WO2012050975A2 - Nouvelles molécules d'arn circulaire de mammifère et utilisations associées - Google Patents

Nouvelles molécules d'arn circulaire de mammifère et utilisations associées Download PDF

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WO2012050975A2
WO2012050975A2 PCT/US2011/054004 US2011054004W WO2012050975A2 WO 2012050975 A2 WO2012050975 A2 WO 2012050975A2 US 2011054004 W US2011054004 W US 2011054004W WO 2012050975 A2 WO2012050975 A2 WO 2012050975A2
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anril
nucleic acid
rna
exons
exon
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WO2012050975A3 (fr
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Norman E. Sharpless
Zefeng Wang
Christin E. Burd
William R. Jeck
Alex P. Siebold
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The University Of North Carolina At Chapel Hill
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • This invention relates generally to the discovery of novel circular mammalian RNA molecules made of exons from ANRIL (CDKN2BAS) and uses of these circular RNA molecules.
  • Atherosclerotic vascular disease is a leading cause of human mortality worldwide [1]. While there are well-recognized risk factors for ASVD such as tobacco use, obesity and hyperlipidemia, the identification of common genetic variants associated with the disease has proven difficult despite strong evidence that susceptibility is heritable. Recently, multiple unbiased genome-wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs) near the INK4/ARF (or CDKN2a/b locus) on chromosome 9p21 to ASVD and other related conditions (i.e., coronary artery disease, stroke, myocardial infarction and aortic aneurysm) [2-12]. These associations have been replicated in multiple independent studies and are not associated with "classical" ASVD risk factors such as hypertension, obesity, tobacco use or lipid levels.
  • SNPs single nucleotide polymorphisms
  • the INK4/ARF tumor suppressor locus plays a principal role in human cancer resistance (reviewed in Kim and Sharpless, [15]). SNPs near this locus have been associated with human malignancies, as have germline polymorphisms associated with reduced function which predispose to several cancer including melanoma, glioblastoma and pancreatic adenocarcinoma. Somatic inactivation of this locus is one of the most frequent events in human malignancy, and regulation of the locus plays a major role in susceptibility to cancer.
  • INK4/ARF locus Moreover, regulation of the INK4/ARF locus is associated with a variety of aging- associated diseases in addition to cancer and atherosclerotic conditions such as ischemic strike, aortic aneurysm and myocardial infarction. SNPs near the INK4/ARF locus have also been associated with susceptibility to Type 2 diabetes mellitus, frailty and human longevity; suggesting the regulation of the INK4/ARF locus plays a general role in the susceptibility to a variety of human diseases associated with aging.
  • INK4b INK4a harboring the risk alleles demonstrating reduced levels of pi 5 , pi 6 , ARF and ANRIL [16]. Decreased expression of such anti-pro liferative molecules could promote pathologic monocytic or vascular proliferation, thus accelerating ASVD development
  • mice lacking pi 6 exhibit increased vascular hyperplasia following intra-arterial injury [19] and ARF deficiency has been implicated in atherosclerotic plaque formation [20]. Additionally, TGF- ⁇ signaling, which induces the
  • pl6 and pl5 are anti-atherogenic in some settings [21-23].
  • excess proliferation of hematopoietic progenitor cells, which is in part controlled by pl6 expression during aging [24] has been associated with atherosclerosis in a murine model [25].
  • mice resulted in severely attenuated expression ⁇ 15 and pi 6 [26]. Although these results suggest that the ASVD-associated 9p21 SNPs control INK4/ARF expression, and that decreased expression of the INK4/ARF tumor suppressors may promote ASVD, it is not known how polymorphisms located ⁇ 120kb away from the locus might influence INK4/ARF expression.
  • ANRIL was first uncovered in a genetic analysis of familial melanoma patients with neural system tumors [27]. Based upon EST assembly, ANRIL has 19 exons with no identified open reading frame [27] ( Figure 9). Although cloning a full-length version of the predicted transcript has proven difficult, a growing number of alternatively spliced ANRIL transcripts have recently been reported in the literature [28,29]. Many of these reports suggest that multiple ANRIL isoforms can be expressed in a single cell type. For example, two ANRIL variants have been reported in testes, five in HUVECs and three in lung [27,28].
  • the present invention provides an isolated and purified nucleic acid encoding a circular RNA comprising two or more ANRIL exons.
  • the circular RNA may comprise ANRIL exons 3i*, 4, 5, 6, 7, 10, 13, 14, 15, 16, 17, 18, or 19.
  • the circular RNA may be a circle where exon 4 is linked to exon 14, e.g., -exon4- exonl3-exonl4- or represented as 4-13-14-4 in Fig. 4B and 4C.
  • Additional examples include -exon4-exon5-exon6-exon3i*-, -exon6-exon5-exon3i*- -exon4-exon5-exon6-exon7-exonl4-, -exon4-exon5-exon6-exon7-, -exon4-exon5-exon6-, -exon6-exon7-exonl4-exon5-, -exon6- exon7-exonl0-exon5-, -exon6-exon7-exon5-, -exon6-exon5-, -exon6-exonl3-exonl4-, or -exonl 6-exonl 7-exon 18-exon 19-exon 13 -exon 14-exon 15-.
  • the invention also provides a method for detecting a level of a nucleic acid encoding a circular RNA comprising two or more ANRIL exons in a sample which comprises contacting the sample with a reagent that selectively enriches the circular RNA and measuring the level of the nucleic acid in the sample.
  • the enriching may be performed by an exonuclease, such as RNase R or RNase H.
  • the method further comprises detecting the nucleic acid encoding the circular RNA with a probe specific for a mis-ordered junction.
  • the mis-ordered junction may be exon5-exon3i*, exon6-exon3i*, exon6-exon5, exon7-exon4, exon7-exon5, exonl0-exon5, exonl4-exon4, exonl4-exon5, or exon 19- exonl3.
  • the method to detect the mis-ordered exon junction may utilize a mass spectrometer.
  • the method may comprise PCR amplification using outward facing primers such only circular nucleic acids will produce an amplicon.
  • the method alternatively may comprise detecting a polyadenonosine end or removing nucleic acids with polyadenosine ends.
  • the invention also provides a method for detecting risk of a vascular disease in a subject which comprises measuring a level of the circular nucleic acids and determining whether or not the subject is at risk for vascular disease.
  • the vascular disease may be abdominal aortic aneurysm, arteriosclerosis, atherosclerosis, coronary artery disease, ischemic stroke, myocardial infarction, peripheral vascular disease, renal artery stenosis, stroke, or thoracic aortic aneurysm.
  • the ischemic stroke may be cardioembolic stroke, large artery stroke, or small vessel stroke.
  • the invention also provides a method for detecting risk of a metabolic disorder in a subject which comprises measuring a level of the circular nucleic acids and determining whether or not the subject is at risk for the metabolic disorder.
  • the metabolic disorder may be diabetes mellitus, metabolic syndrome, or type 2 diabetes.
  • the invention also provides a method for detecting risk of a proliferative disorder in a subject which comprises measuring a level of the circular nucleic acids and determining whether or not the subject is at risk for the proliferative disorder.
  • the proliferative disorder may be cancer or endometriosis.
  • the cancer may be bladder carcinoma, breast cancer, colorectal cancer, endocrinologic cancer, thyroid cancer, glioblastoma, head and neck cancer, leukemia, melanoma, liver cancer, lung cancer, non-small cell lung cancer, pancreatic adenocarcinoma, or skin cancer.
  • the invention also provides a method for detecting risk of an age-associated condition in a subject which comprises measuring a level of the circular nucleic acids and determining whether or not the subject is at risk for the age-associated condition.
  • the age-associated condition may be frailty, life-expectancy or longevity.
  • Kits are also provided.
  • the invention includes a kit comprising: (a) at least one reagent selected from the group consisting of: (i) a nucleic acid probe capable of specifically hybridizing with a nucleic acid encoding a circular RNA comprising two or more ANRIL exons; (ii) a pair of nucleic acid primers capable of PCR amplification of the nucleic acid; and (iii) a probe capable of specifically hybridizing with the nucleic acid; and (b) instructions for use in measuring the nucleic acid in a tissue sample from a subject suspected of having an ANRIL-associated disorder.
  • the ANRIL-associated disorder may be a vascular disease, a metabolic disease, a proliferative disorder or an age-associated condition.
  • the kits may also contain reagent that selectively enriches the circular RNA such as an exonuclease.
  • the invention provides a method of identifying a compound that prevents or treats an ANRIL-associated disorder, the method comprising the steps of: (a) contacting a compound with a sample comprising a cell or a tissue; (b) measuring a level of a nucleic acid encoding a circular RNA comprising two or more ANRIL exons; (c) determining a functional effect of the compound on the level of the nucleic acid; thereby identifying a compound that prevents or treats an ANRIL-associated disorder.
  • Fig. 1A and IB Identification and characterization of ANRIL splice variants.
  • Fig. 1A, 3' and 5' RACE was performed using primers directed against exons 4 and 6 where long stretches of unique sequence were observed (top). The resulting PCR products were cloned and sequenced, revealing several novel exons (10a and 13b) and multiple non-colinear species (13-14-4-5, 13-14-4).
  • Fig. IB Equal quantities of total RNA were harvested from growing cell lines of various tissue types and absolute expression of the indicated transcript was determined. Expression levels are shown in a box- whisker plot on a log 10 scale in 11 of 27 analyzed cell lines which did not harbor homozygous 9p21 deletion. Validated TaqMan® detection strategies for the indicated ANRIL species are shown (top).
  • FIG. 2A and 2B RNA Sequencing of ANRIL transcripts. Coverage plots of RNA sequencing reads derived from short read archive study SRP002274 [45]. Fig. 2A, Top,
  • Fig. 2A Bottom, The grey regions in the top panel were graphed on a truncated scale to better depict ANRIL coverage. Annotations above the larger peaks show the maximum number of reads mapping to these areas.
  • Fig. 2B Maximum peak height at each exon (normalized by overall locus coverage) is displayed from three independent samples: SRP002274 (Brain) and two ENCODE RNA-sequencing replicates of the HeLa cell line (HeLa rep 1 and 2). The inset shows all ANRIL exons on y-axis with peak height of 150 reads.
  • FIG. 3A-3D ANRIL 14-5 and 4-6 are circular RNAs.
  • FIG. 3A Schematic representation of the ANRIL14-5 TaqMan® detection strategy wherein the probe spans the exon 5-exon 14 boundary amplified with outward facing primers.
  • Fig. 3B Expression of the indicated transcripts was quantified in cDNA from Hs68 cells made using the indicate primers (-RT: no reverse transcriptase, H+dT: an equal mix of random hexamers and oligo dT, dT: oligo dT alone, and HEX: random hexamers alone). Error bars represent the standard deviation for three replicates.
  • Fig. 3A Schematic representation of the ANRIL14-5 TaqMan® detection strategy wherein the probe spans the exon 5-exon 14 boundary amplified with outward facing primers.
  • Fig. 3B Expression of the indicated transcripts was quantified in cDNA from Hs68 cells made using the indicate primers (-RT: no reverse transcript
  • RNA harvested from growing Hs68 (top) and IMR90 (bottom) cells was incubated with, or without, RNase R, purified and reverse transcribed.
  • the indicated transcripts were quantified in 'B'.
  • Fig. 3D The average fold enrichment by RNase R for each transcript is shown on a log 10 scale.
  • FIG. 4A-4C ANRIL circular RNAs predominantly contain exons 4-14. Fig.
  • cDNA generated in the presence (+R) or absence (-R) of RNase R as in Figure 3C was subjected to PCR using outward facing primers within the same exon as depicted (left) and separated by gel electrophoresis.
  • Fig. 4B and Fig. 4C The PCR products in 'A' were purified, cloned and sequenced. The resulting sequences are shown for each exon pair.
  • FIG. 5A-5B ANRIL4-6 and 14-5 correlate with INK4/ARF expression and rsl0757278 genotype in human PBTLs.
  • Fig. 5B The relative expression of ANRIL1-2, 14-5 and 18-19 normalized as in 'A' is plotted versus rsl0757278 genotype, p- values were determined by a two-sided t-test.
  • Fig. 6A-6C Deep sequencing of 9p21 in pools of rsl0757278 homozygotes.
  • Fig. 6A The region captured using DNA sequence capture technology is shown on the 'Tiling' track.
  • the 'Unique' track shows the Duke 35bp Uniqueness information as provided in the UCSC Genome Browser.
  • the bar at the top of the figure represents the 53 kb risk interval previously defined by Broadbent et al. [13].
  • Fig. 6B Venn diagrams depicting SNP calls for the AA (left) and GG (right) samples using three different algorithms.
  • Fig. 6C Using the UCSC Genome Browser, SNPs identified by next-generation DNA sequencing are depicted across the captured region of 9p21.
  • the 'Discovered' track shows the polymorphisms identified by two or more algorithms in either the pooled AA or GG sample. SNPs unique to each genotype are shown below.
  • the 'Unique Splice' track depicts the location of the 4 SNPs, unique to the GG sample, which modify cz ' s-acting splice regulation sites (See also Table 1).
  • FIG. 7 Model showing how 9p21 polymorphisms influence ANRIL isoform production to modulate INK4/ARF gene transcription.
  • PcG complexes e.g., PRC-1
  • PRC-1 e.g., PRC-1
  • Nascent ANRIL transcripts are spliced to produce circular ANRIL species (cANRIL).
  • cANRIL circular ANRIL species
  • Causal variants in the ASVD-risk interval modulate ANRIL transcription or splicing to influence INK4/ARF expression. See discussion for further description.
  • Fig. 8A-Fig. 8C Polymorphisms within the INK4/ARF locus linked to age-related diseases.
  • Fig. 8A Schematic diagram of the 9p21 locus depicting the INK4/ARF tumor suppressors, ANRIL and the ASVD risk interval. The captured ("tiling") region for next generation DNA sequencing is indicated.
  • Fig. 8B The localization of SNPs linked in the literature to age-related diseases including T2D- type 2 diabetes, CAD- coronary artery disease, Ml-myocardial infarction, AD- Alzheimer's disease are shown.
  • Fig. 8A Schematic diagram of the 9p21 locus depicting the INK4/ARF tumor suppressors, ANRIL and the ASVD risk interval. The captured ("tiling") region for next generation DNA sequencing is indicated.
  • Fig. 8B The localization of SNPs linked in the literature to age-related diseases including T2D- type 2 diabetes, CAD- coronary artery disease, Ml-my
  • a heatmap depicting the SNP linkage disequilibrium was generated from the Hapmap CEU population using Haploview software [84].
  • the heatmap can be aligned to the depicted 9p21 image as shown in Fig. 8A-8C.
  • FIG. 9 Schematic of previously reported ANRIL variants. All Ensembl (blue) and GenBank (black) records for ANRIL (CDKN2BAS) are shown. Some sequences are derived from cDNA sequencing whereas others were inferred by EST assembly.
  • FIG. 11 Expression of 9p21 transcripts in transformed and non-transformed cell lines. As described in Figure IB, R A was harvested, reverse transcribed and quantitative real-time PCR performed. Bars represent the logio of the average number of molecules detected. The error bars denote the standard deviation between three replicates. The letter 'D' denotes deletion events previously reported in the literature. 'M' indicates gene methylation.
  • Breast cancer cell lines are T-47D, BT-549, BT-474, SUM149, MDA-MB- 231, MDA-MB-436, MDA-MB-468, and MCF7;
  • colorectal cancer cell lines are LS1034, SW480, LS174T, LoVo, T84, and COLO 205;
  • melanoma cell lines are RPMI-8322, WM266- 4, UACC, A2058, and A375;
  • hematological malignancies are Jurkat, Raji, and Ramos cell lines;
  • cervical carcinoma cell line is HeLa;
  • glioblastoma cell line is U87 and non- transformed cells are Hs68, IMR90, and HUVEC.
  • FIG. 12 ANRIL14-5 expression is detected in a wide variety of cell types. cDNA generated as described in Figure IB was assayed for ANRIL14-5 expression using the TaqMan® strategy shown in Figure 3A. Bars represent the logio of the average number of molecules detected. The error bars denote the standard deviation between three replicates. Cell lines and associated diseases are described for Fig. 1 1.
  • Fig. 14A and Fig. 14B are schematics of the location of linear murine ANRIL (mANRIL) and circular murine ANRIL (mcANRIL) products identified from late passage primary wild-type murine embryonic fibroblasts (MEFs) using next-generation sequencing of a cDNA library (RNA-seq). The sequencing included reads bridge several circular junctions. The circular junctions indicate the mcANRIL species.
  • Fig. 14 A is a 493kb region of murine chromosome 4.
  • Fig. 14B shows a region of approximately 250 kb.
  • Fig. 15 shows the PCR validation of mcANRIL circles 1 1, 25, 31, and 40. PCR amplification of mcANRIL circles from wild-type MEF cDNA. In the figure " ⁇ " dots represent validated mcANRIL circles. Additional products likely are due to extensive alternative splicing.
  • Fig. 16 shows quantitative real-time RT-PCR of mANRIL and mcANRIL splice junctions.
  • cDNA was prepared from early passage (P3) and late passage (P8) wild-type (WT) C57B6 MEFs.
  • Relative fold expression of pl6 INK4a with passage (Fig. 16A.) is shown as a positive control.
  • Fig. 16B. Relative expression of a validated linear mANRIL RNA splice junction.
  • Fig. 16C- Fig. 16E Relative fold expression of three non-colinear mcANRIL RNA splice junctions. Fold expression of each target was determined relative to 18S.
  • a negative control no cDNA template
  • yielded undetectable levels of all transcripts (not shown).
  • X-axis label indicates the transcript measured.
  • This invention is useful for detecting a target nucleic acid of interest present in a sample.
  • the target nucleic acid is preferably a circular ANRIL RNA.
  • the methods use relatively few and easily performed steps to isolate and/or concentrate the target nucleic acid from other sample components and to detect the target nucleic acid.
  • Samples are typically one or more cells obtained as a specimen that can be used to determine the presence or abundance of a target nucleic acid that is a biomarker for a disorder.
  • the sample can be one or more blood cells that can be used for determining a disorder wherein a biomarker is present in blood cells.
  • the sample can be one or more biopsied cells that can be used for determining the presence of a disorder wherein a biomarker is present in the tissue or organ from which the biopsy was taken.
  • the methods include isolating the target nucleic acid from the sample. In some instances the target nucleic acid is released from a cell by lysing the cell in which the target nucleic acid is suspected of being present. In some instances, the released target nucleic acid is then isolated away from other components of the sample, which may include cellular debris is the target nucleic acid was released by cell lysis.
  • Isolation can include general nucleic acid isolation, which can be done using kits like the mirVana kits (ABI, Foster City, CA); Trizol LS (Invitrogen, Carlsbad, CA); Micro RNA Isolation kits (Stratagene, La Jolla, CA) and High-Pure miRNA Isolation kits (Roche, Indianapolis, IN).
  • mirVana kits ABSI, Foster City, CA
  • Trizol LS Invitrogen, Carlsbad, CA
  • Micro RNA Isolation kits Stratagene, La Jolla, CA
  • High-Pure miRNA Isolation kits Roche, Indianapolis, IN.
  • the methods of the invention may be used to measure a level of any nucleic acid encoding a circular RNA comprising two or more ANRIL exons, including but not limited to, exons 4, 5, 6, 7, 10, 13, 14, 15, 16, 17, 18 or 19.
  • the methods of the invention may use at least a portion of (i) the nucleotide sequence of mammalian ANRIL (CDKN2BAS), e. g., as derived from whole blood leukocytes or lymphoblastoid cell lines (Pasmant et al., 2007, Cancer Res.
  • CDKN2BAS mammalian ANRIL
  • nucleic acids of the claimed invention include the single nucleotide polymorphisms (SNPs) such as the 130 SNPs that may be found in UCSC Genome Browser on Human Mar. 2006 (NCBI36/hgl8) Assembly or the 131 SNPs UCSC Genome Browser on Human Feb. 2009 (GRCh37/hgl9) Assembly (http://genome.ucsc.edu/).
  • SNPs single nucleotide polymorphisms
  • ANRIL-associated disorder includes: (i) vascular diseases such as aortic aneurism, atherosclerosis, coronary artery disease, ischemic stroke, myocardial infarction, peripheral vascular disease, or stroke including cardioembolic stroke, large artery stroke or small vessel stroke; (ii) metabolic disorders such as diabetes mellitus, metabolic syndrome, or type 2 diabetes; (iii) proliferative disorders such as endometriosis or cancer, e.g., bladder carcinoma, glioblastoma, leukemia, melanoma, non-small cell lung cancer, or pancreatic adenocarcinoma.
  • vascular diseases such as aortic aneurism, atherosclerosis, coronary artery disease, ischemic stroke, myocardial infarction, peripheral vascular disease, or stroke including cardioembolic stroke, large artery stroke or small vessel stroke
  • metabolic disorders such as diabetes mellitus, metabolic syndrome, or type 2 diabetes
  • proliferative disorders such as endo
  • nucleic acid and “nucleic acid molecule” may be used interchangeably throughout the disclosure.
  • X includes nucleic acids of any composition from, such as DNA (e.g., complementary DNA (cDNA), genomic DNA (gDNA) and the like), RNA (e.g., message RNA (mRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA, and the like), and/or DNA or RNA analogs (e.g., containing base analogs, sugar analogs and/or a non-native backbone and the like), RNA/DNA hybrids and polyamide nucleic acids (PNAs), all of which can be in single- or double-stranded form, and unless otherwise limited, can encompass known analogs of natural nucleotides. Examples of nucleic acids are SEQ ID Nos.
  • a template nucleic acid in some embodiments can be from a single chromosome (e.g., a nucleic acid sample may be from one chromosome of a sample obtained from a diploid organism).
  • nucleic acid Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses methylated forms, conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as the sequence explicitly indicated.
  • nucleic acid is used interchangeably with locus, gene, cDNA, and mRNA encoded by a gene.
  • RNA or DNA synthesized from nucleotide analogs include, as equivalents, derivatives, variants and analogs of RNA or DNA synthesized from nucleotide analogs, single- stranded ("sense” or “antisense”, “plus” strand or “minus” strand, "forward” reading frame or “reverse” reading frame) and double-stranded polynucleotides.
  • Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine.
  • the base cytosine is replaced with uracil.
  • Primer refers to oligonucleotides that can be used in an amplification method, such as a polymerase chain reaction (PCR), to amplify a nucleotide sequence based on the polynucleotide sequence corresponding to a particular genomic sequence, e.g., one specific for a particular site in the circular ANRIL RNA. At least one of the PCR primers for amplification of a polynucleotide sequence is sequence-specific for the sequence.
  • PCR polymerase chain reaction
  • oligomer refers to a nucleic acid having generally less than 1,000 nucleotide (nt) residues, including polymers in a range having a lower limit of about 5 nt residues and an upper limit of about 500 to 900 nt residues.
  • oligonucleotides are in a size range having a lower limit of about 12 to 15 nt and an upper limit of about 50 to 600 nt, and other embodiments are in a range having a lower limit of about 15 to 20 nt and an upper limit of about 22 to 100 nt.
  • Oligonucleotides may be purified from naturally occurring sources or may be synthesized using any of a variety of well-known enzymatic or chemical methods.
  • the term oligonucleotide does not denote any particular function to the reagent; rather, it is used generically to cover all such reagents described herein.
  • An oligonucleotide may serve various different functions.
  • it may function as a primer if it is specific for and capable of hybridizing to a complementary strand and can further be extended in the presence of a nucleic acid polymerase, it may provide a promoter if it contains a sequence recognized by an RNA polymerase and allows for transcription (e.g., a promoter-based oligomer), and it may function to prevent hybridization or impede primer extension if appropriately situated and/or modified.
  • complementary or “complementarity of nucleic acids is meant that a nucleotide sequence in one strand of nucleic acid, due to orientation of the functional groups, will hydrogen bond to another sequence on an opposing nucleic acid strand.
  • the complementary bases typically are, in DNA, A with T and C with G, and, in RNA, C with G, and U with A.
  • Substantially complementary means that a sequence in one strand is not completely and/or perfectly complementary to a sequence in an opposing strand, but that sufficient bonding occurs between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., salt concentration and temperature).
  • T m refers to the temperature at which a population of hybridization complexes formed between two nucleic acid strands are 50% denatured. At a temperature below the T m , formation of a hybridization complex is favored, whereas at a temperature above the T m , melting or separation of the strands in the hybridization complex is favored.
  • Nucleic acid sequences are identical when their contiguous nucleotide arrangements are the same. Identical sequences includes those that have a modified residue in one sequence, but not the other, so long as the residue is basically the same (e.g., a 2'-OMe residue in one sequence is still identical to a strand lacking the 2'OMe modification). Substantially identical sequences are those that contain sequence differences between the two strands, but the strands retain similar hybridization properties. Identity and substantial identity between sequences are understood and easily determined by ordinarily skilled artisans.
  • Sequences herein that are at least a certain percent identical or complementary to another sequence means that the sequences includes all rational numbers from the referenced percent identity to 100%. For example, at least 80% means all natural number percentages 80, 81, 82, 82, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100, as well as any fraction in between (e.g., 82.6, 91.1, 97.9, etc). Ordinarily skilled artisans can determine percent complementarity and percent identity.
  • Hybridization condition refers to the cumulative environment in which one nucleic acid strand bonds to a second nucleic acid strand by complementary strand interactions and hydrogen bonding to produce a hybridization complex.
  • Such conditions include the chemical components and their concentrations (e.g., salts, chelating agents, formamide) of an aqueous or organic solution containing the nucleic acids, and the temperature of the mixture.
  • concentrations e.g., salts, chelating agents, formamide
  • Other well-known factors such as the length of incubation time or reaction chamber dimensions may contribute to the environment (e.g., Sambrook et al, Molecular Cloning, A Laboratory Manual, 2nd ed., pp. 1.90-1.91, 9.47-9.51 , 11.47-1 1.57 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)),
  • template refers to any nucleic acid molecule that can be used for amplification in the technology. RNA or DNA that is not naturally double stranded can be made into double stranded DNA so as to be used as template DNA. Any double stranded DNA or preparation containing multiple, different double stranded DNA molecules can be used as template DNA to amplify a locus or loci of interest contained in the template DNA.
  • amplification reaction refers to a process for copying nucleic acid one or more times.
  • the method of amplification includes, but is not limited to, polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q- ⁇ replicase amplification, strand displacement amplification, rolling circle amplification, or splice overlap extension polymerase chain reaction.
  • a single molecule of nucleic acid may be amplified.
  • sensitivity refers to the number of true positives divided by the number of true positives plus the number of false negatives, where sensitivity (sens) may be within the range of 0 ⁇ sens ⁇ 1.
  • method embodiments herein have the number of false negatives equaling zero or close to equaling zero, so that no subject is wrongly identified as not having a disorder when they do indeed have a disorder.
  • an assessment often is made of the ability of a prediction algorithm to classify negatives correctly, a complementary measurement to sensitivity.
  • sensitivity refers to the number of true negatives divided by the number of true negatives plus the number of false positives, where sensitivity (spec) may be within the range of 0 ⁇ spec ⁇ 1.
  • the methods described herein have the number of false positives equaling zero or close to equaling zero, so that no subject is wrongly identified as having an ANRIL- associated disorder when they do not in fact have one.
  • a method that has both sensitivity and specificity equaling one, or 100%, is preferred.
  • RNAi molecule refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA expressed in the same cell as the gene or target gene.
  • siRNA thus refers to the double stranded RNA formed by the complementary strands.
  • the complementary portions of the siRNA that hybridize to form the double stranded molecule typically have substantial or complete identity.
  • siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA.
  • the sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof.
  • the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferably about 20-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • An "antisense" polynucleotide is a polynucleotide that is substantially complementary to a target polynucleotide and has the ability to specifically hybridize to the target polynucleotide.
  • the invention also includes genetically modified organisms that over-express or under-express mammalian circular ANRIL.
  • Methods of making genetically modified model organisms for the study of disease are well-known. Examples include US Patent Nos. 4,736,866 (Leder et al. "oncomouse"), 6,187,991 (Soeller et al.), 6,359, 194 (Galvin et al.), 6,909,030 (Melmed and Wang), 7,288,385 (Ma et al), 7,560,610 (Roberts et al); PCT Patent Pubs.
  • the phrase "functional effects" in the context of assays for testing means compounds that interfere or modulate the function of the circular ANRIL RNAs. This may also be a chemical or phenotypic effect such as altered translation of the circular ANRIL RNAs, or altered activities or the downstream effects cause by the non-coding circular ANRIL RNAs.
  • a functional effect may include transcriptional activation or repression, the ability of cells to proliferate, expression in cells during disease progression, and other characteristics of an ANRIL-associated disorder.
  • “Functional effects” include in vitro, in vivo, and ex vivo activities.
  • determining the functional effect is meant assaying for a compound that increases or decreases the transcription of genes or the translation of proteins that are indirectly or directly under the influence of a circular ANRIL RNA.
  • Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index); hydrodynamic (e.g., shape), chromatographic; or solubility properties for the protein; ligand binding assays, e.g., binding to antibodies; measuring inducible markers or transcriptional activation of the marker; measuring changes in enzymatic activity; the ability to increase or decrease cellular proliferation, apoptosis, cell cycle arrest, measuring changes in cell surface markers.
  • Validation the functional effect of a compound on an ANRIL-associated disease progression can also be performed using assays known to those of skill in the art such as metastasis of cancer cells by tail vein injection of cancer cells in mice.
  • the functional effects can be evaluated by many means known to those skilled in the art, e.g., microscopy for quantitative or qualitative measures of alterations in morphological features, measurement of changes in RNA or protein levels for other genes expressed in cells, measurement of RNA stability, identification of downstream or reporter gene expression (CAT, luciferase, ⁇ -gal, GFP and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, etc.
  • Inhibitors “Inhibitors,” “activators,” and “modulators” of the markers are used to refer to activating, inhibitory, or modulating molecules identified using in vitro and in vivo assays of either the circular ANRIL RNAs, or the expression of genes or the translation proteins encoded thereby modulated by the circular ANRIL RNAs.
  • Inhibitors, activators, or modulators also include naturally occurring and synthetic ligands, antagonists, agonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, RNAi molecules, small organic molecules and the like.
  • Such assays for inhibitors and activators include, e.g., (1) measuring levels of the circular ANRIL RNA, or (2) (a) measuring the mRNA expression, or (b) proteins expressed by genes modulated by circular ANRIL RNA in vitro, in cells, or cell extracts; (3) applying putative modulator compounds; and (4) determining the functional effects on activity, as described above.
  • Samples or assays comprising genes modulated by circular ANRIL RNA that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition.
  • Control samples (untreated with inhibitors) are assigned a relative activity value of 100%.
  • Inhibition of expression, or proteins modulated by circular ANRIL RNA is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%.
  • Activation of expression, or proteins encoded by genes modulated by circular ANRIL RNA is achieved when the activity value relative to the control (untreated with activators) is 1 10%, preferably 150%, more preferably 200- 500% (i.e., two to five fold higher relative to the control), more preferably 1000-3000% higher.
  • test compound or “drug candidate” or “modulator” or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide, small organic molecule, polysaccharide, peptide, circular peptide, lipid, fatty acid, siR A, polynucleotide, oligonucleotide, etc., to be tested for the capacity to directly or indirectly affect genes modulated by circular ANRIL RNA.
  • the test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity.
  • Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties.
  • a fusion partner e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties.
  • new chemical entities with useful properties are generated by identifying a test compound (called a "lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.
  • HTS high throughput screening
  • the compound may be "small organic molecule” that is an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 daltons and less than about 2500 daltons, preferably less than about 2000 daltons, preferably between about 100 to about 1000 daltons, more preferably between about 200 to about 500 daltons.
  • the sample may be from a patient or a subject suspected of having an ANRIL- associated disorder.
  • the biological sample may also be from a subject with an ambiguous diagnosis in order to clarify the diagnosis.
  • the sample may be obtained for the purpose of differential diagnosis, e.g., a healthy subject to confirm the diagnosis.
  • the sample may also be obtained for the purpose of prognosis, i.e., determining the course of the disease and selecting primary treatment options. Tumor staging and grading are examples of prognosis.
  • the sample may also be evaluated to select or monitor therapy, selecting likely responders in advance from non-responders or monitoring response in the course of therapy.
  • the sample may be evaluated as part of post-treatment ongoing surveillance of patients who have an ANRIL-associated disorder.
  • Samples may be obtained using any of a number of methods in the art.
  • a sample may also be a sample of muscosal surfaces, blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, white blood cells, circulating tumor cells isolated from blood, free DNA isolated from blood, and the like), sputum, lymph and tongue tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc.
  • blood and blood fractions or products e.g., serum, plasma, platelets, red blood cells, white blood cells, circulating tumor cells isolated from blood, free DNA isolated from blood, and the like
  • sputum e.g., lymph and tongue tissue
  • cultured cells e.g., primary cultures, explants, and transformed cells, stool, urine, etc.
  • biological samples include those obtained from excised skin biopsies, such as punch biopsies, shave biopsies, fine needle aspirates (FNA), or surgical excisions; or biopsy from non- cutaneous tissues such as lymph node tissue, mucosa, conjuctiva, uvea, or other embodiments.
  • the biological sample can be obtained by shaving, waxing, or stripping the region of interest on the skin.
  • Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy.
  • An "excisional biopsy” refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it.
  • An “incisional biopsy” refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor.
  • a diagnosis or prognosis made by endoscopy or fluoroscopy can require a "core-needle biopsy” of the tumor mass, or a “fine- needle aspiration biopsy” which generally contains a suspension of cells from within the tumor mass.
  • a sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig; rat; mouse; rabbit.
  • a primate e.g., chimpanzee or human
  • cow cow
  • dog cat
  • rodent e.g., guinea pig
  • rat rat
  • mouse rabbit
  • a sample may be treated with a fixative such as formaldehyde and embedded in paraffin (FFPE) and sectioned for use in the methods of the invention.
  • FFPE formaldehyde and embedded in paraffin
  • fresh or frozen tissue may be used.
  • These cells may be fixed, e.g., in alcoholic solutions such as 100% ethanol or 3 : 1 methanokacetic acid.
  • Nuclei can also be extracted from thick sections of paraffin-embedded specimens to reduce truncation artifacts and eliminate extraneous embedded material.
  • biological samples, once obtained, are harvested and processed prior to hybridization using standard methods known in the art. Such processing typically includes protease treatment and additional fixation in an aldehyde solution such as formaldehyde.
  • the invention is also directed to methods to selectively enrich circular RNAs.
  • Such methods are well-known in the art including: (i) deadenylation enzyme(s) followed by (ii) 3 '-> 5' exonuclease(s); or (i) decapping enzyme(s) followed by (ii) 5'-> 3' exonuclease(s).
  • deadenylation enzyme(s) followed by (ii) 3 '-> 5' exonuclease(s)
  • decapping enzyme(s) followed by (ii) 5'-> 3' exonuclease(s) for a review, see Parker and Song, 2004, Nat. Struct. Mol. Biol. 11(2) 121- 126, the contents of which are hereby incorporated by reference in its entirety.
  • Examples include RNase R available from EPICENTER Biotechnologies, Madison, WI.
  • the nucleic acids of the present invention may be detected by primers or probes specific to the mis-ordered exon junction.
  • a labeled probe e.g., a biotinylated probe
  • the resulting bound nucleic acid molecules may be detected using mass spectrometry or a next generation nucleic acid sequencing technology.
  • the nucleic acid molecules encoding the circular RNA are detected using two outward facing PCR primers.
  • nucleic acid amplification is the chemical or enzymatic synthesis of nucleic acid copies which contain a sequence that is complementary to a nucleic acid sequence being amplified (template).
  • the methods and kits of the invention may use any nucleic acid amplification or detection methods known to one skilled in the art, such as those described in U.S. Pat. Nos.
  • the nucleic acids are amplified by PCR amplification using methodologies known to one skilled in the art.
  • amplification can be accomplished by any known method, such as ligase chain reaction (LCR), Q -replicase amplification, rolling circle amplification, transcription amplification, self-sustained sequence replication, nucleic acid sequence-based amplification (NASBA), each of which provides sufficient amplification.
  • LCR ligase chain reaction
  • Q -replicase amplification Q -replicase amplification
  • rolling circle amplification transcription amplification
  • self-sustained sequence replication nucleic acid sequence-based amplification
  • Branched-DNA technology may also be used to qualitatively demonstrate the presence of a circular ANRIL RNA, or to quantitatively determine the amount of this particular nucleic acid in a sample.
  • Nolte reviews branched- DNA signal amplification for direct quantitation of nucleic acid sequences in clinical samples (Nolte, 1998, Adv. Clin. Che
  • PCR process is well known in the art and is thus not described in detail herein.
  • PCR methods and protocols see, e.g., Innis et al, eds., PCR Protocols, A Guide to Methods and Application, Academic Press, Inc., San Diego, Calif. 1990; U.S. Pat. No. 4,683,202 (Mullis); which are incorporated herein by reference in their entirety.
  • PCR reagents and protocols are also available from commercial vendors, such as Roche Molecular Systems.
  • PCR may be carried out as an automated process with a thermostable enzyme. In this process, the temperature of the reaction mixture is cycled through a denaturing region, a primer annealing region, and an extension reaction region automatically. Machines specifically adapted for this purpose are commercially available.
  • Suitable next generation sequencing technologies are widely available. Examples include the 454 Life Sciences platform (Roche, Branford, CT) (Margulies et al. 2005 Nature, 437, 376-380); lllumina's Genome Analyzer (Illumina, San Diego, CA); U.S. Pat. Nos. 6,306,597 and 7,598,035 (Macevicz); 7,232,656 (Balasubramanian et al.)); or DNA Sequencing by Ligation, SOLiD System (Applied Biosystems/Life Technologies; U.S. Pat. Nos.
  • Pyrosequencing is a nucleic acid sequencing method based on sequencing by synthesis, which relies on detection of a pyrophosphate released on nucleotide incorporation.
  • sequencing by synthesis involves synthesizing, one nucleotide at a time, a DNA strand complimentary to the strand whose sequence is being sought.
  • Study nucleic acids may be immobilized to a solid support, hybridized with a sequencing primer, incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5' phosphsulfate and luciferin. Nucleotide solutions are sequentially added and removed.
  • An example of a system that can be used by a person of ordinary skill based on pyrosequencing generally involves the following steps: ligating an adaptor nucleic acid to a study nucleic acid and hybridizing the study nucleic acid to a bead; amplifying a nucleotide sequence in the study nucleic acid in an emulsion; sorting beads using a picoliter multiwell solid support; and sequencing amplified nucleotide sequences by pyrosequencing methodology (e.g., Nakano et al, 2003, J. Biotech. 102, 117- 124).
  • Such a system can be used to exponentially amplify amplification products generated by a process described herein, e.g., by ligating a heterologous nucleic acid to the first amplification product generated by a process described herein.
  • Certain single-molecule sequencing embodiments are based on the principal of sequencing by synthesis, and utilize single-pair Fluorescence Resonance Energy Transfer (single pair FRET) as a mechanism by which photons are emitted as a result of successful nucleotide incorporation.
  • the emitted photons often are detected using intensified or high sensitivity cooled charge-couple-devices in conjunction with total internal reflection microscopy (TIRM). Photons are only emitted when the introduced reaction solution contains the correct nucleotide for incorporation into the growing nucleic acid chain that is synthesized as a result of the sequencing process.
  • TIRM total internal reflection microscopy
  • FRET FRET based single-molecule sequencing or detection
  • energy is transferred between two fluorescent dyes, sometimes polymethine cyanine dyes Cy3 and Cy5, through long-range dipole interactions.
  • the donor is excited at its specific excitation wavelength and the excited state energy is transferred, non-radiatively to the acceptor dye, which in turn becomes excited.
  • the acceptor dye eventually returns to the ground state by radiative emission of a photon.
  • the two dyes used in the energy transfer process represent the "single pair", in single pair FRET. Cy3 often is used as the donor fluorophore and often is incorporated as the first labeled nucleotide.
  • Cy5 often is used as the acceptor fluorophore and is used as the nucleotide label for successive nucleotide additions after incorporation of a first Cy3 labeled nucleotide.
  • the fluorophores generally are within 10 nanometers of each other for energy transfer to occur successfully.
  • An example of a system that can be used based on single-molecule sequencing generally involves hybridizing a primer to a study nucleic acid to generate a complex; associating the complex with a solid phase; iteratively extending the primer by a nucleotide tagged with a fluorescent molecule; and capturing an image of fluorescence resonance energy transfer signals after each iteration (e.g., Braslavsky et al, PNAS 100(7): 3960-3964 (2003); U.S. Pat. No. 7,297,518 (Quake et al) which are incorporated herein by reference in their entirety).
  • Such a system can be used to directly sequence amplification products generated by processes described herein.
  • the released linear amplification product can be hybridized to a primer that contains sequences complementary to immobilized capture sequences present on a solid support, a bead or glass slide for example.
  • Hybridization of the primer-released linear amplification product complexes with the immobilized capture sequences immobilizes released linear amplification products to solid supports for single pair FRET based sequencing by synthesis.
  • the primer often is fluorescent, so that an initial reference image of the surface of the slide with immobilized nucleic acids can be generated. The initial reference image is useful for determining locations at which true nucleotide incorporation is occurring. Fluorescence signals detected in array locations not initially identified in the "primer only" reference image are discarded as non-specific fluorescence.
  • the bound nucleic acids often are sequenced in parallel by the iterative steps of, a) polymerase extension in the presence of one fluorescently labeled nucleotide, b) detection of fluorescence using appropriate microscopy, TIRM for example, c) removal of fluorescent nucleotide, and d) return to step (a) with a different fluorescently labeled nucleotide.
  • the technology may be practiced with digital PCR.
  • Digital PCR was developed by Kalinina and colleagues (Kalinina et al., 1997, Nucleic Acids Res. 25; 1999-2004) and further developed by Vogelstein and Kinzler (1999, Proc. Natl. Acad. Sci. U.S.A. 96; 9236- 9241).
  • the application of digital PCR is described by Cantor et al. (PCT Pub. Nos. WO 2005/023091A2 (Cantor et al.); WO 2007/092473 A2, (Quake et al.)), which are hereby incorporated by reference in their entirety.
  • Digital PCR takes advantage of nucleic acid (DNA, cDNA or RNA) amplification on a single molecule level, and offers a highly sensitive method for quantifying low copy number nucleic acid.
  • Fluidigm® Corporation offers systems for the digital analysis of nucleic acids.
  • nucleotide sequencing may be by solid phase single nucleotide sequencing methods and processes.
  • Solid phase single nucleotide sequencing methods involve contacting sample nucleic acid and solid support under conditions in which a single molecule of sample nucleic acid hybridizes to a single molecule of a solid support. Such conditions can include providing the solid support molecules and a single molecule of sample nucleic acid in a "microreactor.” Such conditions also can include providing a mixture in which the sample nucleic acid molecule can hybridize to solid phase nucleic acid on the solid support.
  • Single nucleotide sequencing methods useful in the embodiments described herein are described in PCT Pub. No. WO 2009/091934 (Cantor).
  • nanopore sequencing detection methods include (a) contacting a nucleic acid for sequencing ("base nucleic acid,” e.g., linked probe molecule) with sequence-specific detectors, under conditions in which the detectors specifically hybridize to substantially complementary subsequences of the base nucleic acid; (b) detecting signals from the detectors and (c) determining the sequence of the base nucleic acid according to the signals detected.
  • the detectors hybridized to the base nucleic acid are disassociated from the base nucleic acid (e.g., sequentially dissociated) when the detectors interfere with a nanopore structure as the base nucleic acid passes through a pore, and the detectors disassociated from the base sequence are detected.
  • a detector also may include one or more regions of nucleotides that do not hybridize to the base nucleic acid.
  • a detector is a molecular beacon.
  • a detector often comprises one or more detectable labels independently selected from those described herein. Each detectable label can be detected by any convenient detection process capable of detecting a signal generated by each label (e.g., magnetic, electric, chemical, optical and the like). For example, a CD camera can be used to detect signals from one or more distinguishable quantum dots linked to a detector.
  • the invention encompasses any method known in the art for enhancing the sensitivity of the detectable signal in such assays, including, but not limited to, the use of cyclic probe technology (Bakkaoui et ah, 1996, BioTechniques 20: 240-8, which is incorporated herein by reference in its entirety); and the use of branched probes (Urdea et ah, 1993, Clin. Chem. 39, 725-6; which is incorporated herein by reference in its entirety).
  • the hybridization complexes are detected according to well-known techniques in the art.
  • Reverse transcribed or amplified nucleic acids may be modified nucleic acids.
  • Modified nucleic acids can include nucleotide analogs, and in certain embodiments include a detectable label and/or a capture agent.
  • detectable labels include, without limitation, fluorophores, radioisotopes, colorimetric agents, light emitting agents, chemiluminescent agents, light scattering agents, enzymes and the like.
  • capture agents include, without limitation, an agent from a binding pair selected from antibody/antigen, antibody/antibody, antibody/antibody fragment, antibody/antibody receptor, antibody/protein A or protein G, hapten/anti -hapten, biotin/avidin, biotin/streptavidin, folic acid/folate binding protein, vitamin B 12/intrinsic factor, chemical reactive group/complementary chemical reactive group (e.g., sulfhydryl/maleimide, sulfhydryl/haloacetyl derivative, amine/isotriocyanate, amine/succinimidyl ester, and amine/sulfonyl halides) pairs, and the like.
  • Modified nucleic acids having a capture agent can be immobilized to a solid support in certain embodiments.
  • Antibody reagents can be used in assays to detect levels of circular ANRIL RNA in patient samples using any of a number of immunoassays known to those skilled in the art. Immunoassay techniques and protocols are generally described in Price and Newman, "Principles and Practice of Immunoassay," 2nd Edition, Grove's Dictionaries, 1997; and Gosling, "Immunoassays: A Practical Approach.” Oxford University Press, 2000. A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used. See, e.g., Self et al, 1996, Curr. Opin. Biotechnol, 7, 60-65.
  • immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction with laser induced fluorescence.
  • EIA enzyme multiplied immunoassay technique
  • ELISA enzyme-linked immunosorbent assay
  • MAC ELISA IgM antibody capture ELISA
  • MEIA microparticle enzyme immunoassay
  • CEIA capillary electrophoresis immunoassay
  • Liposome immunoassays such as flow- injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention. See, e.g., Rongen et al, 1997, J. Immunol Methods, 204, 105-133.
  • nephelometry assays in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the methods of the present invention.
  • Nephelometry assays are commercially available from Beckman Coulter (Brea, CA) and can be performed using a Behring Nephelometer Analyzer (Fink et al, 1989, J. Clin. Chem. Clin. Biochem., 27, 261-276).
  • Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody.
  • An antibody labeled with iodine- 125 125 I can be used.
  • a chemiluminescence assay using a chemiluminescent antibody specific for the nucleic acid is suitable for sensitive, non-radioactive detection of protein levels.
  • An antibody labeled with fluorochrome is also suitable.
  • fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R- phycoerythrin, rhodamine, Texas red, and lissamine.
  • Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ - galactosidase, urease, and the like.
  • a horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm.
  • TMB chromogenic substrate tetramethylbenzidine
  • An alkaline phosphatase detection system can be used with the chromogenic substrate p- nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm.
  • a ⁇ -galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-/3-D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm.
  • An urease detection system can be used with a substrate such as urea- bromocresol purple (Sigma Immunochemicals; St. Louis, MO).
  • a signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of 125 I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength.
  • a quantitative analysis can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, CA) in accordance with the manufacturer's instructions.
  • the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
  • the antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), pieces of a solid substrate material or membrane (e.g., plastic, nylon, paper), and the like.
  • An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.
  • the antibodies may be in an array one or more antibodies, single or double stranded nucleic acids, proteins, peptides or fragments thereof, amino acid probes, or phage display libraries.
  • the invention may further encompass detecting and/or quantitating using fluorescence in situ hybridization (FISH) in a sample, preferably a tissue sample, obtained from a subject in accordance with the methods of the invention.
  • FISH fluorescence in situ hybridization
  • a sample preferably a tissue sample, obtained from a subject in accordance with the methods of the invention.
  • FISH is a common methodology used in the art, especially in the detection of specific chromosomal aberrations in tumor cells, for example, to aid in diagnosis and tumor staging.
  • FISH fluorescence in situ hybridization
  • nucleic acid microarrays Methods for making nucleic acid microarrays are known to the skilled artisan and are described, for example, in Lockhart et al, 1996, Nat. Biotech. 14, 1675-1680, 1996 Schena et al, 1996, Proc. Natl. Acad. Sci. USA, 93, 10614-10619, U.S. Pat. No. 5,837,832 (Chee et al.) and PCT Pub. No. WO 00/56934 (Englert et al), herein incorporated by reference.
  • oligonucleotides may be synthesized or bound to the surface of a substrate using a chemical coupling procedure and an ink jet application apparatus, as described in U.S. Pat. No. 6,015,880 (Baldeschweiler et al), incorporated herein by reference.
  • a gridded array may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedure.
  • kits for measuring nucleic acids encoding the circular ANRTL RNA typically include, in suitable container means, (i) a probe that comprises an antibody or nucleic acid sequence that specifically binds to the polynucleotides of the invention, (ii) a label for detecting the presence of the probe and (iii) instructions for how to measure the level of the circular ANRTL polynucleotide.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe and/or other container into which a first antibody specific for one of the polypeptides or a first nucleic acid specific for one of the polynucleotides of the present invention may be placed and/or suitably aliquoted.
  • the kit will also generally contain a second, third and/or other additional container into which this component may be placed.
  • a container may contain a mixture of more than one antibody or nucleic acid reagent, each reagent specifically binding a different marker in accordance with the present invention.
  • the kits of the present invention will also typically include means for containing the antibody or nucleic acid probes in close confinement for commercial sale. Such containers may include injection and/or blow-molded plastic containers into which the desired vials are retained.
  • kits may further comprise positive and negative controls, as well as instructions for the use of kit components contained therein, in accordance with the methods of the present invention.
  • the various markers of the invention also provide reagents for in vivo imaging such as, for instance, the imaging of circular ANRIL RNAs using labeled reagents that detect them.
  • In vivo imaging techniques may be used, for example, to detect atherosclerosis.
  • reagents that detect the presence of these proteins or genes, such as antibodies may be labeled with a positron-emitting isotope (e.g., 18F) for positron emission tomography (PET), gamma-ray isotope (e.g., 99mTc) for single photon emission computed tomography (SPECT), a paramagnetic molecule or nanoparticle (e.g.,Gd 3+ chelate or coated magnetite nanoparticle) for magnetic resonance imaging (MRI), a near-infrared fluorophore for near- infra red (near-IR) imaging, a luciferase (firefly, bacterial, or coelenterate), green fluorescent protein, or other luminescent molecule for bioluminescence imaging, or a perfluorocarbon- filled vesicle for ultrasound.
  • a positron-emitting isotope e.g., 18F
  • PET positron emission tomography
  • such reagents may include a fluorescent moiety, such as a fluorescent protein, peptide, or fluorescent dye molecule.
  • fluorescent dyes include, but are not limited to, xanthenes such as rhodamines, rhodols and fluoresceins, and their derivatives; bimanes; coumarins and their derivatives such as umbelliferone and aminomethyl coumarins; aromatic amines such as dansyl; squarate dyes; benzofurans; fluorescent cyanines; carbazoles; dicyanomethylene pyranes, polymethine, oxabenzanthrane, xanthene, pyrylium, carbostyl, perylene, acridone, quinacridone, rubrene, anthracene, coronene, phenanthrecene, pyrene, butadiene, stilbene, lanthanide metal chelate complexes, rare-earth
  • Fluorescent dyes are discussed, for example, in U.S. Pat. Nos. 4,452,720 (Harada et al); 5,227,487 (Haugland and Whitaker); and 5,543,295 (Bronstein et al).
  • Other fluorescent labels suitable for use in the practice of this invention include a fluorescein dye.
  • Typical fluorescein dyes include, but are not limited to, 5- carboxyfluorescein, fluorescein-5-isothiocyanate and 6-carboxyfluorescein; examples of other fluorescein dyes can be found, for example, in U.S. Pat. Nos.
  • kits may include a rhodamine dye, such as, for example, tetramethylrhodamine-6- isothiocyanate, 5- carboxytetramethylrhodamine, 5-carboxy rhodol derivatives, tetramethyl and tetraethyl rhodamine, diphenyldimethyl and diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101 sulfonyl chloride (sold under the tradename of TEXAS RED®, and other rhodamine dyes.
  • a rhodamine dye such as, for example, tetramethylrhodamine-6- isothiocyanate, 5- carboxytetramethylrhodamine, 5-carboxy rhodol derivatives, tetramethyl and tetraethyl rhodamine, diphenyl
  • kits may include a cyanine dye, such as, for example, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7.
  • Phosphorescent compounds including porphyrins, phthalocyanines, polyaromatic compounds such as pyrenes, anthracenes and acenaphthenes, and so forth, may also be used.
  • a variety of methods may be used to identify compounds that modulate the levels of the circular ANRIL nucleic acids.
  • an assay that provides a readily measured parameter is adapted to be performed in the wells of multi-well plates in order to facilitate the screening of members of a library of test compounds as described herein.
  • an appropriate number of cells can be plated into the cells of a multi-well plate, and the effect of a test compound on expression levels.
  • the compounds to be tested can be any small chemical compound, or a macromolecule, such as a protein, sugar, nucleic acid or lipid.
  • test compounds will be small chemical molecules and peptides.
  • any chemical compound can be used as a test compound in this aspect of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used.
  • the assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma- Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.
  • high throughput screening methods are used which involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds.
  • Such "combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. In this instance, such compounds are screened for their ability to modulate levels of circular ANRIL nucleic acids.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010, 175 (Rutter and Santi), Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; and Houghton et al, 1991, Nature, 354:84-88).
  • peptide libraries see, e.g., U.S. Pat. No. 5,010, 175 (Rutter and Santi), Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; and Houghton et al, 1991, Nature, 354:84-88.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: U.S. Pat. Nos.
  • nucleic acid libraries see Ausubel, Berger and Sambrook, all supra
  • antibody libraries see, e.g., Vaughn et al, 1996, Nat. Biotech., 14(3):309-314, carbohydrate libraries, e.g., Liang et al, 1996, Science, 274: 1520-1522, small organic molecule libraries (see, e.g., benzodiazepines, Baum, 1993, C&EN, Jan 18, page 33.
  • a variety of nucleic acids may be used to inhibit the function of the circular ANRIL RNAs.
  • Ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy target mRNAs, particularly through the use of hammerhead ribozymes.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA.
  • the target mRNA has the following sequence of two bases: 5'- UG-3'. The construction and production of hammerhead ribozymes is well known in the art.
  • a composition of ribozyme molecules preferably includes one or more sequences complementary to a target mRNA, and the well-known catalytic sequence responsible for mRNA cleavage or a functionally equivalent sequence (see, e.g., U.S. Pat. Nos. 5,093,246 (Cech et al); 5,766,942 (Haseloff et al); 5,856, 188 (Hampel et al) which are incorporated herein by reference in their entirety). Ribozyme molecules designed to catalytically cleave target RNA transcripts can also be used to treat or prevent an ANRIL-associated disorder.
  • ANRIL structure and expression in relation to ASVD-SNP genotype and INK4/ARF expression.
  • ANRIL isoforms containing exons 1-2 or 4-6 correlated with ASVD-SNP genotype, however those containing exons 18-19 did not.
  • ANRIL transcription produces multiple rare, non-coding RNA species
  • ANRIL isoforms have been proposed based upon the assembly of ESTs and the sequencing of cDNA libraries (See Figure 9). To determine which of these isoforms predominates in vivo, we performed RNA ligase mediated (RLM)-RACE in cell lines and primary human peripheral blood T-lymphocytes (PBTL). In addition to using the RLM procedure to maximize the detection of mRNA transcripts, we employed a high-fidelity Taq polymerase capable of amplifying complex DNAs such as those containing SINE, LINE and Alu elements.
  • RLM RNA ligase mediated
  • PBTL primary human peripheral blood T-lymphocytes
  • Primers for 3' and 5' RACE were designed within exons 1, 2, 4, 6, 9, 13, 16 and 18 of the originally reported transcript, NR_003529 [27], but only primers in exons 4 and 6 selectively amplified ANRIL sequences (data not shown). These amplicons were cloned and sequenced to verify the resulting DNA sequences. Using exon 4 and 6 primers, we identified multiple ANRIL variants, including novel splice isoforms that were not previously reported ( Figure 1A, novel exons 10a, 13b). We also detected several transcripts with a peculiar, non-colinear exon sequence, most notably in the HeLa and primary PBTL populations (13-14-4-5, 13-14-4 in Figure 1A).
  • ANRIL RNAs containing exon 15 frequently maintained the canonical exonic structure (e.g., 15-16- 17-18-19). We termed these exons as “distal” because they are located at the 3' end of ANRIL, and those prior to exon 15 as "proximal”.
  • RNA-seq next-generation RNA sequencing
  • ANRIL expression detected in both datasets was comparable to that of other long, non-coding RNAs including HOT AIR and Kcnqlotl (maximal peak heights of 10 and 18, respectively).
  • exon deletions and/or MTAP splicing explain the relative excess transcription of distal ANRIL exons in some cancer cell lines harboring 9p21 deletion, these mechanisms do not explain the decreased transcription of exons 4-12 versus exons 1-3, in any cell line.
  • somatic deletions and MTAP splicing do not explain the uniform decrease of the central exons compared to the proximal and distal exons in cultures of primary cells ( Figure 11).
  • RNA-seq and TaqMan® analyses indicate that ANRIL is a rare, multi-variant RNA species in which transcripts containing exons 1-3 or 13— 19 predominate over those containing exons 4-12.
  • pl6 and pl5 1NK4b into cDNA was efficiently accomplished with either oligo dT or HEX primers alone. Conversely, ANRIL4-6 and 14-5 were effectively primed with HEX but not oligo dT, confirming that these transcripts were not polyadenylated.
  • RNAse R specifically digests both structured and non-structured linear RNAs, but spares RNA circles and lariats [50].
  • RNAse R specifically digests both structured and non-structured linear RNAs, but spares RNA circles and lariats [50].
  • RNA from normal and immortalized human fibroblasts (IMR90 and Hs68, respectively) treated with or without RNAse R we generated cDNA and conducted TaqMan® analysis for ANRIL expression. As expected for linear species, RNAse R
  • INK4a INK4b treatment caused a marked reduction in the number of pi 6 and pi 5 transcripts detected (4.5- and 8.4-fold decrease, respectively), demonstrating that these coding transcripts are predominantly linear ( Figures 3C and 3D).
  • ANRIL4-6 expression also exhibited RNAse R- dependent enrichment in both cell lines.
  • ANRIL1-2 levels decreased consistent with a predominantly linear species, and ANRIL18-19 demonstrated an intermediate behavior consistent with a mix of linear and circular forms ( Figures 3C and 3D). Together, these data provide evidence that ANRIL4-6 and 14-5 are predominantly contained within non- polyadenylated, circular (or lariat) ANRIL (cANRIL) transcripts.
  • Circular ANRIL species are observed in a wide variety of cell types.
  • ESEs exon splicing enhancers
  • ESSs silencers
  • ISEs intronic splicing enhancers
  • ISSs silencers
  • ANRIL Encodes Multiple, Non-abundant Linear and Circular Species
  • INK4/ARF tumor suppressors pi 5 and pi 6 ( Figures IB, 2B and 11) .
  • This low level of expression is comparable to what we observed for other regulatory non-coding RNAs (i.e., HOTAIR and Kcnqlotl) associated with PcG-mediated repression.
  • Other regulatory non-coding RNAs i.e., HOTAIR and Kcnqlotl
  • the discovery of non-colinear ANRIL species whose expression correlated with INK4/ARF transcription suggested that alternative splicing events might modify ANRIL structure leading to changes in PcG-mediated INK4/ARF repression.
  • RNA structure is formed that is sensitive to RNase R digestion [50].
  • exon skipping events generate large lariat structures, which can then undergo cis splicing to create RNase R-resistant circular RNAs.
  • transcripts containing ANRIL4-6 and 14-5 were resistant to RNase R degradation, were not polyadenylated and could be PCR amplified using sets of outward facing primers ( Figures 3 and 4). Therefore, cANRIL species appear to result from exon skipping events occurring during RNA splicing.
  • the ASVD risk interval includes exon 15 where the termination of most exon skipping events that produce cANRIL occur ( Figures 4B, 4C).
  • exon 15 SNPs rs7341786 and rs7341791
  • cANRIL expression reflecting ANRIL splicing
  • INK4/ARF expression which it does, Figures 5A and 13
  • individuals homozygous for the 'A' allele of rs7341786 (and rsl0757278) should exhibit increased production of cANRIL species containing exon 14 but not exon 15 (which they do, see Figure 4B, C) and that these individuals with increased propensity for splicing should exhibit de-repressed INK4/ARF expression (which they do, [16]).
  • a caveat to the splicing model is that the identified exon 15 SNPs need not be the true causal variant(s) influencing ANRIL splicing. It is possible that other polymorphisms not detected by our sequencing strategy could regulate ANRIL splicing. In particular, the sequencing strategy employed would not find differences between the pooled samples in
  • ANRIL harbors several LINE and SINE elements, and such repetitive motifs have been reported to modulate RNA splicing in other systems [65,66]. Therefore, while the exon 15 SNPs appear to be prime candidates to regulate ANRIL splicing, a variety of other classes of polymorphisms could also influence splicing and would not have been observed by the chosen sequencing approach.
  • the splicing and transcriptional models are not mutually exclusive.
  • a single causal variant may influence both processes or there may be multiple causal variants that influence either process within the ASVD risk interval.
  • a single causal variant may influence both processes or there may be multiple causal variants that influence either process within the ASVD risk interval.
  • a third possibility also exists. While circular RNA byproducts of exon skipping have generally been regarded as inconsequential, circular RNAs with catalytic activities (e.g. group I and some group II introns) are well described in bacteria, lower eukaryotes, plants [67].
  • viroids and the hepatitis delta satellite virus have circular RNA genomes [68,69]. Although we are not aware of any endogenously produced circular RNA with discrete function in mammals, clearly circular RNAs species can possess independent functions in non- mammalian species, and we remain open to the possibility that cANRIL itself can directly participate in INK4/ARF regulation. [00111] In summary, this work links ASVD-genotype to ANRIL structure and INK4/ARF regulation, providing evidence for what we believe is a first association between endogenous circular RNA expression and a mammalian phenotype (ASVD).
  • ASVD mammalian phenotype
  • WM266-4, UACC 257, A2058, A375, SUM-149, RRMI-8322 and telomerized Hs68 cells were obtained and grown as previously described [78-80].
  • MDA-MB-468, MDA- MB-436, MDAMB-231, MCF7, BT-474, BT-549, T-47D, COLO 205, T84, LoVo, LS 174T, SW480, LS 1034, HeLa, HUVEC, IMR90, Ramos, Raji, Jurkat and U-87 cells were originally obtained from ATCC and cultured as suggested.
  • CD3 positive T-cells were isolated from human peripheral blood samples as previously described [42]. RNA was generated from proliferating cell lines and isolated human T-cells using the RNAeasy system (Qiagen Inc., Valencia, CA). 3 ' and 5' RACE was performed as described in the Firstchoice RLM-RACE manual (Ambion Inc., Austin, TX). This procedure is optimized for the detection of rare transcripts and provides additional steps to improve the specificity of mRNA amplification. Gene-specific primers were designed within ANRIL exons 4 and 6 as shown in Figure 1A and Table 2; RACE primers for other ANRIL exons tested did not amplify chromosome 9 specific products.
  • PCR reactions were conducted using SuperTaq-Plus (Ambion) in a Bio-Rad DNA Engine thermocycler.
  • SuperTaq-Plus is a high fidelity, long range polymerase with the capability to amplify complex DNAs such as repetitive SINE, LINE and Alu elements. Cycling conditions for 5'RACE were: 94°C 3 min, 34 x [94°C 30s, 60°C 30s, 68°C 3 min], 68°C 5 min (inner reaction) and 94°C 3 min, 34 x [94°C 30s, 62°C 30s, 68°C 3 min], 68°C 5 min (outer reaction).
  • Cycling conditions for 3 'RACE were: 94°C 3 min, 34 x [94°C 30s, 57 or 60°C 30s, 68°C 3 min] (outer reaction) and 94°C 3 min, 34 x [94°C 30s, 60°C 30s, 68°C 3 min] (inner reaction).
  • Cloning of the resulting PCR products was conducted using the TOPO-Blunt cloning kit (Invitrogen). Sequencing of the resulting clones was conducted using both M13F and M13R primers.
  • ANRIL primer sets were designed to span at least one intron and were shown to have high specificity with linear amplification efficiencies between 88 and 94% ( Figure 10). Final primer and probe concentrations were 900 and 250nM, respectively.
  • Products from the ANRIL 1-2, 4-6, 14-5 and 18-19 qRT-PCR reactions were cloned separately into the pBluntll- TOPO vector (Invitrogen) and verified. Real-time PCR was carried out in triplicate on an ABI 7900HT thermocyler.
  • a total of 368,846,235 reads generated on the Illumina platform (study SRP002274) were downloaded from the NCBI Short Read Archive. The reads were first screened for unique 20mers deriving from chromosome 9:21,700,000-22,300,000 using the UCSC genome browser Duke uniqueness mapability table. The resulting reads were mapped using the TopHat spliced aligner (PMID: 19289445) to the reference human genome (hgl 8). The resulting coverage plot was imported into the UCSC genome browser for display. Also analyzed were two independent CalTech ENCODE mRNA-seq datasets (http://bit.ly/af3P4c) from HeLa cells.
  • PCR primers pointing in opposite directions on ANRIL exons 1, 4, 6, 13, 16 and 18 were designed using Primer3 software and analyzed for hairpins using Netprimer (Premier Biosoft and http://frodo.wi.mit.edu/primer3/) (Table 2).
  • PCR reactions were conducted using cDNA representing 15ng of mock or Rnase R-treated RNA. Reactions were performed using Apex Hot Start Taq DNA polymerase and Buffer 2 (Genesee Scientific) in a Bio-Rad DNA Engine thermocycler. The cycling conditions were as follows: 95°C 15 min, 40 x [94°C 30s, 59°C 30s, 72°C 1 min], 72°C 2 min.
  • the resulting PCR products were cloned into the TOPO-Blunt cloning kit (Invitrogen) and sequenced using M13F and M13R primers.
  • Genomic DNA was generated from T-cells of healthy human volunteers of known
  • 2 lug of DNA was pooled from five individuals homozygous for the G-allele of rs 10757278 and another five individuals homozygous for the A-allele.
  • Samples were sent to NimbleGen for sequence capture using a tiled array spanning human chromosome 9 (22,054,888-22, 134, 171).
  • the resulting amplified DNA fragments were analyzed at the UNC Genome Analysis Facility using both Illumina GAII and Roche 454 technology.
  • DNA was randomly sheered and appropriate adapters ligated.
  • Resulting sequences were aligned to the entire human genome (hgl8) using MAQ, SOAP, and gsMapper software [52,53].
  • MAQ was run with default settings and output was translated into BAM format.
  • SOAP alignment was performed allowing up to 10 gap bases and 2 mismatches and also translated into BAM format.
  • Mapping with gsMapper was performed with default settings.
  • SNP calls from MAQ and SOAP were generated using the pileup function of the SAMtools library [81]. Calls were culled to include only those SNPs appearing in > 20 percent of reads.
  • Table 1 Splice site analysis of polymorphisms in the ASVD risk interval near ANRIL exon-intron boundaries. SNPs within 200bp of an ANRIL inton-exon boundary were analyzed for their effects on putative exon splicing enhancer (ESE), exon splicing silencer (ESS), intron splicing enhancer (ISE), and intron splicing silencer (ISS) sequences as described ([56,57] and Z. Wang unpublished data). A score of -1 indicates that the minor allele destroys one cis-element and +1 indicates that the minor allele creates one cis-element. SNPs identified as unique to the AA or GG samples using sequence capture are shown in bold.
  • HapMap3 GG- individuals homozygous for the 'G' allele at rsl0757278.
  • ANRIL 1-2 ABI TaqMan # Hs01390879_ml ANRIL Exons 1-2
  • ANRIL 4/6 R 5'-TCCACCACACCTAACAGTGATGCTTG-3' ANRIL Exon 6 SEQ ID NO.1 1
  • ANRIL 18/19 Probe 5 '-/ 5HEX/TGTGTGTTTCCTTGTGAGCTACTGCA/3BHQ 2/-3 ' SEQ ID N0.15
  • ANRIL 14-5R 5'-TGCTGTTGAATCAGAATGAGG-3' ANRIL Exon 5 SEQ ID N0.17 ANRIL 14-5 Probe 5 56FAM/AGGGACACTAAGTCACTGGTCTGAGTTCTTA/3BHQ 1/-3' SEQ ID NO.18
  • HUMP14-ARF3M1 5'-/FAM CAGCAGCCGCTTCC NFQ/-3' SEQ ID N0.24
  • ANRIL Exon 14 MTAP4 ANRIL 14_Probe 5V56FAM/TCCCTCAAGGAGCCACAAGCTG/3BHQJ/-3' SEQ ID N0.27
  • ANRIL Exon 1 SEQ ID N0.28
  • pl5IN 4b ABI TaqMan # Hs00793225_ml pl5 Exons 1-2
  • An exemplary circular ANRIL species 14-5 (-13-14-5-6-) and human ANRIL exons 5, 6, 13 and 14 may be found below.
  • the human cANRIL (14-5) was cloned from the T47D cell line product. The coding is as follows: italic is exon 13, bold is exon 14, bold italic is exon 5, and large font is exon 6.
  • the TaqMan® primers and probes used for quantitative reverse transcription polymerase PCR are also shown.
  • An exemplary circular ANRIL species 14-5 (exons 13, 14, 5 and 6) and the cloning strategy include SEQ ID Nos. 38-45.
  • TaqMan Probe GGACACTAAGTCACTGGTCTGAGTTCTTAAA
  • Murine ANRIL Exons and Murine Circular ANRIL (mcANRIL)
  • RNA-seq next-generation RNA sequencing
  • Illumina HiSeq 2000 sequencer an Illumina HiSeq 2000 sequencer on an enriched cDNA library from mouse embryonic fibroblasts (MEFs).
  • DNAse treated total RNA was purified from wild-type C57B6 MEFs and RNA quality was assessed using a BioAnalyzer (Agilent).
  • the RNA was then depleted of ribosomal RNA using a RiboMinus Transcriptome Isolation Kit (Invitrogen).
  • a cDNA library was prepared from the RiboMinus treated RNA using a TruSeq RNA Sample Preparation Kit (Illumina). Quality of the cDNA library was measured using a BioAnalyzer (Agilent). The cDNA library was then subjected to an enrichment procedure using a custom designed SureSelect Target Enrichment kit (Agilent). The custom kit selected for cDNA sequences that originated from a 500 kb region on mouse chromosome 4 (88,838,000-89,350,000) that is homologous to the human INK4/ARF locus. The enriched cDNA library was tested for quality on a BioAnalyzer (Agilent) and paired-end sequencing of the enriched cDNA library was performed on the Illumina HiSeq 2000.
  • Paired-end sequencing reads were mapped to the mm9 release of the murine genome.
  • the mapped reads were analyzed using MapSplice (PMID: 20802226), an algorithm that analyzes next-generation RNA-seq data to identify splicing between annotated and novel exonic sequences.
  • MapSplice PMID: 20802226
  • This analysis identified linear (murine ANRIL (mANRIL)) and non- colinear (murine circular ANRIL (mcANRIL)) RNA species that are similar in exonic architecture to human ANRIL and cANRIL.
  • murine ANRIL exons and circular ANRIL were isolated (see the description of Fig. 14A and 14B).
  • Table 3 displays the identified exons that comprise mANRIL/mc ANRIL species.
  • Table 4 displays intron/exon splice junctions associated with linear mANRIL splice variants, and Table 5 displays non-colinear splice junctions that comprise the four most predominant mcANRIL species. Cloning and Sanger sequencing of splice junctions from one linear transcript (shown in boldface in Table 4) and the four predominant non-colinear mANRIL/mcANRIL transcripts was performed to validate the MapSplice analysis. The murine circular ANRIL results (mcANRIL 25, 40, 31 and 11) were validated using standard PCR techniques. The results are shown in Figure 15.
  • the complete structure for mcANRIL is -3a-3-8- (see Table 3 below for the locations of the mANRIL exons).
  • a Taqman probe was designed to span the validated linear mANRIL splice junction between exons 2 and 3. Three additional Taqman probes were designed which span three of the four validated non-colinear mcANRIL splice junctions that comprise mcANRIL_Circle_25, mcANRIL_Circle_40, and mcANRIL_Circle_31.
  • Taqman quantitative RT-PCR was performed from freshly prepared cDNA from both early and late passage wild-type C57B6 MEFs to assess the relative difference in expression of each transcript with passage (Fig. 16). Relative expression of pl6 INK4a with passage (Fig. 16A) is shown as a positive control.
  • Table 3 Exons identified from analysis of RNA-seq data that comprise mANRIL and mcANRIL species.
  • Table 4 Intron/Exon splice junctions identified by MapSplice from RNA-seq data that associate with the linear splice variants of mANRIL. The validated linear mANRIL transcript is shown in bold.
  • Table 6 Sequences of primers and Taqman probes used to clone and measure mANRIL and mcANRIL species (SEQ ID NO.46-56).
  • chr4 89034424 89034443 mAN IL3 GGTTGCCATTTCCTCTGACA chr4 88942553 88942572 mANRILex2-3F i GCTGAGGCCTCTTTCTGTTG chr4 89066491 89066513 mcAN IL_AIILate_F i ACCTGAACTGAGCGTTGCTTTCC chr4 89033392 89033414 mcANRIL_lR TCCCAGTTGTGTACAAGGAAGAA chr4 89047041 89047060 mcANRIL_2R ILLAILLLI 11 ILLLAGl IL chr4 89053416 89053440 mcANRIL_3R CGATGTTAATTCAACAGTCAGCTTT i chr4 89049928 89049946 mcANRIL_4R GCCAGCCTTGGCTTTGTTA
  • Boehm M Nabel EG (2003) The cell cycle and cardiovascular diseases. Prog Cell Cycle Res 5: 19-30.
  • MTAP methylthioadenosine phosphorylase
  • Zaphiropoulos PG (1996) Circular RNAs from transcripts of the rat cytochrome P450 2C24 gene: correlation with exon skipping. Proc Natl Acad Sci U S A 93: 6536-6541.

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Abstract

La présente invention concerne des acides nucléiques isolés et purifiés codant pour un ARN circulaire comprenant un ou plusieurs exons ANRIL, des procédés de détection de ces acides nucléiques, et leurs utilisations. L'invention concerne des procédés de détection de troubles associés à ANRIL tels que l'athérosclérose, des kits associés, et des procédés de criblage de composés destinés à prévenir ou traiter ces troubles.
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