and are termed pre-miRNAs. Cofactors join the pre-miRNP and unwind the pre-miRNAs into single-stranded miRNAs, and pre-miRNP is then transformed to miRNP. miRNAs can recognize regulatory targets while part of the miRNP complex. There are several similarities between miRNP and the RNA-induced silencing complex, RISC, including similar sizes and both containing RNA helicase and the PPD proteins. It has there- fore been proposed that miRNP and RISC are the same RNP with multiple functions (Ke et al. 2003, Curr.Opin. Chem. Biol. 7:516-523). Different effectors direct miRNAs into diverse pathways. The structure of pre-miRNAs is consistent with the observation that 22 nt RNA duplexes with 2 or 3 nt overhangs at the 3' ends are beneficial for reconstitution of the protein complex and might be required for high affinity binding of the short RNA duplex to the protein components (for review, see Ke et al. 2003, Curr.Opin. Chem. Biol. 7:516-523).
Growing evidence suggests that miRNAs play crucial roles in eukaryotic gene regulation. The first miRNAs genes to be discovered, lin-4 and let-7, base-pair incompletely to repeated elements in the 3' untranslated regions (UTRs) of other heterochronic genes, and regulate the translation directly and negatively by antisense RNA-RNA interaction (Lee et al. 1993, Cell 75:843-854; Reinhart ef a/. 2000, Nature 403: 901- 906). Other miRNAs are thought to interact with target mRNAs by limited complementary and suppressed translation as well (Lagos-Quintana et al. 2001, Science 294: 853-858; Lee and Ambros 2001 , Science 294: 858-862). Many studies have shown, however, that given a perfect complementarity between miRNAs and their target RNA, could lead to target RNA degradation rather than inhibit translation (Hut- vanger and Zamore 2002, Science 297: 2056-2060), suggesting that the degree of complementarity determines their functions. By identifying sequences with near com- plementarity, several targets have been predicted, most of which appear to be potential transcriptional factors that are crucial in cell growth and development. The high percentage of predicted miRNA targets acting as developmental regulators and the conservation of target sites suggest that miRNAs are involved in a wide range of organism development and behaviour and cell fate decisions (for review, see Ke et al. 2003, Curr.Opin. Chem. Biol. 7:516-523).
MicroRNAs and human disease
Analysis of the genomic location of miRNAs indicates that they play important roles in human development and disease. Several human diseases have already been pinpointed in which miRNAs or their processing machinery might be implicated. One of them is spinal muscular atrophy (SMA), a paediatric neurodegenerative disease caused by reduced protein levels or loss-of-function mutations of the survival of motor neurons (SMN) gene (Paushkin et al. 2002, Curr.Opin.Cell Biol. 14: 305-312). Two proteins (Gemin3 and Gemin4) that are part of the SMN complex are also com- ponents of miRNPs, whereas it remains to be seen whether miRNA biogenesis or function is dysregulated in SMA and what effect this has on pathogenesis. Another neurological disease linked to mi/siRNAs is fragile X mental retardation (FXMR) caused by absence or mutations of the fragile X mental retardation protein (FMRP)(Nelson et al. 2003, TIBS 28: 534-540), and there are additional clues that miRNAs might play a role in other neurological diseases. Yet another interesting finding is that the miR-224 gene locus lies within the minimal candidate region of two different neurological diseases: early-onset Parkinsonism and X-linked mental retardation (Dostie et al. 2003, RNA: 9: 180-186). Links between cancer and miRNAs have also been recently described. The most frequent single genetic abnormality in chronic lymphocytic leukaemia (CLL) is a deletion localized to chromosome 13q14 (50% of the cases). A recent study determined that two different miRNA (miR15 and miR16) genes are clustered and located within the intron of LEU2, which lies within the deleted minimal region of the B-cell chronic lymphocytic leukaemia (B-CLL) tu- mour suppressor locus, and both genes are deleted or down-regulated in the majority of CLL cases (Calin et al. 2002, Proc. Natl. Acad. Sci.U.S.A. 99: 15524-15529). It has been anticipated that connections between miRNAs and human diseases will only strengthen in parallel with the knowledge of miRNAs and the gene networks that they control. Moreover, the understanding of the regulation of RNA-mediated gene ex- pression is leading to the development of novel therapeutic approaches that will be likely to revolutionize the practice of medicine (Nelson et al. 2003, TIBS 28: 534-540).
Small interfering RNAs and RNAi
Some of the recent attention paid to small RNAs in the size range of 21 to 25 nt is due to the phenomenon RNA interference (RNAi), in which double-stranded RNA leads to the degradation of any RNA that is homologous (Fire et al. 1998, Nature 391 : 806-811). RNAi relies on a complex and ancient cellular mechanism that has probably evolved for protection against viral attack and mobile genetic elements. A crucial step in the RNAi mechanism is the generation of short interfering RNAs (siRNAs), double-stranded RNAs that are about 22 nt long each. The siRNAs lead to the degradation of homologous target RNA and the production of more siRNAs against the same target RNA (Lipardi et al. 2001 , Cell 107: 297-307). The present view for the mRNA degradation pathway of RNAi is that antiparallel Dicer dinners cleave long double-stranded dsRNAs to form siRNAs in an ATP-dependent manner. The siRNAs are then incorporated in the RNA-induced silencing complex (RISC) and ATP-dependent unwinding of the siRNAs activates RISC (Zhang et al. 2002, EMBO J. 21: 5875-5885; Nykanen et al. 2001, Cell 107: 309-321). The active RISC complex is thus guided to degrade the specific target mRNAs.
Detection and analysis of microRNAs and siRNAs
The current view that miRNAs may represent a newly discovered, hidden layer of gene regulation has resulted in high interest among researchers around the world in the discovery of miRNAs, their targets and mechanism of action. Detection and analysis of these small RNAs is, however not trivial. Thus, the discovery of more than 700 miRNAs to date has required taking advantage of their special features. First, the research groups have used the small size of the miRNAs as a primary criterion for isolation and detection. Consequently, standard cDNA libraries would lack miRNAs, primarily because RNAs that small are normally excluded by sixe selection in the cDNA library construction procedure. Total RNA from fly embryos, worms or HeLa cells have been size fractionated so that only molecules 25 nucleotides or smaller would be captured (Moss 2002, Curr.Biology 12: R138-R140). Synthetic oligomers have then been ligated directly to the RNA pools using T4 RNA ligase. Then the sequences have been reverse-transcribed, amplified by PCR1 cloned and sequenced (Moss 2002, Curr.Biology 12: R138-R140). The genome databases have subsequently been queried with the sequences, confirming the origin of the miRNAs from these organisms as well as placing the miRNA genes physically in the context of other genes in the genome. The vast majority of the cloned sequences have been located in intronic regions or between genes, occasionally in clusters, suggesting that the tandemly arranged miRNAs are processed from a single transcript to allow coordinate regulation. Furthermore, the genomic sequences have revealed the fold-back structures of the miRNA precursors (Moss 2002, Curr.Biology 12: R138-R140).
The size and sometimes low level of expression of different miRNAs require the use of sensitive and quantitative analysis tools. Due to their small size of 21-25 nt, the use of quantitative real-time PCR for monitoring expression of mature miRNAs is excluded. Therefore, most miRNA researchers currently use Northern blot analysis combined with polyacrylamide gels to examine expression of both the mature and pre-miRNAs (Reinhart et al. 2000, Nature 403: 901-906; Lagos-Quintana et al. 2001, Science 294: 853-858; Lee and Ambros 2001 , Science 294: 862-864). Primer extension has also been used to detect the mature miRNA (Zeng and Cullen 2003, RNA 9: 112-123). The disadvantage of all the gel-based assays (Northern blotting, primer extension, RNase protection assays etc.) as tools for monitoring miRNA expression includes low throughput and poor sensitivity. DNA microarrays would appear to be a good alternative to Northern blot analysis to quantify miRNAs since microarrays have excellent throughput. However, the drawbacks of microarrays are the requirement of high concentrations of input target for efficient hybridization and signal generation, poor sensitivity for rare targets, and the necessity for post-array validation using more sensitive assays such as real-time quantitative PCR, which is not feasible. A recent report used cDNA microarrays to monitor the expression of miRNAs during neuronal development with 5 to 10 μg aliquot of input total RNA as target, but the mature miRNAs had to be separated from the miRNA precursors using micro concentrators prior to microarray hybridizations (Krichevsky et al. 2003, RNA 9: 1274-1281). A PCR approach has also been used to determine the expression levels of mature miRNAs (Grad et al. 2003, MoI. Cell 11 : 1253-1263). This method is useful to clone miRNAs, but highly impractical for routine miRNA expression profiling, since it involves gel isolation of small RNAs and ligation to linker oligonucleotides. Schmittgen et al. (2004, Nucleic Acids Res. 32: e43) describe an alternative method to Northern blot analysis, in which they use real-time PCR assays to quantify the expression of miRNA precursors. The disadvantage of this method is that it only allows quantification of the pre- cursor miRNAs, which does not necessarily reflect the expression levels of mature miRNAs. In order to fully characterize the expression of large numbers of miRNAs, it is necessary to quantify the mature miRNAs, such as those expressed in human disease, where alterations in miRNA biogenesis produce levels of mature miRNAs that are very different from those of the precursor miRNA. For example, the precursors of 26 miRNAs were equally expressed in non-cancerous and cancerous colorectal tissues from patients, whereas the expression of mature human miR143 and miR145 was greatly reduced in cancer tissues compared with non-cancer tissues, suggesting altered processing for specific miRNAs in human disease (Michael et al. 2003, MoI. Cancer Res. 1: 882-891). On the other hand, recent findings in maize with miR166 and miR165 in Arabidopsis thaliana, indicate that microRNAs act as signals to specify leaf polarity in plants and may even form movable signals that emanate from a signalling centre below the incipient leaf (Juarez et al. 2004, Nature 428: 84-88; Kidner and Martienssen 2004, Nature 428: 81-84).
In conclusion, the biggest challenge in measuring the mature miRNAs as well as siRNAs using real-time quantitative PCR is their small size of the of 21-25 nt. The described method of invention addresses the aforementioned practical problems in detection and quantification of small RNA molecules, miRNAs as well as siRNAs, and aims at ensuring the development of flexible, convenient and inexpensive assays for accurate and specific quantification of miRNA and siRNA transcripts.
RNA editing and alternative splicing
RNA editing is used to describe any specific change in the primary sequence of an RNA molecule, excluding other mechanistically defined processes such as alternative splicing or polyadenylation. RNA alterations due to editing fall into two broad categories, depending on whether the change happens at the base or nucleotide level (Gott 2003, C. R. Biologies 326 901-908). RNA editing is quite widespread, occurring in mammals, viruses, marsupials, plants, flies, frogs, worms, squid, fungi, slime molds, dinoflagellates, kinetoplastid protozoa, and other unicellular eukaryotes. The current list most likely represents only the tip of the iceberg; based on the distribution of homologues of known editing enzymes, as RNA editing almost certainly oc- curs in many other species, including all metazoa. Since RNA editing can be regulated in a developmental or tissue-specific manner, it is likely to play a significant role in the etiology of human disease (Gott 2003, C. R. Biologies 326 901-908).
A common feature for eukaryotic genes is that they are composed of protein- encoding exons and introns. lntrons are characterized by being excised from the pre-mRNA molecule in RNA splicing, as the sequences on each side of the intron are spliced together. RNA splicing not only provides functional mRNA, but is also responsible for generating additional diversity. This phenomenon is called alternative splicing, which results in the production of different mRNAs from the same gene. The mRNAs that represent isoforms arising from a single gene can differ by the use of alternative exons or retention of an intron that disrupts two exons. This process often leads to different protein products that may have related or drastically different, even antagonistic, cellular functions. There is increasing evidence indicating that alternative splicing is very widespread (Croft et al. Nature Genetics, 2000). Recent studies have revealed that at least 80% of the roughly 35,000 genes in the human genome are alternatively spliced (Kampa et al. 2004, Genome Research 14: 331- 342). Clearly, by combining different types of modifications and thus generating different possible combinations of transcripts of different genes, alternative splicing together with RNA editing are potent mechanisms for generating protein diversity. Analysis of the alternative splice variants and RNA editing, in turn, represents a novel approach to functional genomics, disease diagnostics and pharmacogenomics.
Misplaced control of alternative splicing as a causative agent for human disease
The detection of the detailed structure of the transcriptional output is an important goal for molecular characterization of a cell or tissue. Without the ability to detect and quantify the splice variants present in one tissue, the transcript content or the protein content cannot be described accurately. Molecular medical research shows that many cancers result in altered levels of splice variants, so an accurate method to detect and quantify these transcripts is required. Mutations that produce an aberrant splice form can also be the primary cause of such severe diseases such as spinal muscular dystrophy and cystic fibrosis.
Much of the study of human disease, indeed much of genetics is based upon the study of a few model organisms. The evolutionary stability of alternative splicing patterns and the degree to which splicing changes according to mutations and environ- mental and cellular conditions influence the relevance of these model systems. At present, there is little understanding of the rates at which alternative splicing patterns or RNA editing change, and the factors influencing these rates.
Previously, other analysis methods have been performed with the aim of detecting either splicing of RNA transcripts per se in yeast, or of detecting putative exon skip- ping splicing events in rat tissues, but neither of these approaches had sufficient resolution to estimate quantities of splice variants, a factor that could be essential to an understanding of the changes in cell life cycle and disease. Thus, improved methods are needed for nucleic acid amplification, hybridization, and quantification. The present method of invention enables to distinguish between mRNA splice variants as well as RNA-edited transcripts and quantify the amount of each variant in a nucleic acid sample, such as a sample derived from a patient.
Antisense transcription in eukaryotes
RNA-mediated gene regulation is widespread in higher eukaryotes and complex genetic phenomena like RNA interference, co-suppression, transgene silencing, im- printing, methylation, and possibly position-effect variegation and transvection, all involve intersecting pathways based on or connected to RNA signalling (Mattick 2001; EMBO reports 2, 11: 986-991). Recent studies indicate that antisense transcription is a very common phenomenon in the mouse and human genomes (Oka- zaki et al. 2002; Nature 420: 563-573; YeNn et al. 2003, Nature Biotechnol.). Thus, antisense modulation of gene expression in eukaryotic cells, e.g. human cells appear to be a common regulatory mechanism. In light of this, the present invention provides a method for quantification of non-coding antisense RNAs, as well as a method for highly accurate mapping of the overlapping regions between sense-antisense transcriptional units.
Summary of the Invention
The challenges of establishing genome function and understanding the layers of information hidden in the complex transcriptomes of higher eukaryotes call for novel, improved technologies for detection, analysis and quantification of RNA molecules in complex nucleic acid samples. Thus, it would be highly desirable to be able to detect and quantify the expression of mature microRNAs, siRNAs, RNA-edited transcripts as well as highly homologous splice variants in the transcriptomes of eukaryotes using methods based on specific and sensitive oligonucleotide detection probes in a homogeneous assay system.
The present invention solves the current problems faced by conventional approaches to homogeneous assays outlined above by providing a method for the design, synthesis and combined use of novel oligonucleotide tagging probes and detection probes with sufficient sequence specificity and high affinity to short nucleic acid targets, e.g. RNA target sequences- so that they are unlikely to detect a random RNA target molecule and also unlikely to detect pre-mature RNA molecules. Such tagging probes contain a sequence, anchored to the tagging probes, essential as priming sites for subsequent amplification of the nucleic acids by polymerase chain reaction in real-time quantitative PCR assays. The method of invention utilizes two anchored tagging probes, each designed in combination to detect a complementary target sequence, e.g. a short RNA sequence, where the first tagging probe hybridizes to a first region within a target sequence and the second tagging probe hybridizes to a second region within the same complementary target sequence, e.g. a short RNA target sequence that is adjacent to the first region. In the preferred mode, one of the tagging probes is 5' phosphorylated enabling covalent coupling of the two contiguous tagging oligonucleotide probes hybridized to the complementary target sequence by a ligase to form a single oligonucleotide sequence. The background in the hybridization to the target RNA sequence in complex nucleic acid samples is eliminated by the use of two tagging probes, where the hybridization of both probes to the complementary target sequence, e.g. short RNA target sequence is required for the covalent joining of the two probes. The method furthermore takes the advantage of substitution of the recognition sequences with high-affinity nucleotide analogues, e.g. LNA, for sensitive and specific hybridization to short target sequences, e.g. miRNAs or siRNAs. The ligation reaction is followed by quantitative real-time PCR of the target sequence, e.g. ribonucleic acid-tern plated, covalently joined oligonucleotide molecules using the anchor sequences attached to the tagging probes as priming sites for the PCR primers and a short detection probe with sufficient duplex stability to allow binding to the am- plicon, and employing any of a variety of detection principles used in homogeneous assays. In the preferred mode, the detection probe is substituted with duplex- stabilizing, high-affinity nucleotide analogues, e.g. LNA, and preferably oxy-LNA, to allow use of short detection probes in the real-time quantitative PCR assay.
In another approach the covalent joining of the tagging probes hybridized to the target ribonucleic acid in the nucleic acid sample is carried out using a thermostable ligase, which allows repetitive cycles of denaturation, annealing and ligation at ele- vated temperatures to be carried out in the target sequence tagging reaction, thus generating a plurality of covalently joined template molecules for the subsequent real-time quantitative PCR assay. In the preferred mode the annealing temperature is adjusted so as to allow discrimination between highly homologous target ribonucleic acids in complex nucleic acid samples. In another aspect the annealing temperature is adjusted to allow single mismatch discrimination between highly homologous target sequences.
In yet another approach the recognition sequence of the first tagging probe is complementary to a sequence in the target ribonucleic acid sequence, e.g. to the 3'-end of the mature microRNA or siRNA or to a sequence located 3' to the RNA edited nu- cleotide, splice junction, single nucleotide polymorphism or point mutation in the target ribonucleic acid sequence.. The said first tagging probe, designated as RT tagging probe, is used as an anchored primer in a reverse transcription reaction to generate a primer extension product, complementary to the target RNA sequence using a reverse transcriptase enzyme. The second tagging probe, designated as 2nd strand tagging probe, is designed so that its recognition sequence is complementary to the reverse transcriptase-extended nucleotide sequence corresponding to the 5'-end of the mature microRNA or siRNA or located 5' to the RNA edited nucleotide, splice junction, single nucleotide polymorphism or point mutation in the ribonucleic acid tar- get sequence The 2nd strand tagging probe is used as anchored primer to generate the second strand complementary to the primer extension product. The specificity of the reaction is based on the sequential use of the two anchored tagging probes, hybridising to complementary 3'-end and 5'-end regions of the target RNA and complementary DNA sequences, respectively. In a preferred mode the recognition se- quence of the RT tagging probe is modified with duplex-stabilizing, high-affinity nucleotide analogues e.g. LNA, and preferably oxy-LNA, to allow use of high-stringency primer annealing conditions. In yet another preferred mode the recognition sequences of both tagging probes are modified with duplex-stabilizing, high-affinity nucleotide analogues e.g. LNA, and preferably oxy-LNA, to allow use of high-stringency primer annealing conditions in both the reverse transcription and second strand synthesis reactions, respectively.The second strand reaction is followed by quantitative real-time PCR of the resulting double-stranded target sequence, corresponding to an anchored target ribonucleic acid sequence, e.g. a microRNA sequence, using the anchor sequences attached to the tagging probes as priming sites for the PCR prim- ers and a short detection probe with sufficient duplex stability to allow binding to the amplicon, and employing any of a variety of detection principles used in homogeneous assays. In the preferred mode, the detection probe is substituted with duplex- stabilizing, high-affinity nucleotide analogues, e.g. LNA, and preferably oxy-LNA, to allow use of short detection probes in the real-time quantitative PCR assay. In yet another preferred mode, the detection probe is furthermore substituted with duplex- stabilizing LNA diaminopurine or LNA 2-thio-T high-affinity analogues in combination with LNA monomers.
The present methods of invention are highly useful and applicable for detection and quantification of individual small RNA molecules in complex mixtures composed of hundreds of thousands of different nucleic acids, such as detecting mature miRNAs or siRNAs, when combined with a miRNA or siRNA target specific tagging probe set and a miRNA or a siRNA detection probe. The recognition sequences in the tagging probe set as well as the detection probe are synthesized by substitution of high affinity nucleotide analogues, e.g. LNA, and preferably oxy-LNA, allowing highly sensitive and specific hybridization and ligation to occur at elevated temperatures. By the use of short detection probes of the invention, substituted with high affinity nucleotide analogues, e.g. LNA1 LNA diaminopurine and LNA 2-thio-thymidine, short amplicons corresponding to mature miRNAs or siRNAs, including the anchor primer sites from the tagging probe set can be monitored directly in standard real-time quantitative PCR assays. The present method is furthermore highly useful in the detection and quantification of non-coding RNAs other than miRNAs or siRNAs, antisense RNA transcripts, RNA-edited transcripts or highly homologous, alternatively spliced transcripts in complex nucleic acid samples, such as the human, mouse, rat, C. elegans, Drosophila melanogaster, Arabidopsis thaliana, rice and maize transcriptomes com- posed of hundreds of thousands of different ribonucleic acids in their respective transcriptomes. The method is also directly applicable to detecting, testing, diagnosing or quantifying miRNAs, siRNAs, other non-coding RNAs1 RNA-edited transcripts or alternative mRNA splice variants implicated in or connected to human disease in complex human nucleic acid samples, e.g. from cancer patients.
"Brief Description Of The Drawings
Fig. 1 shows a schematic presentation of one method of the invention for quantification of microRNAs by sequence-specific real-time quantitative RT-PCR.
Fig. 2 shows a dilution series for the human miR-145 real-time quantitative PCR assay. The GSP-RT primer for human miR-145 microRNA was used in first strand syn- thesis, where the human miR-145 template concentration was 100 (open triangles), 10 (open diamonds), 1 (open squares), or 0.1 fmol (crosses), respectively. The 0.5 μg Brain total RNA is depicted by (open circles), the 10 fmol synthetic miR-145 template by solid diamonds. The negative first strand synthesis without any RNA template is depicted by solid triangles. The cDNA templates were subsequently detected using real-time PCR by the universal PCR primers, the miR-specific forward primer, and the LNA-modified dual-labelled detection probe EQ20317 for the miR-145 mi- croRNA using a minus template as a negative control (solid squares).
Fig. 3: Comparing Real time qPCR performed with either a gene specific qPCR primer approach or by using universal primers. Dilution series were performed with spike-in of synthetic miR-16 in a total RNA background Fig. 4 shows a schematic presentation of one method of the invention for quantification of microRNAs by sequence-specific real-time quantitative RT-PCR.comprising the following steps:
a) prior to the shown steps, a the target ribonucleic acid sequence is ligated with an RNA oligonucleotide adaptor using ligase enzyme.
b) in the next event taking place prior to the shown steps the RNA oligonucleotide adaptor sequence is contacted with an oligonucleotide RT-primer, wherein the 3'- recognition nucleotide sequence is complementary to a sequence in the RNA oligonucleotide adaptor sequence.
c) in the next event taking place prior to the shown steps, a DNA strand complementary to the target ribonucleic acid is synthesized by reverse transcription using a reverse transcriptase enzyme and the 3'-nucleotide sequence in the RNA oligonucleotide adaptor sequence as primer binding site for an oligonucleotide RT-primer.
d) in the first of the shown steps, a second strand synthesis is performed as a first step of a real-time PCR to amplify the reverse transcription product produced in the reverse transcription reaction taking place prior to the real time PCR. This first step of the real-time PCR uses a gene specific forward primer with a 3'-recognition nucleotide sequence, which is sequence identical with the target ribonucleic acid sequence, here the mature miRNA. The 5'-sequence of the gene specific forward primer con- tains a universal forward primer sequence. Quantification of the resulting nucleic acids by real-time PCR is performed using a universal forward primer and so called scorpion primer as the reverse primer. The universal forward primer contains sequence identical to the 5'-sequence of the gene specific forward primer and the 3'- region of the scorpion primer contains a sequence stretch complementary to the RNA oligonucleotide adaptor ligated to the target ribonucleic acid sequence. The 5'-region of the scorpion primer contains a detection moiety consisting of a sequence stretch complementary to the target ribonucleic acid sequence.
e) amplification of a mature miRNA results in a short amplicon where the short distance between the scorpion primer and target ribonucleic acid sequence of the ma- ture miRNA facilitates binding of the scorpion primer detection moiety to the target ribonucleic acid sequence. This causes unfolding of the scorpion primer and thereby increased fluorescence.
f) amplification of a precursor miRNA results in a amplicon where the longer distance between the scorpion primer and target ribonucleic acid sequence of the mature miRNA prevents binding of the scorpion primer detection moiety to the target ribonu- cleic acid sequence. Therefore unfolding of the scorpion primer is prevented and fluorescence remains unchanged.
Fig. 5 shows a schematic presentation of one method of the invention for quantification of microRNAs by sequence-specific real-time quantitative RT-PCR.comprising the following steps:
a) contacting the target ribonucleic acid sequence with an oligonucleotide RT-primer, wherein the 3'-recognition nucleotide sequence is complementary to a sequence in the target sequence. The 5'-sequence of the oligonucleotide RT-primer contain a universal reverse primer binding site.
b) synthesis of a complementary DNA strand to the target ribonucleic acid by reverse transcription using a reverse transcriptase enzyme and the 3'-nucleotide sequence in the target sequence as primer binding site for the oligonucleotide RT-primer.
c) performing a second strand synthesis as a first step of a real-time PCR using a Forward primer with a 3'-recognition nucleotide sequence which is sequence identi- cal with the target ribonucleic acid sequence. The 5'-sequence of the Forward primer contain a universal forward primer binding site.
d) quantifying the resulting nucleic acids by real-time PCR using a set of universal forward and reverse primers and a labelled detection probe comprising a target recognition sequence and a detection moiety.
e) the Forward primer in c) is present in a low concentration to prevent mis-priming whereas the set of universal forward and reverse primers in d) are present in normal concentration. Fig. 6 shows a schematic presentation of one method of the invention for quantification of microRNAs by sequence-specific real-time quantitative RT-PCR.comprising the following steps:
a) Ligating the target ribonucleic acid sequence with an RNA oligonucleotide adaptor using ligase enzyme.
b) contacting the RNA oligonucleotide adaptor sequence with an oligonucleotide RT- primer, wherein the 3'-recognition nucleotide sequence is complementary to a sequence in the RNA oligonucleotide adaptor sequence. The 5'-sequence of the oligonucleotide RT-primer contain a universal reverse primer binding site.
c) synthesis of a complementary DNA strand to the target ribonucleic acid by reverse transcription using a reverse transcriptase enzyme and the 3'-nucleotide sequence in the RNA oligonucleotide adaptor sequence as primer binding site for the oligonucleotide RT-primer.
d) performing a second strand synthesis as a first step of a real-time PCR using a Forward primer with a 3'-recognition nucleotide sequence which is sequence identical with the target ribonucleic acid sequence. The 5'-sequence of the Forward primer contain a universal forward primer binding site.
e) quantifying the resulting nucleic acids by real-time PCR using a set of universal forward and reverse primers and a labelled detection probe comprising a target rec- ognition sequence and a detection moiety.
f) the Forward primer in d) is present in a low concentration to prevent mis-priming whereas the set of universal forward and reverse primers in e) are present in normal concentration.
Fig. 7 Bioanalyzer profiles of real-time PCR end-products resulting from use of For- ward primers with different lengths of the sequence which is complementary to the first strand synthesis consisting of reverse transcribed miRNA sequence - here hsa- mir-92 - ligated to RNA adaptor. The used forward primer had 9, 13, or 17 nucleotides of their sequence complementary to the reverse transcription product. The Bioanalyzer profile shows that the priming with the forward primer having only 9 nucleo- tides of complementarity is unspecific with two bands being visible. In contrast, specificity is obtained by increasing the number of complementary nucleotides from 9 to 13 and 17. The increased specificity is shown by the presence of a single band of correct size on the Bioanalyzer profile.
Definitions
For the purposes of the subsequent detailed description of the invention the following definitions are provided for specific terms, which are used in the disclosure of the present invention:
In the following, "dNTP" means a mixture of 2'-deoxyadenosine-5'-triphosphate, 2'- deoxycytidine-5'-triphosphate, 21-deoxyguanosine-5'-triphosphate, and 2'- deoxythymidine-5'-triphosphate
"RT-primer" refers to a primer, comprising a recognition sequence, complementary to a sequence in the target deoxyribonucleic and/or ribonucleic acid sequence, e.g. to the 3'-end of the mature microRNA or siRNA, or to an RNA-DNA chimerical moiety, or to a sequence located 3' to a RNA-edited nucleotide, splice junction, single nucleotide polymorphism or point mutation in the target ribonucleic acid sequence, and an anchor sequence essential for subsequent capture or amplification by PCR. The said RT-primer is used as an anchored primer in a reverse transcription reaction to generate a primer extension product, complementary to the target RNA sequence using a reverse transcriptase enzyme.
The term "Capture probes" or "capture probe" refer to a probe(s), comprising a recognition sequence, complementary to the target sequence, e.g. a short RNA target sequence, and an anchor sequence essential for subsequent capture, reverse tran- scription reaction, or amplification by PCR. The anchor sequence function as priming sites for the RT- or PCR primers in subsequent reverse transcription reaction, realtime PCR, or as tags for capture assays.
In the present context, the term "linker" means a thermochemically and photochemi- cally non-active distance-making group that is used to join two or more different nu- cleotide moieties of the types defined above. Linkers are selected on the basis of a variety of characteristics including their hydrophobicity, hydrophilicity, molecular flexibility and length (e.g. see Hermanson et. al., "Immobilized Affinity Ligand Techniques", Academic Press, San Diego, California (1992), p. 137-ff). Generally, the length of the linkers is less than or about 400 angstroms, in some applications preferably less than 100 angstroms. The linker, thus, comprises a chain of carbon atoms optionally interrupted or terminated with one or more heteroatoms, such as oxygen atoms, nitrogen atoms, and/or sulphur atoms. Thus, the linker may comprise one or more amide, ester, amino, ether, and/or thioether functionalities, and optionally aro- matic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as poly-(3-alanine, polyglycine, polylysine, and peptides in general, oligosaccharides, oligo/polyphosphates. Moreover the linker may consist of combined units thereof. The length of the linker may vary, taking into consideration the desired or necessary positioning and spatial orientation of the "ac- tive/functional" part of the group in question in relation to the 5- or 6-membered ring. In particularly interesting embodiments, the linker includes a chemically cleavable group. Examples of such chemically cleavable groups include disulphide groups cleavable under reductive conditions, peptide fragments cleavable by peptidases, etc.
In the present context a "solid support" may be chosen from a wide range of polymer materials e.g. CPG (controlled pore glass), polypropylene, polystyrene, polycarbonate or polyethylene and is may take a variety of forms such as a tube, a microtiter well plate, a stick, a bead, a particle, a filter etc. The oligonucleotide may be immobilized to the solid support via its 5'- or 3'-end (or via the terminus of a linker attached to the 5'- or 3'-end) by a variety of chemical or photochemical methods usually employed in the immobilization of oligonucleotides or by non-covalent coupling e.g. via binding of a biotinylated oligonucleotide to immobilized streptavidin.
A looped primer" refers to a probe, comprising a recognition sequence, complementary to a sequence in the target deoxyribonucleic acid sequence which recognition sequence is complementary to the reverse transcriptase-extended nucleotide sequence corresponding to the 5'-end of the mature microRNA or siRNA or located 5' to the RNA edited nucleotide, splice junction, single nucleotide polymorphism or point mutation in the initial ribonucleic acid target sequence, and an anchor sequence essential for subsequent capture or amplification by PCR. The said looped primer is used as an anchored primer to generate the second nucleic acid strand, which is complementary to the primer extension product. Another aspect of the looped primer is that the anchor sequence forms an intramolecular hairpin structure at the chosen assay temperature mediated by complementary sequences at the 5'- and the 3'-end of the oligonucleotide. The specificity of the reaction is based on the sequential use of the two anchored tagging probes with non-overlapping recognition sequences, hybridising to complementary 3'-end and 5'-end regions of the target RNA and complementary DNA sequences, respectively.
A "hairpin structure" refers to an intramolecular structure of an oligonucleotide at the chosen assay temperature mediated by hybridization of complementary sequences at the 5'- and the 3'-end of the oligonucleotide.
"U" refers to a enzyme unit defined as the amount of enzyme required to convert a given amount reactants to a product using a defined time and temperature.
In the present context "ligand" means something, which binds. Ligands comprise bio- tin and functional groups such as: aromatic groups (such as benzene, pyridine, naph- talene, anthracene, and phenanthrene), heteroaromatic groups (such as thiophene, furan, tetrahydrofuran, pyridine, dioxane, and pyrimidine), carboxylic acids, carboxylic acid esters, carboxylic acid halides, carboxylic acid azides, carboxylic acid hy- drazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, C1-C20 alkyl groups optionally interrupted or terminated with one or more heteroatoms such as oxygen atoms, nitrogen atoms, and/or sulphur atoms, optionally containing aromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as poly-β-alanine, polyglycine, polylysine, peptides, oligo/polysaccharides, oligo/polyphosphates, toxins, antibiotics, cell poisons, and steroids, and also "affinity ligands", i.e. functional groups or biomolecules that have a specific affinity for sites on particular proteins, antibodies, poly- and oligosaccharides, and other biomolecules.
The singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. The term "a nucleic acid molecule" includes a plurality of nucleic acid molecules.
"Transcriptome" refers to the complete collection of transcriptional units of the genome of any species. In addition to protein-coding mRNAs, it also represents non- coding RNAs, such as small nucleolar RNAs, siRNAs, microRNAs and antisense RNAs, which comprise important structural and regulatory roles in the cell.
The term "amplicon" refers to small, replicating DNA fragments.
"Sample" refers to a sample of cells, or tissue or fluid isolated from an organism or organisms, including but not limited to, for example, skin, plasma, serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs, tumours, and also to samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, recombinant cells and cell components).
An "organism" refers to a living entity, including but not limited to, for example, hu- man, mouse, rat, Drosophila, C. elegans, yeast, Arabidopsis thaliana, maize, rice, zebra fish, primates, domestic animals, etc.
"Tagging probes" or "tagging probe" refer to a probe(s), comprising a recognition sequence, complementary to the target sequence, e.g. a short RNA target sequence, and an anchor sequence essential for subsequent capture or amplification by PCR. "Two tagging probes" or a "Pair of tagging probes" refer to two anchored tagging probes, each designed to detect in combination a short complementary target sequence, e.g. a short RNA sequence, where the recognition sequence of the first tagging probe hybridizes to a first region within a target sequence and the recognition sequence of the second tagging probe hybridizes to a second region within the same complementary target sequence, e.g. a short RNA target sequence that is adjacent to the first region. In the method of invention, one of the tagging probes is 5' phos- phorylated enabling covalent coupling of the two contiguous, non-overlapping tagging oligonucleotide probes hybridized to the complementary target sequence by a ligase to form a single oligonucleotide sequence. The anchor sequences attached to the tagging probes are designed so that they do not cross-hybridize to any target nucleic acid in a given transcriptome or to each other under the hybridization conditions used in the method of invention. The anchor sequences function as priming sites for the PCR primers in subsequent real-time quantitative PCR or as tags for capture assays.
"RT tagging probe" refers to a probe, comprising a recognition sequence, complementary to a sequence in the target ribonucleic acid sequence, e.g. to the 3'-end of the mature microRNA or siRNA or to a sequence located 3' to a RNA-edited nucleotide, splice junction, single nucleotide polymorphism or point mutation in the target ribonucleic acid sequence, and an anchor sequence essential for subsequent capture or amplification by PCR. The said RT tagging probe is used as an anchored primer in a reverse transcription reaction to generate a primer extension product, complementary to the target RNA sequence using a reverse transcriptase enzyme. "2nd strand tagging probe" refers to an anchored tagging probe, which recognition sequence is complementary to the reverse transcriptase-extended nucleotide sequence corresponding to the 5'-end of the mature microRNA or siRNA or located 5' to the RNA edited nucleotide, splice junction, single nucleotide polymorphism or point muta- tion in the initial ribonucleic acid target sequence. The 2nd strand tagging probe is used as anchored primer to generate the second nucleic acid strand, which is complementary to the primer extension product. The specificity of the reaction is based on the sequential use of the two anchored tagging probes with non-overlapping recognition sequences, hybridising to complementary 3'-end and 5'-end regions of the target RNA and complementary DNA sequences, respectively.
"Two tagging probes" or a "Pair of tagging probes" refer to two anchored tagging probes, each designed to detect in combination a short complementary target sequence, e.g. a short RNA sequence, where the recognition sequence of the first tagging probe hybridizes to a first region within a target sequence and the recognition sequence of the 2nd strand tagging probe recognizing a sequence is complementary to the reverse transcriptase-extended nucleotide sequence corresponding to the 5'- end of the mature microRNA or siRNA or located 51 to the RNA edited nucleotide, splice junction, single nucleotide polymorphism or point mutation in the initial ribonucleic acid target sequence. The 2nd strand tagging probe is used as anchored primer to generate the second nucleic acid strand, which is complementary to the primer extension product.
The anchor sequences attached to each of the two tagging probes are designed so that they do not cross-hybridize to any target nucleic acid in a given transcriptome or to each other under the hybridization conditions used in the method of invention. The anchor sequences function as priming sites for the PCR primers in subsequent realtime quantitative PCR or as tags for capture assays.
The term "primer" may refer to more than one primer and refers to an oligonucleotide, whether occurring naturally, as in a purified restriction digest, or produced syntheti- cally, which is capable of acting_as a point of initiation of synthesis along a complementary strand when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is catalyzed. Such conditions include the presence of four different deoxyribonucleoside triphosphates and a polymerization-inducing agent such as DNA polymerase or reverse transcriptase, in a suitable buffer ("buffer" includes substituents which are cofactors, or which affect pH, ionic strength, etc.), and at a suitable temperature. The primer is preferably single-stranded for maximum efficiency in amplification by a polymerase or reverse transcriptase, in a suitable buffer ("buffer" includes substituents which are cofactors, or which affect pH, ionic strength, etc.), and at a suitable temperature. The primer is preferably single-stranded for maximum efficiency in amplification.
The primer concentration:
The amount of primer used in PCR may depend on the experiment. Generally, the two amplification primers should be used in equal concentrations and normal amounts vary from 0.1 μM to 1 μM equivalent to 5 - 50 pmol of each primer in a 50 μl reaction volume.
In the present invention the forward primer is present in a low concentration. Accordingly the concentration ratio between forward primer and the other amplification primer(s) is equal to or less than 0.5, provided that the other amplification primer(s) are present in a normal amount. Preferably the ratio is equal to or less than 0.2 or 0.1 or 0.05 or even less than 0.01.
In a preferred embodiment of the invention the ratio is 0.2, i.e. the forward primer concentration is 80 nM and the concentration of the other amplification primer is 400 nM.
The terms "Detection probes" or "detection probe" refer to labelled oligonucleotide, which forms a duplex structure with a sequence within the amplified target nucleic acid, e.g. short RNA target sequence, due to complementarity of the probe with a sequence in the target region. The detection probe, preferably, does not contain a se- quence complementary to sequence(s) used to prime the polymerase chain reaction. Generally the 3' terminus of the probe will be "blocked" to prohibit incorporation of the probe into a primer extension product. "Blocking" may be achieved by using non- complementary bases or by adding a chemical moiety such as biotin or a phosphate group to the 31 hydroxyl of the last nucleotide, which may, depending upon the selected moiety, serve a dual purpose by also acting as a label.
The terms "miRNA" and "microRNA" refer to 21-25 nt non-coding RNAs derived from endogenous genes. They are processed from longer (ca 75 nt) hairpin-like precursors termed pre-miRNAs. MicroRNAs assemble in complexes termed miRNPs and recognize their targets by antisense complementarity. If the microRNAs match 100 % their target, i.e. the complementarity is complete, the target mRNA is cleaved, and the miRNA acts like a siRNA. If the match is incomplete, i.e. the complementarity is partial, then the translation of the target mRNA is blocked.
The terms "Small interfering RNAs" or "siRNAs" refer to_21-25 nt RNAs derived from processing of linear double-stranded RNA. siRNAs assemble in complexes termed RISC (RNA-induced silencing complex) and target homologous RNA sequences for endonucleolytic cleavage. Synthetic siRNAs also recruit RISCs and are capable of cleaving homologous RNA sequences
The term "RNA interference" (RNAi) refers to a phenomenon where double-stranded RNA homologous to a target mRNA leads to degradation of the targeted mRNA. More broadly defined as degradation of target mRNAs by homologous siRNAs.
The term "Recognition sequence" refers to a nucleotide sequence that is complementary to a region within the target nucleotide sequence essential for sequence- specific hybridization between the target nucleotide sequence and the recognition sequence. The tagging probes as well as the detection probes of invention contain a target sequence-specific recognition sequence.
The term "Anchor sequences" refer to two nucleotide sequences contiguously attached to the pair of tagging probes, which anchor sequences are designed so that they do not cross-hybridize with each other or with a target nucleotide sequence or any nucleotide sequence in the nucleic acid sample, containing the target nucleotide sequence. The term "label" as used herein refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetric, X-ray diffraction or absorption, magnetism, enzymatic activ- ity, and the like.
A label is a reporter group detectable either by itself or as a part of a detection series. Examples of functional parts of reporter groups are biotin, digoxigenin, fluorescent groups (groups which are able to absorb electromagnetic radiation, e.g. light or X- rays, of a certain wavelength, and which subsequently reemits the energy absorbed as radiation of longer wavelength; illustrative examples are DANSYL (5- dimethylamino)-1-naphthalenesulfonyl), DOXYL (N-oxyl-4,4-dimethyloxazolidine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6- tetramethylpiperidine), dinitrophenyl, acridines, coumarins, Cy3 and Cy5 (trademarks for Biological Detection Systems, Inc.), erythrosine, coumaric acid, umbelliferone, Texas red, rhodamine, tetramethyl rhodamine, Rox, 7-nitrobenzo-2-oxa-1-diazole (NBD), pyrene, fluorescein, Europium, Ruthenium, Samarium, and other rare earth metals), radio isotopic labels, chemiluminescence labels (labels that are detectable via the emission of light during a chemical reaction), spin labels (a free radical (e.g. substituted organic nitroxides) or other paramagnetic probes (e.g. Cu2+, Mg2+) bound to a biological molecule being detectable by the use of electron spin resonance spectroscopy). Especially interesting examples are biotin, fluorescein, Texas Red, rhodamine, dinitrophenyl, digoxigenin, Ruthenium, Europium, Cy5, Cy3, etc.
"Ligation" or "covalent coupling" refers to covalent coupling of two adjacent nucleotide sequences, e.g. the tagging oligonucleotide probe sequences of the invention, to form a single nucleotide sequence. The reaction is catalyzed by the enzyme ligase, which forms a phosphodiester bond between the 5'-end of one nucleotide sequence and the 3'-end of the adjacent nucleotide sequence, e.g. between the two adjacent tagging probes of invention, annealed to their complementary, target nucleic acid sequence.
"RNA-templated oligonucleotide ligation" refers to covalent coupling of two adjacent oligonucleotide probe sequences annealed to a complementary RNA target sequence, to form a single nucleotide sequence. The reaction is catalyzed by the enzyme ligase, which forms a phosphodiester bond between the 5'-end of one nucleo- tide sequence and the 3'-end of the adjacent nucleotide sequence, e.g. between the two adjacent tagging probes of invention.
The terms "PCR reaction", "PCR amplification", "PCR", "pre-PCR" and "real-time quantitative PCR" are interchangeable terms used to signify use of a nucleic acid amplification system, which multiplies the target nucleic acids being detected. Examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system. Other methods recently described and known to the person of skill in the art are the nucleic acid sequence based amplification (NASBA™, Cangene, Mississauga, Ontario) and Q Beta Replicase systems. The products formed by said amplification reaction may or may not be monitored in real time or only after the reaction as an end point measurement.
As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" refer to primers, probes, oligomer fragments to be detected, oligomer controls and unla- belled blocking oligomers and shall be generic to polydeoxyribonucleotides (contain- ing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), and to any other type of polynucleotide which is an N glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases. There is no intended distinction in length between the term "nucleic acid", "polynucleotide" and "oligonucleotide", and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single stranded RNA. The oligonucleotide is comprised of a sequence of approximately at least 3 nucleotides, preferably at least about 6 nucleotides, and more preferably at least about 8 - 30 nucleotides corresponding to a region of the designated target nucleotide sequence. "Corresponding" means identical to or com- plementary to the designated sequence. The oligonucleotide is not necessarily physically derived from any existing or natural sequence but may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription or a combination thereof.
The terms "oligonucleotide" or "nucleic acid" intend a polynucleotide of genomic DNA or RNA, cDNA, semi synthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature; and (3) is not found in nature. Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5'-phosphate of one mononucleotide pentose ring is attached to the 3" oxygen of its neighbour in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the "51 end" if its 51 phosphate is not linked to the 3' oxygen of a mononucleotide pen- tose ring and as the "3' end" if its 3' oxygen is not linked to a 5" phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have a 51 and 3' ends. When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, the 3' end of one oligonucleo- tide points toward the 5' end of the other; the former may be called the "upstream" oligonucleotide and the latter the "downstream" oligonucleotide.
By the term "SBC nucleobases" is meant "Selective Binding Complementary" nu- cleobases, i.e. modified nucleobases that can make stable hydrogen bonds to their complementary nucleobases, but are unable to make stable hydrogen bonds to other SBC nucleobases. As an example, the SBC nucleobase A', can make-a stable hydrogen bonded pair with its complementary unmodified nucleobase, T. Likewise, the SBC nucleobase T' can make a stable hydrogen bonded pair with its complementary unmodified nucleobase, A. However, the SBC nucleobases A' and T' will form an unstable hydrogen bonded pair as compared to the base pairs A'-T and A-T'. Likewise, a SBC nucleobase of C is designated C and can make a stable hydrogen bonded pair with its complementary unmodified nucleobase G1 and a SBC nucleobase of G is designated G' and can make a stable hydrogen bonded pair with its complementary unmodified nucleobase C, yet C and G' will form an unstable hydrogen bonded pair as compared to the base pairs C-G and C-G'. A stable hydrogen bonded pair is ob- tained when 2 or more hydrogen bonds are formed e.g. the pair between A' and T, A and T1, C and G', and C and G. An unstable hydrogen bonded pair is obtained when 1 or no hydrogen bonds is formed e.g. the pair between A' and T', and C and G'. Especially interesting SBC nucleobases are 2,6-diaminopurine (A', also called D) together with 2-thio-uracil (U1, also called 2SU)(2-thio-4-oxo-pyrimidine) and 2-thio- thymine (T', also called 2ST)(2-thio-4-oxo-5-methyl-pyrimidine). "SBC LNA oligomer" refers to a "LNA oligomer" containing at least one LNA monomer where the nucleobase is a "SBC nucleobase". By "LNA monomer with an SBC nucleobase" is meant a "SBC LNA monomer". Generally speaking SBC LNA oligomers include oligomers that besides the SBC LNA monomer(s) contain other modified or naturally occurring nu- cleotides or nucleosides. By "SBC monomer" is meant a non-LNA monomer with a SBC nucleobase. By "isosequential oligonucleotide" is meant an oligonucleotide with the same sequence in a Watson-Crick sense as the corresponding modified oligonucleotide e.g. the sequences agTtcATg is equal to agTscD2SUg where s is equal to the SBC DNA monomer 2-thio-t or 2-thio-u, D is equal to the SBC LNA monomer LNA-D and 2SU is equal to the SBC LNA monomer LNA 2SU.
The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 51 end of one sequence is paired with the 3' end of the other, is in "antiparallel association." Bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention include, for example, inosine and 7-deazaguanine. Complementarity may not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, percent concentration of cytosine and guanine bases in. the oligonucleotide, ionic strength, and incidence of mismatched base pairs.
The melting temperature, or "Tm" measures stability of a nucleic acid duplex. The Tm of a particular nucleic acid duplex under specified conditions is the temperature at which half of the duplexes have disassociated.
As defined herein, "5'→3' nuclease activity" or "51 to 3' nuclease activity" refers to that activity of a template-specific nucleic acid polymerase including either a 5'→3' ex- onuclease activity traditionally associated with some DNA polymerases whereby nucleotides are removed from the 51 end of an oligonucleotide in a sequential manner, (i.e., E. coli DNA polymerase I has this activity whereas the Klenow fragment does not), or a 5'→3' endonuclease activity wherein cleavage occurs more than one nucleotide from the 51 end, or both.
"Thermostable nucleic acid polymerase" refers to an enzyme which is relatively stable to heat when compared, for example, to polymerases from E. coli and which catalyzes the polymerization of nucleosides. Generally, the enzyme will initiate synthesis at the 3'-end of the primer annealed to the target sequence, and will proceed in the 5'-direction along the template, and if possessing a 51 to 3' nuclease activity, hydro- lyzing or displacing intervening, annealed probe to release both labelled and unla- belled probe fragments or intact probe, until synthesis terminates. A representative thermostable enzyme isolated from Thermus aquaticus (Taq) is described in U.S. Pat. No. 4,889,818 and a method for using it in conventional PCR is described in Saiki et al., (1988), Science 239:487.
"Thermostable Reverse transciptase" refers to a reverse transcriptase enzyme, which is more heat-stable compared to, for example the Avian Myeloma Virus (AMV) reverse transcriptase or the Moloney Monkey Leukaemia Virus (MMLV) reverse transcriptase.
The term "nucleobase" covers the naturally occurring nucleobases adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) as well as non-naturally occurring nucleobases such as xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7- deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin, N6,N6-ethano-2,6- diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynyl-cytosine, 5-fluorouracil, 5- bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanine, inosine and the "non-naturally occurring" nucleobases described in Benner et al., U.S. Patent No. 5,432,272 and Susan M. Freier and Karl-Heinz
Altmann, Nucleic Acid Research,25: 4429-4443, 1997. The term "nucleobase" thus includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Further naturally and non naturally occurring nucleobases include those disclosed in U.S. Patent No. 3,687,808; in chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993; in Englisch, et al., Angewandte Chemie, International Edition, 30: 613-722, 1991 (see, especially pages 622 and 623, and in the Concise Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed., John Wiley & Sons, pages 858-859, 1990, Cook, Anti-Cancer DrugDesign 6: 585-607, 1991 , each of which are hereby incorporated by reference in their entirety).
The term "nucleosidic base" or "nucleobase analogue" is further intended to include heterocyclic compounds that can serve as like nucleosidic bases including certain "universal bases" that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases. Especially mentioned as a universal base is 3-nitropyrrole or a 5-nitroindole. Other preferred compounds include pyrene and pyridyloxazole derivatives, pyrenyl, pyrenylmethylglycerol derivatives and the like. Other preferred universal bases include, pyrrole, diazole or triazole derivatives, including those universal bases known in the art. "Universal base" refers to a naturally-occurring or desirably a non-naturally occurring compound or moiety that can pair with at least one and preferably all natural bases (e.g., adenine, guanine, cytosine, uracil, and/or thymine), and that has a Tm differential of 15, 12, 10, 8, 6, 4, or 2oC or less as described herein.
By "oligonucleotide," "oligomer," or "oligo" is meant a successive chain of monomers (e.g.; glycosides of heterocyclic bases) connected via intemucleoside linkages. The linkage between two successive monomers in the oligo consist of 2 to 4, desirably 3, groups/atoms selected from -CH2-, -O-, -S-, -NRH-, >C=O, >C=NRH, >C=S, -Si(R")2-, -SO-, -S(O)2-, -P(O)2-, -PO(BH3)-, -P(O.S)-, -P(S)2-, -PO(R11)-, -PO(OCH3)-, and -PO(NHRH)-, where RH is selected from hydrogen and C1^-alkyl, and R" is selected from C1-6-alkyl and phenyl. Illustrative examples of such linkages are -CH2-CH2-CH2-, -CH2-CO-CH2-, -CH2-CHOH-CH2-, -O-CH2-O-, -O-CH2-CH2-, -O-CH2-CH= (including R5 when used as a linkage to a succeeding monomer), -CH2-CH2-O-, -NRH-CH2-CH2-, -CH2-CH2-NRH-, -CH2-NRH-CH2-, -O-CH2-CH2- NRH-, -NRH-CO-O-, -NRH-CO-NRH-, -NRH-CS-NRH-, -NRH-C(=NRH)-NRH-,
-NRH-CO-CH2-NRH-, -O-CO-O-, -O-CO-CH2-O-, -O-CH2-CO-O-, -CH2-CO-NRH-, - O-CO-NRH-, -NRH-CO-CH2-, -O-CH2-CO-NRH-, -O-CH2-CH2-NRH-, -CH=N-O-, -CH2-NRH-O-, -CH2-O-N= (including R5 when used as a linkage to a succeeding monomer), -CH2-O-NRH-, -CO-NRH-CH2-, -CH2-NRH-O-, -CH2-NRH-CO-, -O-NRH-CH2-, -O-NRH-, -O-CH2-S-, -S-CH2-O-, -CH2-CH2-S-, -O-CH2-CH2-S-, -S- CH2-CH= (including R5 when used as a linkage to a succeeding monomer), -S-CH2- CH2-, -S-CH2-CH2-O-, -S-CH2-CH2-S-, -CH2-S-CH2-, -CH2-SO-CH2-, -CH2- SO2-CH2-, -O-SO-O-, -O-S(O)2-O-, -O-S(O)2-CH2-, -O-S(O)2-NRH-, -NRH-S(O)2-CH2-, -O-S(O)2-CH2-, -O-P(O)2-O-, -0-P(O1S)-O-, -0-P(S)2-0-, -S-P(0)2-0-, -S-P(O1S)-O-, -S-P(S)2-0-, -0-P(0)2-S-, -0-P(O1S)-S-, -0-P(S)2-S-, -S-P(0)2-S-, -S-P(O1S)-S-, -S-P(S)2-S-, -O-PO(R")-O-, -0-P0(0CH3)-0-, -0-P0- (OCH2CH3)-O-, -O-PO(OCH2CH2S-R)-O-, -0-P0(BH3)-0-, -0-PO(NHRN)-O-, -O- P(0)2-NRH-, -NRH-P(0)2-0-, -0-P(O1NRH)-O-, -CH2-P(O)2-O-, -O-P(O)2-CH2-, and -O-Si(R")2-O-; among which -CH2-C0-NRH-. -CH2-NRH-0-, -S-CH2-0-, -O- P(0)2-0-. -0-P(O1S)-O-, -0-P(S)2-0-, -NRH-P(0)2-0-, -0-P(O1NRH)-O-,
-O-PO(R")-O-, -0-P0(CH3)-0-, and -0-PO(NHRN)-O-, where RH is selected form hydrogen and C1-4-alkyl, and R" is selected from C1-6-alkyl and phenyl, are especially desirable. Further illustrative examples are given in Mesmaeker et. a/., Current Opinion in Structural Biology 1995, 5, 343-355 and Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol 25, pp 4429-4443. The left-hand side of the internucleoside linkage is bound to the 5-membered ring as substituent P* at the 3'-position, whereas the right-hand side is bound to the 5'-position of a preceding monomer.
By "LNA" or "LNA monomer" (e.g., an LNA nucleoside or LNA nucleotide) or an LNA oligomer (e.g., an oligonucleotide or nucleic acid) is meant a nucleoside or nucleotide analogue that includes at least one LNA monomer. LNA monomers as disclosed in PCT Publication WO 99/14226 are in general particularly desirable modified nucleic acids for incorporation into an oligonucleotide of the invention. Additionally, the nucleic acids may be modified at either the 3' and/or 5' end by any type of modification known in the art. For example, either or both ends may be capped with a protecting group, attached to a flexible linking group, attached to a reactive group to aid in attachment to the substrate surface, etc. Desirable LNA monomers and their method of synthesis also are disclosed in US 6,043,060, US 6,268,490, PCT Publications WO 01/07455, WO 01/00641 , WO 98/39352, WO 00/56746, WO 00/56748 and WO 00/66604 as well as in the following papers: Morita et al., Bioorg. Med. Chem. Lett. - 12(1 ):73=76, 2002; Hakansson et al., Bioorg. Med. Chem. Lett. 11(7):935-938, 2001; Koshkin et al., J. Org. Chem. 66(25):8504-8512, 2001 ; Kvaerno et al., J. Org. Chem. 66(16):5498-5503, 2001; Hakansson et al., J. Org. Chem. 65(17):5161-5166, 2000; Kvaerno et al., J. Org. Chem. 65(17):5167-5176, 2000; Pfundheller et al., Nucleo- sides Nucleotides 18(9):2017-2030, 1999; and Kumar et al., Bioorg. Med. Chem. Lett. 8(16):2219-2222, 1998.
Preferred LNA monomers, also referred to as "oxy-LNA" are LNA monomers which include bicyclic compounds as disclosed in PCT Publication WO 03/020739 wherein the bridge between R4 and R2 as shown in formula (I) below together designate - CH2-O- or -CH2-CH2-O-.
By "LNA modified oligonucleotide" or "LNA substituted oligonucleotide" is meant a oligonucleotide comprising at least one LNA monomer of formula (I), described infra, having the below described illustrative examples of modifications:
Figure imgf000031_0001
wherein X is selected from -O-, -S-, -N(RN)-, -C(R6R6*)-, -0-C(R7R7*)-, -C(R6R6*)-O-, - S-C(R7R7*)-, -C(R6R6*)-S-, -N(RN*)-C(R7Rr)-, -C(R6R6*)-N(RN*)-, and -C(R6R6*)- C(R7R7*).
B is selected from a modified base as discussed above e.g. an optionally substituted carbocyclic aryl such as optionally substituted pyrene or optionally substituted pyrenylmethylglycerol, or an optionally substituted heteroalicylic or optionally substituted heteroaromatic such as optionally substituted pyridyloxazole, optionally substituted pyrrole, optionally substituted diazole or optionally substituted triazole moieties; hydrogen, hydroxy, optionally substituted C1-4-alkoxy, optionally substituted C^-alkyl, optionally substituted C1-4-acyloxy, nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.
P designates the radical position for an internucleoside linkage to a succeeding monomer, or,a 5'-terminal group, such internucleoside linkage or 5'-terminal group optionally including the substituent R5. One of the substituents R2, R2*, R3, and R3* is a group P* which designates an internucleoside linkage to a preceding monomer, or a 273'-terminal group. The substituents of R1*, R4*, R5, R5*, R6, R6*, R7, R7*, RN, and the ones of R2, R2*, R3, and R3* not designating P* each designates a biradical comprising about 1-8 groups/atoms selected from -C(RaRb)-, -C(Ra)=C(Ra)-, -C(Ra)=N-, - C(Ra)-O-, -O-, -Si(Ra)2-, -C(Ra)-S, -S-, -SO2-, -C(Ra)-N(Rb)-, -N(Ra)-, and >C=Q, wherein Q is selected from -O-, -S-, and -N(Ra)-, and Ra and Rb each is independently selected from hydrogen, optionally substituted Ci.i2-alkyl, optionally substituted C2-i2-alkenyl, optionally substituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, QM2- alkenyloxy, carboxy, C1-12-alkoxycarbonyl, d.^-alkylcarbonyl, formyl, aryl, aryloxy- carbonyl, aryloxy, arylcarbonyl, heteroaryl, hetero-aryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(Ci-6-alkyl)-amino-carbonyl, amino-Ci^-alkyl-aminocarbonyl, mono- and di(C1-6- alky^amino-Ci-e-alkyl-aminocarbonyl, Ci-6-alkyl-carbonylamino, carbamido, Ci-6- alkanoyloxy, sulphono, C^-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents Ra and Rb together may designate optionally substituted methylene (=CH2), and wherein two non- geminal or geminal substituents selected from Ra, Rb, and any of the substituents R1*, R2, R2*, R3, R3*, R4*, R5, R5*, R6 and R6*, R7, and R7* which are present and not involved in P1 P* or the biradical(s) together may form an associated biradical selected from biradicals of the same kind as defined before; the pair(s) of non-geminal sub- stituents thereby forming a mono- or bicyclic entity together with (i) the atoms to which said non-geminal substituents are bound and (ii) any intervening atoms.
Each of the substituents Rr,.R2, R2*, R3, R4*, R5, R5*, R6 and R6*, R7, and R7* which are present and not involved in P, P* or the biradical(s), is independently selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-i2-alkenyl, optionally substituted C2-i2-alkynyl, hydroxy, C^^-alkoxy, C2-i2-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, d.^-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, aryl- carbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and
Figure imgf000033_0001
carbamoyl, mono- and di(C1-6-alkyl)-amino- carbonyl, amino-C^-alkyl-aminocarbonyl, mono- and di(Ci^-alkyl)amino-Ci-6-alkyl- aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido,
Figure imgf000033_0002
sulphono, Ci- 6-alkylsulphonyloxy, nitro, azido, sulphanyl, C^-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene, or together may form a spiro biradical consisting of a 1-5 carbon atom(s) alkylene chain which is optionally interrupted and/or terminated by one or more heteroatoms/groups selected from -O-, -S-, and -(NRN)- where RN is selected from hydrogen and Ci-4-alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and RN\ when present and not involved in a biradical, is selected from hydrogen and C1-4- alkyl; and basic salts and acid addition salts thereof.
Exemplary 51, 31, and/or 2' terminal groups include -H, -OH, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., phenyl or benzyl), alkyl (e.g., methyl or ethyl), alkoxy (e.g., methoxy), acyl (e.g. acetyl or benzoyl), aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, ary- loxycarbonyl, aralkoxycarbonyl, acylamino, aroylamino, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, arylthio, het- eroarylthio, aralkylthio, heteroaralkylthio, amidino, amino, carbamoyl, sulfamoyl, al- kene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trityl(triphenylmethyl)), linkers (e.g., a linker containing an amine, ethylene glycol, quinone such as anthraquinone), detectable labels (e.g., radiolabels or fluorescent labels), and biotin.
It is understood that references herein to a nucleic acid unit, nucleic acid residue, LNA monomer, or similar term are inclusive of both individual nucleoside units and nucleotide units and nucleoside units and nucleotide units within an oligonucleotide.
A "modified base" or other similar terms refer to a composition (e.g., a non-naturally occurring nucleobase or nucleosidic base), which can pair with a natural base (e.g., adenine, guanine, cytosine, uracil, and/or thymine) and/or can pair with a non- naturally occurring nucleobase or nucleosidic base. Desirably, the modified base provides a Tm differential of 15, 12, 10, 8, 6, 4, or 20C or less as described herein. Exemplary modified bases are described in EP 1 072 679 and WO 97/12896.
The term "chemical moiety" refers to a part of a molecule. "Modified by a chemical moiety" thus refer to a modification of the standard .molecular structure by inclusion of an unusual chemical structure. The attachment of said structure can be covalent or non-covalent.
The term "inclusion of a chemical moiety" in an oligonucleotide probe thus refers to attachment of a molecular structure. Such as chemical moiety include but are not limited to covalently and/or non-covalently bound minor groove binders (MGB) and/or intercalating nucleic acids (INA) selected from a group consisting of asymmetric cya- nine dyes, DAPI, SYBR Green I, SYBR Green II, SYBR Gold, PicoGreen, thiazole orange, Hoechst 33342, Ethidium Bromide, 1-O-(1-pyrenylmethyl)glycerol and Hoechst 33258. Other chemical moieties include the modified nucleobases, nucleosidic bases or LNA modified oligonucleotides.
The term "Dual-labelled probe" refers to an oligonucleotide with two attached labels. In one aspect, one label is attached to the 5' end of the probe molecule, whereas the other label is attached to the 3' end of the molecule. A particular aspect of the invention contain a fluorescent molecule attached to one end and a molecule which is able to quench this fluorophore by Fluorescence Resonance Energy Transfer (FRET) attached to the other end. 5' nuclease assay probes and some Molecular Beacons are examples of Dual labelled probes. "5' nuclease assay probe" refers to a dual labelled probe which may be hydrolyzed by the 5'-3" exonuclease activity of a DNA polymerase. A 5' nuclease assay probes is not necessarily hydrolyzed by the 5'-3' exonuclease activity of a DNA polymerase under the conditions employed in the particular PCR assay. The name "5' nuclease assay" is used regardless of the degree of hydrolysis observed and does not indicate any expectation on behalf of the experimenter. The term "5' nuclease assay probe" and "5' nuclease assay" merely refers to assays where no particular care has been taken to avoid hydrolysis of the involved probe. "5' nuclease assay probes" are often referred to as a "TaqMan assay probes", and the "5' nuclease assay" as "TaqMan assay". These names are used interchangeably in this application.
"Oligonucleotide analogue" refers to a nucleic acid binding molecule capable of recognizing a particular target nucleotide sequence. A particular oligonucleotide analogue is peptide nucleic acid (PNA) in which the sugar phosphate backbone of an oligonucleotide is replaced by a protein like backbone. In PNA, nucleobases are at- tached to the uncharged polyamide backbone yielding a chimeric pseudopeptide- nucleic acid structure, which is homomorphous to nucleic acid forms.
"Molecular Beacon" refers to a single or dual labelled probe which is not likely to be affected by the 5'-3' exonuclease activity of a DNA polymerase. Special modifications to the probe, polymerase or assay conditions have been made to avoid separation of the labels or constituent nucleotides by the 5'-3" exonuclease activity of a DNA polymerase. The detection principle thus rely on a detectable difference in label elicited signal upon binding of the molecular beacon to its target sequence. In one aspect of the invention the oligonucleotide probe forms an intramolecular hairpin structure at the chosen assay temperature mediated by complementary sequences at the 5'- and the 3'-end of the oligonucleotide. The oligonucleotide may have a fluorescent molecule attached to one end and a molecule attached to the other, which is able to quench the fluorophore when brought into close proximity of each other in the hairpin structure. In another aspect of the invention, a hairpin structure is not formed based on complementary structure at the ends of the probe sequence instead the detected signal change upon binding may result from interaction between one or both of the labels with the formed duplex structure or from a general change of spatial conformation of the probe upon binding - or from a reduced interaction between the labels after binding. A particular aspect of the molecular beacon contain a number of LNA residues to inhibit hydrolysis by the 5'-3' exonuclease activity of a DNA polymerase. "High affinity nucleotide analogue" refers to a non-naturally occurring nucleotide analogue that increases the "binding affinity" of an oligonucleotide probe to its complementary recognition sequence when substituted with at least one such high-affinity nucleotide analogue.
As used herein, a probe with an increased "binding affinity" for a recognition sequence compared to a probe which comprises the same sequence but does not comprise a stabilizing nucleotide, refers to a probe for which the association constant (Ka) of the probe recognition segment is higher than the association constant of the complementary strands of a double-stranded molecule. In another preferred em- bodiment, the association constant of the probe recognition segment is higher than the dissociation constant (Kd) of the complementary strand of the recognition sequence in the target sequence in a double stranded molecule.
Monomers are referred to as being "complementary" if they contain nucleobases that can form hydrogen bonds according to Watson-Crick base-pairing rules (e.g. G with C, A with T or A with U) or other hydrogen bonding motifs such as for example dia- minopurine with T, 5-methyl C with G, 2-thiothymidine with A, inosine with C, pseu- doisocytosine with G, etc.
The term "succeeding monomer" relates to the neighbouring monomer in the 51- terminal direction and the "preceding monomer" relates to the neighbouring monomer in the 31 -terminal direction.
The term "target nucleic acid" or "target ribonucleic acid" refers to any relevant nucleic acid of a single specific sequence, e. g., a biological nucleic acid, e. g., derived from a patient, an animal (a human or non-human animal), a plant, a bacteria, a fungi, an archae, a cell, a tissue, an organism, etc. For example, where the target ribonucleic acid or nucleic acid is derived from a bacteria, archae, plant, non-human animal, cell, fungi, or non-human organism, the method optionally further comprises selecting the bacteria, archae, plant, non-human animal, cell, fungi, or non-human organism based upon detection of the target nucleic acid. In one embodiment, the target nucleic acid is derived from a patient, e.g., a human patient. In this embodi- ment, the invention optionally further includes selecting a treatment, diagnosing a disease, or diagnosing a genetic predisposition to a disease, based upon detection of the target nucleic acid. "Target sequence" refers to a specific nucleic acid sequence within any target nucleic acid.
The term "stringent conditions", as used herein, is the "stringency" which occurs within a .range from about Tm-5° C. (5° C. below the melting temperature (Tm) of the probe) to about 20° C. to 25° C. below Tm. As will be understood by those skilled in the art, the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences. Hybridization techniques are generally described in Nucleic Acid Hybridization, A Practical Approach, Ed. Hames, B. D. and Higgins, S. J., IRL Press, 1985; Gall and Pardue, Proc. Natl. Acad. ScL, USA 63: 378-383, 1969; and John, et al. Nature 223: 582-587, 1969.
The present invention also provides a kit for the isolation, purification, amplification, detection, identification, quantification, or capture of natural or synthetic nucleic acids, where the kit comprises a reaction body and one or more LNA modified oligonucleotides (oligomer) as defined herein. The LNA modified oligonucleotides are pref- erably immobilised onto said reactions-body.. - -
For the kits according to the invention, the reaction body is preferably a solid support material, e.g. selected from borosilicate glass, soda-lime glass, polystyrene, polycarbonate, polypropylene, polyethylene, polyethyleneglycol terephthalate, polyvinylace- tate, polyvinylpyrrolidinone, polymethylmethacrylate and polyvinylchloride, preferably polystyrene and polycarbonate. The reaction body may be in the form of a specimen tube, a vial, a slide, a sheet, a film, a bead, a pellet, a disc, a plate, a ring, a rod, a net, a filter, a tray, a microtitre plate, a stick, or a multi-bladed stick.
A written instruction sheet stating the optimal conditions for use of the kit typically accompanies the kits.
Detailed Description of the Invention
The present invention relates to the use of an oligonucleotide for the isolation, purification, amplification, detection, identification, quantification, or capture of microRNA or small interfering RNAs characterized in that the oligonucleotide contains a number of nucleoside analogues. More particular the present invention provides methods for detection and quantification of microRNA or small interfering RNAs having a high sensitivity and good selectivity. According to the invention the quantification of microRNA and small interfering RNAs is detectable at levels of from 10 fmol to 10 amol RNA target or less (10 zmol) in the sample corresponding to RNA target concentration of from 100 pM to 10 fM or less (1O aM).
The detection of binding is either direct by a measurable change in the properties of one or more of the labels following binding to the target (e.g. a molecular beacon type assay with or without stem structure) or indirect by a subsequent reaction follow- ing binding, e.g. cleavage by the 5' nuclease activity of the DNA polymerase in 5' nuclease assays. The detection probe is yet another novel component of the present invention. It comprises a short oligonucleotide moiety which sequence has been selected to enable specific detection of the short amplified DNA molecules corresponding to the target sequence in the core segment and the anchored sequences used as annealing sites for the PCR primers.
The novel, short detection probes designed to detect target sequences, for example different mature miRNA target molecules, are enabled by the discovery that very short 8 - 12-mer LNA-DNA chimeric, mix-mer probes are compatible with real-time PCR based assays. In one aspect of the present invention modified or nucleobase analogues, nucleosidic bases or nucleotides are incorporated in the tagging probes as well as the detection probe, possibly together with minor groove binders and other modifications, that all aim to stabilize the duplex formed between the probes and the target molecule so that the shortest possible probe sequences can be used to hybridized and detect the target molecules. In a preferred aspect of the invention the modi- fications are incorporation of LNA residues to reduce the length of the detection probe to 8 or 9 or 10 or 11 or 12 to 14 nucleotides while maintaining sufficient stability of the formed duplex to be detectable under standard real-time PCR assay conditions. In another preferred aspect of the invention, the target recognition sequences in one or both tagging probes for the ligation reaction or the recognition sequence in the RT tagging probe or the recognition sequences in both the RT tagging probe and the 2nd strand tagging probe for the RT-PCR reaction, are substituted with LNA monomers at every second, every third or every fourth nucleotide position with at least one DNA nucleotide at the 3'-ends of both probes, respectively, allowing highly specific and sensitive hybridization even at elevated temperatures due to the in- creased duplex stability of LNA modified oligonucleotide probes to their complementary target molecules, particularly target RNA molecules.
In cells, microRNA molecules occur both as longer (over 70 nucleotides) pricursor and precursor molecules as well as in the active form of mature miRNAs (17-25 nu- cleotides). One challenge in the detection of microRNA molecules is to detect the mature form of the molecule only, which is a 17-25 bp long single strand RNA molecule.
In a preferred embodiment of the present invention, the mature miRNA functions as a primer, i.e. the miRNA is hybridized to a template and extended by an enzyme capa- ble of RNA-primed DNA-directed DNA synthesis. Secondly the detection relies on the occurence of this extension and furthermore the occurence of extension relies on having an -OH termination at the 3'end of the miRNA available at the expected distance from the annealing site to the template, which is used to ensure detection of processed mature miRNA molecules only. The principle of using the target (in this case miRNAs) as a primer in the detection reaction can be applied to other detection formats using other targets (both DNA and RNA).
General aspect of the invention
Many non-coding RNA molecules, such as microRNA molecules are very short and do not accommodate placement of primers for both reverse transcriptase, PCR am- plification and optionally a labelled detection probe for amplification and detection by PCR. One solution for accommodating this is, according to the present invention, to append additional sequence to the microRNA, preferably by a method that enables the design of mature-specific assays.
As described (cf. the Examples), such sequence(s) may be appended by means of providing (by sequence specific hybridisation) a template for a polymerase-reaction to the microRNA, and providing a polymerase (e.g. a Klenow polymerase) and nucleotides to allow extension, leading to the appending to the mature microRNA of a sequence similar in part to that of the provided template. Such appended sequences may accommodate in part primers for reverse transcriptase, for PCR amplification or for a labelled detection probe, alone or in combination with the nucleic acid sequence of the microRNA. Another means of appending additional sequence may be that of a ligation reaction. In such a reaction, an adaptor nucleic acid sequence may be attached to either the 3'-end, the 5'- end or both ends of the microRNA molecule by means of a ligation reaction. Such ligation reaction may be assisted by providing a "bridging" nucleic acid sequence comprising a nucleotide sequence specific for a terminal part of a mature target RNA sequence and a nucleotide sequence specific for terminal part of said adapter molecule such that the mature RNA target and said adaptor molecule are place in close vicinity to each other upon sequence specific hybridisation. Such sequence appended by ligation may accommodate in part primers for reverse transcrip- tase, for PCR amplification or for a labelled detection probe, alone or in combination with the nucleic acid sequence of the microRNA.
Yet another means of appending additional sequence to a target small RNA molecule may be that of a template-independent polymerase reaction. In one such an embodiment a sample of small target RNA molecules are subjected to a polymerase re- action, providing a polyA tail to all microRNAs present in the sample. This could for example be performed by using a polyA polymerase. In another such embodiment a sample of small target RNA molecules are subjected to a terminal transferase enzyme reaction, capable of providing an A, C, G or T polynucleotide tail to all microR- NAs present in the sample when respective dATP, dCTP, dGTP or dTTPs are added. Such a microRNA sample provided with a nucleotide tail of similar nucleotides may be converted to cDNA by using a primer comprising the complementary similar nucleotides in a reverse transcriptase reaction, hence providing a cDNA sample of mi- croRNAs with an appended polynucleotide tail of similar nucleotides. By overlapping part of the micro RNA sequence the RT-primer may also be specific for a specific mi- croRNA or a group or family of microRNAs. Such a cDNA sample may subsequently serve a template for a PCR amplification reaction using primers specific for specific microRNA sequences, encompassed within the mature microRNA sequence or partly overlapping the sequence appended by means of a template independent polymerase reaction.
A broad aspect of the invention thus relates to a method for quantitative determination of a short-length RNA (which can be any of the small RNA types described herein), which has a length of at most 100 nucleotides, comprising a) preparing, from a sample comprising said short-length RNA, a template polynucleotide which consists of 1) a single stranded target sequence consisting of the se- quence of said short-length RNA, its correponding DNA sequence or a nucleotide sequence complementary to the sequence of said short-length RNA and 2) a 5' and/or a 3' adjacent nucleotide sequence, b) using said template polynucleotide in a reverse transcription or a nucleotide po- lymerization to obtain a strand of cDNA, and c) performing a quantitative real-time PCR (qPCR) including as template(s) said cDNA and optionally the template polynucleotide.
This aspect of the invention reflects the underlying concept of the invention, namely that specific detection of short-length RNA can be accomplished by ensuring a rela- tively high degree of specificity in all of steps a to c and that the specificity in each step adds to the general specificity of the method. One main characteristic is the provision of the template polynucleotide in step a, where said template includes appended sequences which can serve as "handles" for primers in the subsequent steps, thus providing space for all primers necessary and for the detection probes used. As will appear from the description herein, these "handles" can be both specific and non-specific for the short-length RNA one desires to quantify - in the case of specific sequences, these are appended in a reaction that preferentially or specifically will add the sequences to the short-length RNA but not to sequences which include the short-length RNA.
When using the term "corresponding to" is in the present context meant that a nucleotide sequence that corresponds to a reference nucleotide sequence is either identical to the reference sequence or constitutes a sequence that is hybridizes stringently to a sequence complementary to the reference nucleotide sequence. Typically, this means that an RNA sequence can correspond to a DNA sequence if the complementary sequence to the DNA sequence can be transcribed to the RNA sequence in question.
The term "cDNA" in this context means a DNA fragment which is obtained by means of either reverse transcription of the template polynucleotide or by means of nucleotide polymerization (such a DNA polymerization) based on the template nucleotide.
The short-length RNA is as mentioned at most 100 nucleotides, but much shorter RNA can be determined by means of the method. RNA having lengths of at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, and at most 25 nucleotide residues can conveniently be determined by means of the present methods and kits, but even shorter RNAs such as those having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 nucleotide residues. Preferably, the short-length RNAs have lengths between 16 and 25 nucleotide residues.
The primers used for the qPCR in step c are in one embodiment selected from
- at least 2 oligonucleotides, wherein at least one of said oligonucleotides corresponds to or is complementary to a sequence in the 5' or 3' adjacent nucleotide sequence - an embodiment which, especially if both primers relate to the adjacent sequences, benefits from the existence in steps a and b of sequence specific (for the short-length RNA or a sequence derived therefrom) appending of the 5' and/or 3' sequences and/or that step b has utilised an approach specific for the short-length RNA;
- at least 2 oligonucleotides, wherein at least one of said oligonucleotides corresponds to or is complementary to a contiguous sequence in the template polynucleo- - tide constituted by part of the single stranded target sequence and part of the adjacent 5' or 3' nucleotide sequence - an embodiment, where a relatively high degree of specificity is present in step c due to the specific recognition of part of the short- length RNA (or a sequence derived therefrom) and where it may be advantageous that the 5' or 31 nucleotide sequence has been appended based on a sequence spe- cific approach and/or that step b has utilised an approach specific for the short-length RNA; and
- at least 2 oligonucleotides, wherein one corresponds to a first nucleotide sequence in the single stranded target sequence and the other is complementary to a second nucleotide sequence in the single stranded target sequence - an embodiment, where a high degree of specificity is present in step c due to the specific recognition of the short-length RNA (or a sequence derived therefrom).
Said primers used for the qPCR may each independently include a detectable label.
In another embodiment, the reaction in step (b) utilises a reverse transcription primer or a DNA poymerization primer which corresponds to or is complementary to the sin- gle stranded target sequence or which corresponds to or is complementary to a contiguous sequence in the template polynucleotide constituted by part of the single stranded target sequence and part of the adjacent 5' or 3' nucleotide sequence. It is preferred that the reverse transcription primer or nucleotide polymerization primer is specific for at least one short-length RNA; this reflects the fact that a number of short- length RNAs falls in certain families having a high degree of sequence identity.
The appended 5' and/or a 3' adjacent nucleotide sequence is in some embodiments a polynucleotide consisting of identical nucleotides (an effect which can be attained by utilising terminal transferase enzymes for appending the sequence or, alternatively by utilising a polymerase which adds identical nucleotide residues).
At any rate, the single stranded target sequence and the 5' and/or a 3' adjacent nucleotide sequence(s) may be covalently joined but also non-covalently joined - the important issue is whether the template sequence can be subjected to reverse tran- scription or nucleotide polemerization in step b.
The 5' and/or a 3' adjacent nucleotide sequence in some embodiments include(s) a detectable label, thus facilitating subsequent detection.
In most embodiments the 5' and/or 3' adjacent nucleotide sequence is joined to the single stranded target sequence by an enzymatic reaction, but also non-enzymatic reactions are envisaged.
Useful enzymes for adding identical nucleotides include, using the IUBMB Enzyme Nomenclature are provided in the following:
Transferases: EC 2.7.7.19 (polynucleotide adenylyltransferase), EC 2.7.7.52 (RNA uridylyltransferase), and EC 2.7.7.31 (DNA nucleotidylexotransferase).
Ligases: EC 6.5.1.1 (DNA ligase (ATP)), EC 6.5.1.2 (DNA ligase (NAD+)), and EC 6.5.1.3 (RNA ligase (ATP)).
In certain embodiments, the 5' and/or 31 adjacent nucleotide sequence does not occur naturally in the organism from where the sample RNA is derived. This is believed to reduce the risk of detecting irrelevant sequences in the sample. It is preferred that the 51 and/or 3' adjacent nucleotide sequence is non-mammalian.
In other embodiments, step (a) comprises preparation of the template polynucleotide by ligation of the 5' and/or 31 adjacent nucleotide sequence to the short-length RNA1 or step (a) comprises preparation of the template polynucleotide by joining the 5' and/or 3' adjacent nucleotide sequence to the short-length RNA in a terminal transferase reaction, preferably in a poly-A transferase reaction. The ligation can be both sequence specific (e.g. overhang ligation) and blunt-end ligation, but it is preferred to utilise overhang ligation. In a preferred version of overhang ligation, the method involves annealing, to the short-length RNA, an oligonucleotide in part complementary to the ligase-reactive end of the 5' or 3' adjacent nucleotide sequence and in part complementary to the ligase-reative end of the short-length RNA molecule so as to position the ligase-reactive end of the 5' or 3' adjacent nucleotide sequence directly adjacent to the ligase-reative end of the small RNA molecule to allow overhang ligation.
One main advantage of using ligation or terminal transferases is that all RNA in the sample can be rendered useful for the subsequent steps (which then, on the other hand, should be highly specific). This enables creation of e.g. a non-specific cDNA library which can later be used for the more specific steps in b and c.
Typically, ligation or the terminal transferase reaction is only performed at the 3' end of the target sequence, but ligation to the 51 end of the target sequence can be performed by phosphorylating the 5' end of the target sequence prior to the ligation reaction. At any rate, in order to avoid "self-ligation" of the adjacent nucleotide se- quences, it is preferred to block one of the termini (since ligases require 3'-hydroxyl and 5'-phosphate in the molecules to be ligated, this is a fairly easy task for the skilled person). Hence, the 5' adjacent nucleotide sequence is blocked at its 5' terminus and the 3' adjacent nucleotide sequence is blocked at its 31 terminus prior to ligation, and since these two nucleotide sequences are normally added in separate steps, it is avoided that they self-ligate.
The 5' and/or 3' adjacent nucleotide sequence(s) is/are preferentially or exclusively joined to a defined processing state of said short-length RNA in step (a). This is to mean that the means for appending the adjacent nucleotide sequence utilises a sequence coupling step which depends on the presence of a free 3' or 5' end in the short-length RNA (whereby discrimination is introduced over e.g. a pre-mature RNA that includes the same sequence but not in its relevant terminus). It is preferred that the defined processing state of said RNA is the mature state. Instead of utilising ligation or terminal transferases, step (a) may comprise a step of nucleotide polymerization to attach the adjacent nucleotide sequences. The polymerase used for this purpose can be both a template-independent and a template- dependent polymerase. Typcically employed polymerases are DNA polymerases.
-Even though preferred embodiments utilise polymerization which is template specific, the polymerization may also consist in addition of a poly-A, poly-G, poly-T or a poly-C tail to the 3' end of the target sequence.
However, as mentioned, the currently preferred embodiments entail use of template specific approaches. In the cases of detection of microRNA, it is one object of the invention to be able to discriminate between mature and pre-mature microRNA, and in this context it is important to look at two different situations: the situation where the microRNA is situated in the 3' terminus of its premature precursor and the situation where the microRNA is situated in the 5' terminus of the premature precursor. To discriminate the mature forms from each of thes precursors, different approaches _ have to be used.
In accordance with the description of this general aspect of the invention, the present invention also relates to a kit useful in the quantitative determination of mature short- length RNA having a length of at most 100 nucleotides, said kit comprising - the minimum number of reverse transcription primers and/or nucleotide polymeriza- tion primers and/or primers for qPCR and/or oligonucleotide capture probes and/or helper oligonucleotides and/or oligonucleotide probes, which are used in a method described herein, wherein the reverse transcription primers, nucleotide polymerization primers, primers for qPCR, oligonucleotide capture probes, helper oligonucleotides, and oligonucleotide probes share the features described above; and
- instructions for quantitative determination of the mature short-length RNA using the the reverse transcription primers and/or nucleotide polymerization primers and/or primers for qPCR and/or oligonucleotide capture probes and/or helper oligonucleotides and/or oligonucleotide probes. All disclosures relating to the provision of kits apply mutatis mutandis do this kit.
The kit may further comprise one or more enzymes and other reagents as described herein. As an example os such a "minimal kit", the following is provided (the reference primers and probes are optional):
The miR-specific assay
Biotinyleret LNA capture probe • . . miR-specific reverse primer miR-specific forward and reverse primers miR-specific dual-labeled probe
• RNA control oligonucleotide
• DNA control oligonucleotide
The reference U6 snoRNA assay
Reference U6 snoRNA RT primer/random hexamer primer Reference U6 snoRNA primers and dual-labeled probe
Oligonucleotide amount: 1 assay 10 assays concentration ._ volume
Biotinylated LNA capture probe 0.5 pmol 5 pmol 0.5 μM 1 μL miR-specific reverse primer 0.1 pmol 1 pmol 0.1 μM 1 μL miR-specific forward primer 2.025 pmol 20.25 pmol 0.9 μM 2.25 μL miR-specific reverse primer 2.025 pmol 20.25 pmol 0.9 μM 2.25 μL miR-specific dual- labeled probe 0.3125 pmol 3.125 pmol 0.25 μM 1.25 μL
RNA control oligonucleotide 0.01 pmol 0.1 pmol 0.01 μM 1 μL
DNA control oligonucleotide 0.01 pmol 0.1 pmol 0.01 μM 1 μL
Reference U6 snoRNA RT primer/random hexamer primer 2 pmol 20 pmol 2 μM 1 μL
Reference U6 snoRNA forward primer 2.025 pmol 20.25 pmol 0.9 μM 2.25 μL
Reference U6 snoRNA reverse primer 2.025 pmol 20.25 pmol 0.9 μM 2.25 μL
Reference U6 snoRNA dual- labeled probe 0.3125 pmol 3.125 pmol 0.25 μM 1.25 μL Further aspects of the invention
Once the appropriate target sequences have been selected, LNA substituted tagging probes and detection probes are preferably chemically synthesized using commercially available methods and equipment as described in the art (Tetrahedron 54: 3607-30, 1998). For example, the solid phase phosphoramidite method can be used to produce short LNA probes (Caruthers, et al., Cold Spring Harbor Symp. Quant. Biol. 47:411-418, 1982, Adams, et al., J. Am. Chem. Soc. 105: 661 (1983).
LNA-containing-probes are typically labelled during synthesis. The flexibility of the phosphoramidite synthesis approach furthermore facilitates the easy production of LNAs carrying all commercially available linkers, fluorophores and labelling- molecules available for this standard chemistry. LNA may also be labelled by enzymatic reactions e.g. by kinasing.
Detection probes according to the invention can comprise siηgle labels or a plurality of labels. In one aspect, the plurality of labels comprise a pair of labels which interact with each other either to produce a signal or to produce a change in a signal when hybridization of the detection probe to a target sequence occurs.
In another aspect, the detection probe comprises a fluorophore moiety and a quencher moiety, positioned in such a way that the hybridized state of the probe can be distinguished from the unhybridized state of the probe by an increase in the fluo- rescent signal from the nucleotide. In one aspect, the detection probe comprises, in addition to the recognition element, first and second complementary sequences, which specifically hybridize to each other, when the probe is not hybridized to a recognition sequence in a target molecule, bringing the quencher molecule in sufficient proximity to said reporter molecule to quench fluorescence of the reporter molecule. Hybridization of the target molecule distances the quencher from the reporter molecule and results in a signal, which is proportional to the amount of hybridization.
In another aspect polymerization of strands of nucleic acids can be detected using a polymerase with 5' nuclease activity. Fluorophore and quencher molecules are incorporated into the probe in sufficient proximity such that the quencher quenches the signal of the fluorophore molecule when the probe is hybridized to its recognition sequence. Cleavage of the probe by the polymerase with 5' nuclease activity results in separation of the quencher and fluorophore molecule, and the presence in increasing amounts of signal as nucleic acid sequences
Suitable samples of target nucleic acid molecules may comprise a wide range of eu- karyotic and prokaryotic cells, including protoplasts; or other biological materials, which may harbour target nucleic acids. The methods are thus applicable to tissue culture animal cells, animal cells (e.g., blood, serum, plasma, reticulocytes, lymphocytes, urine, bone marrow tissue, cerebrospinal fluid or any product prepared from blood or lymph) or any type of tissue biopsy (e.g. a muscle biopsy, a liver biopsy, a kidney biopsy, a bladder biopsy, a bone biopsy, a cartilage biopsy, a skin biopsy, a pancreas biopsy, a biopsy of the intestinal tract, a thymus biopsy, a mammae biopsy, a uterus biopsy, a testicular biopsy, an eye biopsy or a brain biopsy, e.g., homogenized in lysis buffer), archival tissue nucleic acids, plant cells or other cells sensitive to osmotic shock and cells of bacteria, yeasts, viruses, mycoplasmas, protozoa, rickettsia, fungi and other small microbial cells and the like.
Various amplifying reactions are well known to one of ordinary skill in the art and include, but are not limited to PCR, RT-PCR, LCR, in vitro transcription, rolling circle PCR, OLA and the like. Multiple primers can also be used in multiplex PCR for detecting a set of specific target molecules.
Preferably, the tagging probes as well as the detection probes of the invention are modified in order to increase the binding affinity of the probes for the target sequence by at least two-fold compared to probes of the same sequence without the modification, under the same conditions for hybridization or stringent hybridization conditions. The preferred modifications include, but are not limited to, inclusion of nucleobases, nucleosidic bases or nucleotides that have been modified by a chemical moiety or replaced by an analogue to increase the binding affinity. The preferred modifications may also include attachment of duplex-stabilizing agents e.g., such as minor-groove- binders (MGB) or intercalating nucleic acids (INA). Additionally, the preferred modifications may also include addition of non-discriminatory bases e.g., such as 5- nitroindole, which are capable of stabilizing duplex formation regardless of the nu- cleobase at the opposing position on the target strand. Finally, multi-probes composed of a non-sugar-phosphate backbone, e.g. such as PNA, that are capable of binding sequence specifically to a target sequence are also considered as a modification. All the different binding affinity-increasing modifications mentioned above will in the following be referred to as "the stabilizing modification(s)", and the tagging probes and the detection probes will in the following also be referred to as "modified oligonucleotide". More preferably the binding affinity of the modified oligonucleotide is at least about 3-fold, 4-fold, 5-fold, or 20-fold higher than the binding of a probe of the same sequence but without the stabilizing modification(s).
Most preferably, the stabilizing modification(s) is inclusion of one or more LNA nucleotide analogs. Probes from 6 to 30 nucleotides according to the invention may comprise from 1 to 8 stabilizing nucleotides, such as LNA nucleotides. When at least two LNA nucleotides are included, these may be consecutive or separated by one or more non-LNA nucleotides. In one aspect, LNA nucleotides are alpha and/or xylo LNA nucleotides.
The invention also provides a probe library comprising tagging probes and detection probes with stabilizing modifications as defined above. Preferably, the detection probes are less than about 20 nucleotides in length and more preferably less than 15 nucleotides, and most preferably about 7 or 8 or 9 or 10 or 11 nucleotides. Also, preferably, the tagging probes are less than about 40 nucleotides in length and more preferably less than 35 nucleotides, and most preferably about 20 or 30 nucleotides. Also, preferably, the tagging probes ligation reaction and the RT tagging probe and the 2nd strand tagging probe for the RT-PCR reaction are composed of a high-affinity tagging recognition sequence of less than about 15 nucleotides in length and more preferably less than 14 nucleotides, and most preferably between 6 and 13 nucleotides, and furthermore of an anchored sequence as a primer site for PCR primers of less than about 30 nucleotides in length and more preferably less than 25 nucleotides, and most preferably between 15 to 20 nucleotides. The probe libraries contain- ing labelled detection probes may be used in a variety of applications depending on the type of detection element attached to the recognition element. These applications include, but are not limited to, dual or single labelled assays such as 5' nuclease assay, molecular beacon applications (see, e.g., Tyagi and Kramer Nat. Biotechnol. 14: 303-308, 1996) and other FRET-based assays.
The problems with existing quantification assays for microRNAs, siRNAs, RNA- edited transcripts, alternative splice variants and antisense non-coding RNAs as outlined above are addressed by the use of the probes of the invention in combination with any of the methods of the invention consisting of a set of RNA tagging probes and detection probes or sets of RNA RT tagging probes combined with 2nd strand tagging probes and detection probes, selected so as to recognize or detect a majority of all discovered and detected miRNAs, RNA-edited transcripts, siRNAs, alternative splice variants and antisense non-coding RNAs in a given cell type from a given or- ganism. In one aspect, the probe library comprises probes that tag and detect mammalian mature miRNAs, e.g., such as mouse, rat, rabbit, monkey, or human miRNAs. By providing a cost-efficient useful method for quantitative real-time and end-point PCR assays for mature miRNAs, RNA-edited transcripts, siRNAs, alternative splice variants and antisense non-coding RNAs, the present invention over- comes the limitations discussed above especially for conventional miRNA assays and siRNA assays. The detection element of the detection probes according to the invention may be single or double labelled (e.g. by comprising a label at each end of the probe, or an internal position). Thus, probes according to the invention can be adapted for use in 5' nuclease assays, molecular beacon assays, FRET assays, and other similar assays. In one aspect, the detection probe comprises two labels capable of interacting with each other to produce a signal or to modify a signal, such that a signal ora change in a signal may be detected when the probe hybridizes to a target sequence. A particular aspect is when the two labels comprise a quencher and a reporter molecule.
In another aspect, the probe comprises a target-specific recognition segment capable of specifically hybridizing to a target molecule comprising the complementary recognition sequence. A particular detection aspect of the invention referred to as a "molecular beacon with a stem region" is when the recognition segment is flanked by first and second complementary hairpin-forming sequences which may anneal to form a hairpin. A reporter label is attached to the end of one complementary sequence and a quenching moiety is attached to the end of the other complementary sequence. The stem formed when the first and second complementary sequences are hybridized (i.e., when the probe recognition segment is not hybridized to its target) keeps these two labels in close proximity to each other, causing a signal produced by the reporter to be quenched by fluorescence resonance energy transfer (FRET). The proximity of the two labels is reduced when the probe is hybridized to a target sequence and the change in proximity produces a change in the interaction between the labels. Hybridization of the probe thus results in a signal (e.g. fluorescence) being produced by the reporter molecule, which can be detected and/or quantified. In yet another aspect, the target detection probe comprises a reporter and a quencher molecule at opposing ends of the short target recognition sequence, so that these moieties are in sufficient proximity to each other, that the quencher substantially reduces the signal produced by the reporter molecule. This is the case both when the probe is free in solution as well as when it is bound to the target nucleic acid. A particular detection aspect of the invention referred to as a "5' nuclease assay" is when the detection probe may be susceptible to cleavage by the 5' nuclease activity of the DNA polymerase. This reaction may possibly result in separation of the quencher molecule from the reporter molecule and the production of a detectable signal. Thus, such probes can be used in amplification-based assays to detect and/or quantify the amplification process for a target nucleic acid.
The invention also provides a method, system and computer program embedded in a computer readable medium ("a computer program product") for designing tagging probes and detection probes comprising at least one stabilizing nucleobase. The method comprises querying a database of target sequences (e.g., such as the - miRNA registry at http://www.sanger.ac.uk/Software/Rfam/mirna/index.shtml ) and designing probes which: i) have sufficient binding stability to bind their respective target sequence under stringent hybridization conditions, ii) have limited propensity to form duplex structures with itself, and iii) are capable of binding to and detect- ing/quantifying at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% of all the target sequences in the given database of.
Capture probe design program.
The invention also provides a method, system and computer program embedded in a computer readable medium ("a computer program product") for designing the se- quence of nucleotides to implement the capture probe.
The method consists of the following steps:
a) Initial guess of one or mores sequence(s) of nucleotides to implement the capture probe(s).
b) Iterative improvement of the initial guesses based on the fulfillment of conditions and aims. c) Stopping the algorithm when there is a sufficient fulfillment of the conditions and aims also including the computing time used on the current method.
The melting temperature is designated "Tm".
Detailed description of the three steps:
A) The initial guess is based on the miRNA sequence to match a list of suitable reverse primers found by using a primer finding software (primer3). Random sequences are generated to fill up not initialized parts of the capture probe. The random generation is guided by the use of di-nucleotide Tm tables to ensure sequences with Tm in the neighborhood of the aimed Tm value.
B) The iterative improvement will be directed by a scoring function based on the aims and conditions and of di-nucleotide Tm tables. Random changes are made to avoid suboptimal iteration.
C) The algorithm stops when a scoring function based on the aims, conditions and computation time is fulfilled.
The aims to obtain the primer and probe conditions listed below:
1. The melting temperature condition for the hybridization of the capture probe towards the miRNA
The melting temperature of the duplex formed by the capture probe and the miRNA is extended to be suitable for a DNA polymerase extension reaction. The oligonu- cleotide length within this duplex ought to satisfy the Tm condition for a DNA polymerase extension reaction mentioned above. The miRNA hybridized to the 3'-end of the capture probe. 2. The melting temperature condition for the duplex formed by the capture probe and the DNA polymerase-extended miRNA
The Tm of the duplex formed by the capture probe and the DNA polymerase- extended miRNA target is not allowed to exceed the temperature by means of which the heteroduplex can be denatured without destroying the RNA-DNA target.
3. The relationship between the capture probe and the reverse transcription (RT) primer
The RT primer is sequence identical to the 51 end of the capture probe and hybridizes to the 3"-end of the DNA polymerase-extended miRNA. The Tm for this duplex formed by RT primer and DNA polymerase-extended miRNA has to be suitable for a first strand synthesis using a reverse transcriptase.
4. The differentiation between the mature and precursor miRNA.
The 3'-end of the precursor miRNA is not allowed to hybridize with a significant amount of oligonucleotides to the capture probe under the given hybridization condi- tions for the capture reaction. Likewise the preceding monomers after the mature miRNA sequence motive within the precursor miRNA sequence are not allowed to hybridize to the non-miRNA-related capture probe sequence.
A general condition for every designed probe and primers is the requirement of low self-annealing and low self-hybridization.
Dual-labeled probe design program.
The invention also provides a method, system and computer program embedded in a computer readable medium ("a computer program product") for designing nucleotide sequences to implement into the dual-labeled probe. The dual-labeled probe is used for detection of a particular miRNA or a particular family of miRNA's with maximal specificity.
The dual-labeled probe must fulfill the following conditions: a) A requirement of low self-annealing and low self-hybridization.
b) Must anneal to the target by having a suitable Tm to function in the PCR reaction.
c) _ Must not anneal to the primers in the PCR reaction.
The method consist of the following steps:
A) A design of probes with maximal specificity toward miRNA or a family of miRNA's. The preferred probes that fulfil the conditions, called dual-labeled probe matches, are investigated by the ability of the dual-labeled probes to bind to other miRNA's. A dual-labeled probe match is then assigned a specificity score according to a scoring function. A sequence match, length of the sequence, and the use of LNA-modified nucleotides in the sequence determine a dual-labeled probe match.
B) Dual-labeled probe matches are scored by how well they fulfil the conditions above. The dual-labeled probes are scored by how well they fulfil the conditions above according to the scoring functions. The specificity score and the scores from the conditions are then used to deside the best nucleotide sequence of dual-labeled probe.
The quencher is preferably selected from dark quencher as disclosed in EP Application No. 2004078170.0, in particular compounds selected from 1 ,4-bis-(3-hydroxy- propylamino)-anthraquinone, 1-(3-(4,4'-dimethoxy-trityloxy)propylamino)-4-(3- hydroxypropylamino)-anthraquinone, 1-(3-(2- cyanoethoxy(diisopropylamino)phosphinoxy)propylamino)-4-(3-(4,4'-dimethoxy- trityloxy)propylamino)-anthraquinone (#Q1 ), 1 ,5-bis-(3-hydroxy-propylamino)- anthraquinone, 1-(3-hydroxypropylamino)-5-(3-(4,4'-dimethoxy- trityloxy)propylamino)-anthraquinone, 1-(3- (cyanoethoxy(diisopropylamino)phosphinoxy)propylamino)-5-(3-(4,4'-dimethoxy- trityloxy)propylamino)-anthraquinone (#Q2), 1 ,4-bis-(4-(2-hydroxyethyl)phenylamino)- anthraquinone, 1-(4-(2-(4,4'-dimethoxy-trityloxy)ethyl)phenylamino)-4-(4-(2- hydroethyl)phenylamino)-anthraquinone, 1-(4-(2-(2-cyanoethoxy(diisopropylamino)- phosphinoxy)ethyl)phenylamino)-4-(4-(2-(4,4'-dimethoxy-trityloxy)ethyl)phenylamino)- anthraquinone, and 1 ,8-bis-(3-hydroxy-propylamino)-anthraquinone; or alternatively selected from 6-methyl-Quinizarin, 1,4-bis(3-hydroxypropylamino)-6-methyl- anthraquinone, 1-(3-(4,4'-dimethoxy-trityloxy)propylamino)-4-(3-hydroxypropyl- amino)-6(7)-methyl-anthraquinone, 1-(3-(2-cyanoethoxy(diisopropylamino)- phosphinoxy)propylamino)-4-(3-(4,4'-dimethoxy-trityloxy)propylamino)-6(7)-methyl- anthraquinone, 1,4-bis(4-(2-hydroethyl)phenylamino)-6-methyl-anthraquinone, 1,4- Dihydroxy-2,3-dihydro-6-carboxy-anthraquinone, 1 ,4-bis(4-methyl-phenylamino)-6- carboxy-anthraquinone, 1 ,4-bis(4-methyl-phenylamino)-6-(N-(6,7-dihydroxy-4-oxo- heptane-1-yl))carboxamido-anthraquinone, 1,4-bis(4-methyl-phenylamino)-6-(N-(7- dimethoxytrityloxy-6-hydroxy-4-oxo-heptane-1-yl))carboxamido-anthraquinone, 1,4- Bis(4-methyl-phenylamino)-6-(N-(7-(2-cyanoethoxy(diisopropylamino)phosphinoxy)- 6-hydroxy-4-oxo-heptane-1 -yl))carboxamido-anthraquinone, 1 ,4-bis(propylamino)-6- carboxy-anthraquinone, 1 ,4-bis(propylamino)-6-(N-(6,7-dihydroxy-4-oxo-heptane-1- yl))carboxamido-anthraquinone, 1 ,4-bis(propylamino)-6-(N-(7-dimethoxytrityloxy-6- hydroxy-4-oxo-heptane-1 -yl))carboxamido-anthraquinone, 1 ,5-bis(4-(2-hydroethyl)- phenylamino)-anthraquinone, 1-(4-(2-hydroethyl)phenylamino)-5-(4-(2-(4,4'- dimethoxy-trityloxy)ethyl)phenylamino)-anthraquinone, 1-(4-(2-(cyanoethoxy- (diisopropylamino)phosphinoxy)ethyl)phenylamino)-5-(4-(2--(4,4'-dimethoxy- trityloxy)ethyl)phenylamino)-anthraquinone, 1,8-bis(3-hydroxypropylamino)- anthraquinone, 1-(3-hydroxypropylamino)-8-(3-(4,4'-dimethoxy-trityloxy)- propylamino)-anthraquinone, 1 ,8-bis(4-(2-hydroethyl)phenylamino)-anthraquinone, and 1 -(4-(2-hydroethyl)phenylamino)-8-(4-(2-(4,4'-dimethoxy- trityloxy)ethyl)phenylamino)-anthraquinone.
One preferred method for covalent coupling of oligonucleotides on different solid supports is photochemical immobilization using a photochemically active anthraquinone attached to the 5'- or 3'-end of the oligonucleotide as described in WO 96/31557 or in WO 99/14226.
In another preferred embodiment the high affinity and specificity of LNA modified oligonucleotides is exploited in the sequence specific capture and purification of natural or synthetic nucleic acids. In one aspect, the natural or synthetic nucleic acids are contacted with the LNA modified oligonucleotide immobilised on a solid surface. In this case hybridisation and capture occurs simultaneously. The captured nucleic acids may be, for instance, detected, characterised, quantified or amplified directly on the surface by a variety of methods well known in the art or it may be released from the surface, before such characterisation or amplification occurs, by subjecting the immobilised, modified oligonucleotide and captured nucleic acid to dehybridising conditions, such as for example heat or by using buffers of low ionic strength.
In another aspect the LNA modified oligonucleotide carries a ligand covalently attached to either the 5' or 3' end. In this case the LNA modified oligonucleotide is con- tacted with the natural or synthetic nucleic acids in solution whereafter the hybrids formed are captured onto a solid support carrying molecules that can specifically bind the ligand.
In one preferred aspect, the target sequence database comprises nucleic acid sequences corresponding to human, mouse, rat, Drosophila melanogaster, C. elegans, Arabidopsis thaliana, maize, fugu, zebrafish, Gallus Gallus, vira or rice miRNAs.
In another aspect, the method further comprises calculating stability based on the assumption that the recognition sequence comprises at least one stabilizing nucleotide, such as an LNA molecule. In one preferred aspect the calculated stability is " used to eliminate probes with inadequate stability from the database of virtual candi- date probes prior to the initial query against the database of target sequence to initiate the identification of optimal probe recognition sequences.
In another aspect, the method further comprises calculating the capability for a given probe sequence to form a duplex structure with itself based on the assumption that the sequence comprises at least one stabilizing nucleotide, such as an LNA mole- cule. In one preferred aspect the calculated propensity is used to eliminate probe sequences that are likely to form probe duplexes from the database of virtual candidate probes.
A preferred embodiment of the invention are kits for the detection or quantification of target miRNAs, siRNAs, RNA-edited transcripts, non-coding antisense transcripts or alternative splice variants comprising libraries of tagging probes and target detection probes. In one aspect, the kit comprises in silico protocols for their use. In another aspect, the kit comprises information relating to suggestions for obtaining inexpensive DNA primers. The probes contained within these kits may have any or all of the characteristics described above. In one preferred aspect, a plurality of probes com- prises at least one stabilizing nucleotide, such as an LNA nucleotide. In another aspect, the plurality of probes comprises a nucleotide coupled to or stably associated with at least one chemical moiety for increasing the stability of binding of the probe. The kits according to the invention allow a user to quickly and efficiently develop an assay for different miRNA targets, siRNA targets, RNA-edited transcripts, non-coding antisense transcripts or alternative splice variants.
5 . In general, the invention features the design of high affinity oligonucleotide probes that have duplex stabilizing properties and methods highly useful for a variety of target nucleic acid detection, amplification, and quantification methods (e.g., monitoring expression of microRNAs or siRNAs by real-time quantitative PCR). Some of these oligonucleotide probes contain novel nucleotides created by combining specialized
10 synthetic nucleobases with an LNA backbone, thus creating high affinity oligonucleotides with specialized properties such as reduced sequence discrimination for the complementary strand or reduced ability to form intramolecular double stranded structures. The invention also provides improved methods for detecting and quantifying nucleic acids in a complex nucleic acid sample. Other desirable modified bases
15 have decreased ability to self-anneal or to form duplexes with oligonucleotide probes ~~ ~-~ containing one or more modified bases. -
EXAMPLES
The invention will now be further illustrated with reference to the following examples. It will be appreciated that what follows is by way of example only and that modifica- 20 tions to detail may be made while still falling within the scope of the invention.
In the following Examples probe reference numbers designate the LNA- oligonucleotide sequences shown in the synthesis examples below.
Assessment of sensitivity and specificity of the real-time quantitative PCR assays for the human miR-15a microRNA target sequence.
25 Materials and methods
1. Design and synthesis of the oligonucleotide tagging probes and detection probes for microRNA detection and quantification. Table I: The design of the microRNA tagging probes, synthetic transcription templates and detection probes.
Figure imgf000058_0001
aLNA (upper cases), DNA (lower cases), RNA (italic and lower cases), 5-methyl C (mC); Fluorescein (6-FITC (Glenn Research, Prod. Id. No. 10-1964)), #Q1 (see above), z (5-nitroindole (Glenn Research, Prod. Id. No. 10-1044)), and Phosphate (P).
The human miR-15a microRNA tagging probe with the 3'-end recognition sequence was enzymatically 5'-phosphorylated in a 50 μl_ reaction using 10 U T4 polynucleo- tide kinase (New England Biolabs (NEB) USA), 400 pmol hsa-miR-15a microRNA probe 1 (EQ15848), and 1χ T4 DNA ligase buffer (NEB1 USA). The reaction was incubated 30 min at 37°C and heat inactivated 10 min at 7O0C. The kinase was removed by adding 50 μl_ DECP-treated H2O and filtering the reaction through an YM-30 Microcon spin column (Millipore, USA) 3 min 14000* g. The concentration of the phosphorylated tagging probe was determined on a NanoDrop ND-1000 (Nano- Drop technologies, USA).
2. microRNA-templated ligation reactions
The ligation reaction was performed in 20 μl_ consisting of 120 nM miR-15a RNA template (EQ15885), 120 nM of each microRNA tagging probe (phosphylated EQ15848 (see above) and EQ15849), 10 mM Tris-HCI pH 7.0 (Ambion.USA), 10 mM MgCI2 (PE Biosystems, USA), 0.05* T4 DNA ligase buffer [2.5 mM TRIS-HCI1 0.5 mM MgCI2, 0.5 mM DTT, 50 μM ATP, 1.25 μg/mL BSA, pH 7.5 @ 25°C; (NEB, USA)]. The reactions were pre-incubated for 15 min at 37°C and 800 U T4 DNA Ii- gase was added and incubated for additional 2 hours at 370C. Finally the reactions were heat-inactivated 20 min at 65°C. The ligation reaction was repeated using miR- 15a DNA (EQ15852), miR-16 RNA (EQ15886) as target or no template instead of the miR-15a RNA target. In addition to the 1:1 molar ratio of the target: microRNA tagging probes the ratios 5:1 and 1:5 were used in separate ligation reactions.
The ligation reaction performed using the Quick ligation kit (NEB, USA) was carried out according to the supplier's instructions. In brief, the oligonucleotides were the same as described above, In a 20 μl_ reaction mixture, the oligonucleotides and 1 * quick ligation buffer (NEB, USA) were incubated 15 min at 25°C and 1 μl_ Quick T4 DNA ligase (NEB, USA) was added and the incubation was prolonged for additional 30 min. The enzyme was heat-inactivated for 20 min at 65°C.
3. Real-time polymerase chain reaction (PCR) assays
3.1. MicroRNA real-time PCR assays using SYBR green detection
The reaction comprised (50 μl_) 1 x SYBR® Green PCR Master Mix (Applied Biosystems, USA) 200 nM of M13 forward primer (EQ7396), 200 nM M13 reverse primer (EQ7655) and 2.5 μl_ ligation reaction (described above). Cycling procedure: 10 min 95°C, 50 cycles of 15 sec 95°C, 1 min 45°C, 1 min 6O0C, and finally dissociation 20 min from 600C to 95°C in an ABI Prism® 7000 Sequence Detection System.
3.2. MicroRNA real-time PCR assays using LNA-modified detection probes
The reaction (50 μl_) was 1 * QuantiTect Probe PCR master mix (Qiagen, Germany) 200 nM hsa miR-15a M13 forward primer (EQ15887), 200 nM hsa miR-15a M13 reverse primer (EQ15888), 100 nM LNA sequence-specific probe (EQ15866 or EQ15867), 2.5μl_ ligation reaction (described above). Cycling procedure: 15 min 950C, 50 cycles of 20 sec 95°C, 1 min 6O0C in an ABI Prism® 7000 Sequence Detection System. In the following, dUTP means 21-deoxyuridine-5'-triphosphate
Example 1.: Gene specific first strand synthesis of microRNAs and real-time quantitative PCR detection
1. Gene specific priming and reverse transcription
The reverse transcription (RT) reaction was performed in 20 μL consisting of 0.5 μg Brain Total RNA template (Ambion, USA) spiked with 100, 10, 1, or 0.1 fmol synthetic hsa-miR-145 (EQ16901) template, respectively. 1 μM Gene Specific Reverse Transcription Primer (EQ24021), 1 * Incubation buffer (50 mM Tris-HCI, 40 mM KCI, 6 mM MgCI2, 10 mM DTT; pH 8.3 370C) (Roche, Germany), 0.5 mM of each of dNTP (Ap- plied Biosystems, USA), 20 U Protector RNase Inhibitor (Roche, Germany), and 40 U M-MuLV reverse transcriptase (Roche, Germany). Three control samples with 0.5 μg Brain total RNA, only, 10 fmol synthetic hsa-miR-145 template, and without RNA were included. The RNA templates and the GSP-RT primer were mixed and heated 2 min at 950C and quenched on ice. The thermocycler DYAD™ (MJ Research DNA engine, USA) was heated to the reaction start temperature. Temperature profile was 30 min 16 0C, 30 min 37 0C, 5 min 85 0C and cooled down to 4 0C, finally. The sample recovered after centrifugation was diluted to five times the originally RT starting volume (100 μL in total).
2. GSP microRNA real-time quantitative PCR assay using LNA-modified detection probes.
The real-time PCR reaction (50 μL) was performed in 1* QuantiTect Probe PCR Master Mix (Qiagen, Germany), 400 nM Universal forward primer (EQ 15809, Table M), 400 nM Universal reverse primer (EQ15810, Table II), 80 nM miR-specific forward primer (EQ 24037, Table II), 200 nM hsa-miR 145-Probe1 (EQ20317, Table II), 5 μL of the reverse transcription (RT) reaction (described above), and 0.5 U Uracil DNA Glycosylase (Invitrogen, USA). The following temperature cycling program was used; 10 min at 37 0C, 15 min at 95 0C, 1 min at 50 0C, 39 cycles of 20 s at 94 0C and 1 min at 60 0C. The real-time RT-PCR analysis was performed on an Opticon real-time PCR instrument (MJ Research, USA). 3. Results
The hsa-miR-145 (ace. no. MIMAT0000437, miRBase, Sanger Institute) RT reactions were subsequently detected using real time PCR as described above, universal PCR primers, miR-specific forward primer, and LNA-modified dual-labelled detection probe for the human miR-145 using a no template reaction as a negative control. The Ct values using 100, 10, 1, and 0.1 fmol hsa-miR 145 template were 9.2, 12.6, 16.2, and 20.4 for the LNA-modified dual-labelled detection probe (EQ20317), respectively (Fig. 2). The two positive control samples with 0.5 μg Brain total RNA, 10 fmol synthetic miR-145 template gave 23.5 and 12.9, respectively whereas no Ct values were detectable for the negative control experiments (no RNA and no cDNA template).
Example 2.: Application of Universal Primers for real-time PCR amplification of reverse transcribed miRNA using the adaptor ligation approach. __
1. Adaptor ligation.
For each ligation reaction 1 μg of total RNA was mixed with 100 pmol of activated RNA adaptor* (EQ23336) and 20 U of T4 DNA Ligase (New England Biolabs, USA) in a total volume of 10 μL consisting of 1X T4 DNA Ligase Reaction Buffer (50 mM Tris-HCI pH 8.0 at 25°C, 10 mM MgCI2, 3.3 mM dithiothreitol, 10 μg/ml BSA, and 8.3 % glycerol). Ligation was performed by incubation for 15 min at 37CC followed by heating for 10 min at 65 0C to terminate the reaction. A series of reactions were made to which were added different amounts of a synthetic miRNA (EQ15886) corresponding to the mature sequence of hsa-miR-16. The different reactions contained hsa-miR-16 in 10-fold dilutions starting with 100 fmol, 10 fmol, 1 fmol, 0.1 fmol, and 0 fmol as well as a reaction containing 10 fmol without a total RNA background and a reaction without either synthetic or total RNA.
*: The adaptor was activated as described in Lau et al. (Nelson C. Lau, Lee P. Lim, Earl G. Weinstein, and David P. Bartel, Science 26 October 2001 294: 858-862. An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis ele- gans.) 2a. Reverse transcription with RT-primer facilitating PCR with universal primers.
Reverse transcription was performed in a reaction volume of 20 μL consisting of the following components: the 10 μL of the terminated ligation reaction described above, 35 pmol of DNA RT primer (EQ23791), 1* First strand buffer (50 mM Tris-HCI pH 8.3 at 20 0C, 75 mM KCI, 3 mM MgCI2; Invitrogen, USA), 1.25 mM of each of dNTP (Applied Biosystems, USA, 10 mM DTT (Invitrogen, USA), 20 U SUPERase-ln (Ambion, USA), and 200 U Superscript Il reverse transcriptase (Invitrogen, USA). The reaction was incubated for 1 h at 42 0C followed by heating for 15 min at 70 0C to terminate the reaction. The volume of the reaction was adjusted to 100 μL by adding 80 μL of DEPC H2O.
2b. Reverse transcription with RT-primer facilitating PCR with gene specific (GSP) primers.
Reverse transcription was performed as described above but RT primer (EQ23791) was replaced with 35 pmol of DNA RT primer (EQ20172).
3a. Real-time PCR with universal primers
Real-time PCR was performed in 50 μL consisting of 1 * QuantiTect Probe PCR Master Mix (Qiagen, Germany), 80 nM GSP F-PRIMER (EQ23753), 400 nM of each of the two Universal primers (EQ15509/EQ 15510), 200 nM Probe (EQ20300), 5 μL of the reverse transcription (RT) reaction (described above), and 0.5 U Uracil DNA GIy- cosylase (Invitrogen, USA). The following temperature cycling program was used: 10 min at 37 0C, 15 min at 95 0C, 1 min at 500C, and 39 cycles of 20 s at 94 0C and 1 min at 60 0C. The real-time RT-PCR analysis was performed on a Opticon real-time PCR instrument (MJ Research, USA) with detection at the 60 0C step.
3b. Real-time PCR with GSP primers
Real-time PCR was performed as described above but with 400 nM of the two GSP primers EQ22965/EQ20565. 4. Results
Performing the experiment with the GSP primers results in a fall in signal at the low concentrations of spiked-in synthetic miRNA in a total RNA background (Fig. 3). The final signal obtained for 1 fmol synthetic miRNA in 1 μg total RNA background was 5- 6 times lower than the signal obtained with 100 fmol in 1 μg total RNA background. In contrast, when using the universal primers, the signal only dropped around 20% at 1 fmol synthetic miRNA in 1 μg total RNA background relative to signal obtained at 100 fmol in 1 μg total RNA. No signal was detectable for the negative control experiments (no RNA and no PCR template (qPCR/NTC)).
Example 3.: Scorpion primer mediated detection of reverse transcribed miRNAs obtained with the gene specific first strand synthesis approach or with the adaptor ligation approach.
A method for detection of miRNAs using a Scojpion primer based detection format is described. The total RNA sample containing miRNAs should be reverse transcribed using either the gene specific first strand synthesis (described in example 001) or the adaptor ligation approach (described in example 002). For the adaptor ligation approach, RNA adaptor (EQ23336) should be used together with RT-primer (EQ23791) or alternatively RNA adaptor (EQ24076) should be used together with RT-primer (EQ24077). Both RNA adaptor/RT-primer sets facilitate PCR with universal primers.
Real-time PCR
Real-time PCR should be performed in 50 μl_ consisting of 1 * QuantiTect Probe PCR Master Mix (Qiagen, Germany), 80 nM GSP F-PRIMER^5 μl_ of the reverse transcription (RT) reaction (described above), and 0.5 U Uracil DNA Glycosylase (Invi- trogen, USA). One of the following primer pairs may be used for detection in a concentration of 400 nM each:
EQ24080/EQ15509 and GSP F-PRIMER EQ23745 for detection of hsa-miR-21
EQ24084/EQ 15509 and GSP F-PRIMER EQ23753 for detection of hsa-miR-16 EQ24088/EQ15510 and GSP F-PRIMER EQ23757 for detection of hsa-miR-27a
The following temperature cycling program may be used; 10 min at 37 0C, 15 min at 95 0C, 1 min at 500C, and 39 cycles of 20 s at 94 0C and 1 min at 60 0C.
The real-time RT-PCR analysis may be performed on an Opticon real-time PCR in- strument (MJ Research, USA) or other real-time PCR instruments that are able to detect the FITC fluorophore.
Example 4.: Discrimination of mature and precursor forms of mi RNAs using the Scorpion primer mediated detection format.
A method for discrimination of mature and precursor forms of miRNAs using a Scor- pion primer based detection format is described.
The total RNA sample containing miRNAs should be reverse transcribed using the adaptor ligation approach (described in example 002) using the RNA adaptor (EQ23336) together with RT-primer (EQ23791) or RNA adaptor (EQ24076) together with RT-primer (EQ24077). Both RNA adaptor/RT-primer sets facilitate PCR with universal primers.
The real-time PCR is performed as described in Example 3 and the assays are expected to be able to discriminate between mature and precursor forms of miRNAs based on the difference in distance between the 3'-extension site of the Scorpion primer and the probe-binding site on the extended Scorpion primer. This distance will be relatively long and suboptimal when Scorpion primers detect precursor forms whereas is it will be short and optimal when Scorpion primers detect mature forms of the miRNAs (Fig. 4).
Table II: The design of the microRNA tagging probes, synthetic transcription templates and detection probes.
Figure imgf000064_0001
Figure imgf000065_0001
aLNA (uppercases), DNA/RNA (lowercases), mC: 5 methyl C; FITC: Fluorescein (Glenn Research, Prod.ld.No. 10-1964), #Q2: quencher, z: 5-nitroindole (Glenn Research, Prod. Id. No. 10-1044), P: Phosphate.
Example 5.: Increasing specificity of the real-time PCR amplification using primers with an increased number of nucleotides complementary to the target.
1. Adaptor ligation.
In a ligation reaction, 1 μg of total RNA was ligated with 100 pmol of activated RNA adaptor* (EQ23336) using 20 U of T4 DNA Ligase (New England Biolabs, USA) in a total volume of 10 μL consisting of 1X T4 DNA Ligase Reaction Buffer (50 mM Tris- HCI pH 8.0 at 250C1 10 mM MgCI2, 3.3 mM dithiothreitol, 10 μg/ml BSA, and 8.3 % glycerol). Ligation was performed by incubation for 15 min at 370C followed by heating for 10 min at 65 0C to terminate the reaction.
*: the adaptor was activated as described in Lau et al. 2001
2. Reverse transcription
Reverse transcription was performed in a reaction volume of 20 μL consisting of the following components: the 10 μL of the terminated ligation reaction described above, 35 pmol of DNA RT primer (EQ20172), 1 * First strand buffer (50 mM Tris-HCI pH 8.3 at 20 0C, 75 mM KCI1 3 mM MgCI2; Invitrogen, USA), 1.25 mM of each of dNTP (Applied Biosystems, USA, 10 mM DTT (Invitrogen, USA), 20 U SUPERase-ln (Ambion, USA), and 200 U Superscript Il reverse transcriptase (Invitrogen, USA). The reaction was incubated for 1 h at 42 0C followed by heating for 15 min at 70 0C to terminate the reaction. The volume of the reaction was adjusted to 100 μL by adding 80 μL of -DEPC H2O:
3. Real-time PCR different forward primers
Real-time PCR to detect hsa-mir-92 was performed in 50 μL consisting of 1* Quan- tiTect Probe PCR Master Mix (Qiagen, Germany), 400 nM of the reverse primer (EQ22965) and forward primer (see below),-200 nM Probe, 5 μL of the reverse transcription (RT) reaction (described above), and 0.5 U Uracil DNA Glycosylase (Invitrogen, USA). Three different gene specific forward primers were used (EQ20573, EQ23439, EQ23440) together with three different probes (EQ20834, EQ23457, EQ23458), each in a separate PCR. Forward primer EQ20573 together with probe EQ20834; forward primer EQ23439 together with probe EQ23457; and forward primer EQ23440 together with probe EQ23458. The three forward primers EQ20573, EQ23439, and EQ23440 had 9, 13 or 17 nucleotides, respectively, complementary to the reverse transcription product produced above in (2), and the probes were designed not to interfere with the forward primers. The following temperature cycling program was used: 10 min at 37 0C, 15 min at 95 0C, 1 min at 500C, and 39 cycles of 20 s at 94 0C and 1 min at 60 0C. The real-time RT-PCR analysis was performed on a Opticon real-time PCR instrument (MJ Research, USA) with detection at the 60 0C step. 4. Results
End products produced in the real time PCR experiment, were analyzed by electrophoresis using the Agilent Bioanalyzer. The resulting profiles are shown in Fig. 7. The Bioanalyzer profiles demonstrated that using the forward primer with only 9 nu- cleotides of complementary sequence relative to the reverse transcription product resulted in an unspecific PCR with more than one amplicon being produced. In contrast, increasing the number of complementary nucleotides in the forward primers to 13 and 17 resulted in specific PCR, which was indicated by Bioanalyzer profiles with only one band of correct size suggesting that only a single PCR amplicon were pro- duced with these primers.

Claims

Claims
1. A method for for the isolation, purification, amplification, detection, identification, quantification, or capture of non-coding RNAs, such as microRNA or small interfering RNA (siRNA) characterized in using an oligonucleotide containing a number of nucleoside analogues.
2. The method according to claim 1 comprising the following steps:
a) a target ribonucleic acid sequence is ligated with an RNA oligonucleotide adaptor using ligase enzyme,
b) the RNA oligonucleotide adaptor sequence is contacted with an oligonucleotide RT-primer, wherein the 3'-recognition nucleotide sequence optionally is complementary to a sequence in the RNA oligonucleotide adaptor sequence,
c) a DNA strand complementary to the target ribonucleic acid is synthesized by reverse transcription using a reverse transcriptase enzyme and the 3'-nucleotide sequence in the RNA oligonucleotide adaptor sequence as primer binding site for an oligonucleotide RT-primer,
d) a second strand synthesis is performed as a first step of a real-time PCR to amplify the reverse transcription product produced in the reverse transcription reaction taking place prior to the real time PCR using a gene specific forward primer with a 3'- recognition nucleotide sequence, which is sequence identical with the target ribonu- cleic acid sequence and wherein the 5'-sequence of the gene specific forward primer optionally contains a universal forward primer sequence,
~ e) quantifying of the resulting nucleic acids by real-time PCR performed using a universal forward primer and a scorpion primer as the reverse primer wherein the universal forward primer optionally contains a sequence identical to the 5'-sequence of the gene specific forward primer and the 3'-region of the scorpion primer optionally contains a sequence stretch complementary to the RNA oligonucleotide adaptor ligated to the target ribonucleic acid sequence and wherein the 5'-region of the scorpion primer optionally contains a detection moiety consisting of a sequence stretch complementary to the target ribonucleic acid sequence.
3. The method according to claim 1 comprising the following steps:
a) contacting the target ribonucleic acid sequence with an oligonucleotide RT-primer, wherein the 3'-recognition nucleotide sequence is complementary to a sequence in the target sequence and wherein the 5'-sequence of the oligonucleotide RT-primer optionally contains a universal reverse primer binding site,
b) synthesising of a complementary DNA strand to the target ribonucleic acid by reverse transcription using a reverse transcriptase enzyme and the 3'-nucleotide sequence in the target sequence as primer binding site for the oligonucleotide RT- primer,
c) performing a second strand synthesis as a first step of a real-time PCR using a forward primer with a 3'-recognition nucleotide sequence which is sequence identical with the target ribonucleic acid sequence, and
d) quantifying the resulting nucleic acids by real-time PCR using a set of universal forward and reverse primers and a labelled detection probe comprising a target rec- ognition sequence and a detection moiety.
4. The method of claim 3 wherein the forward primer in step c) is present in a low concentration.
5. The method according to claim 1 comprising the following steps:
a) ligating the target ribonucleic acid sequence with an RNA oligonucleotide adaptor using ligase enzyme,
b) contacting the RNA oligonucleotide adaptor sequence with an oligonucleotide RT- primer, wherein the 3'-recognition nucleotide sequence optionally is complementary to a sequence in the RNA oligonucleotide adaptor sequence and wherein the 5'- sequence of the oligonucleotide RT-primer optionally contain a universal reverse primer binding site,
c) synthesising of a complementary DNA strand to the target ribonucleic acid by reverse transcription using a reverse transcriptase enzyme and the 3'-nucleotide se- quence in the RNA oligonucleotide adaptor sequence as primer binding site for the oligonucleotide RT-primer,
d) performing a second strand synthesis as a first step of a real-time PCR using a forward primer with a 3'-recognition nucleotide sequence which optionally is se- quence identical with the target ribonucleic acid sequence and wherein the 5'- sequence of the Forward primer optionally contains a universal forward primer binding site, and
e) quantifying the resulting nucleic acids by real-time PCR using a set of universal forward and reverse primers and a labelled detection probe comprising a target rec- ognition sequence and a detection moiety.
6. The method of claim 5 wherein the forward primer in step d) is present in a low concentration.
~-7: The method according to any one of claims 3 or 4, wherein step (c) comprises use of a detection probe comprising modified nucleotides.
8. The method according to claim 7, wherein the modified nucleotides are LNA nucleotides.
~~ 9. The method according to claim 7 or 8, wherein the detection probe corresponds to or is complementary to a sequence in the short-length RNA.
10. The method according to any one of claims 1 to 6 wherein the nucleoside ana- logue is LNA.
11. A kit for the isolation, purification, amplification, detection, identification, quantification, or capture of non-coding RNAs, such as microRNA or small interfering RNA (siRNA), where the kit comprises a reaction body and one or more modified oligonucleotides.
12. A kit according to claim 11 , wherein the modified oligonucleotides contains a number of oligonucleoside analogues, optionally being LNA.
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