JP2008500039A - Viral miRNA and virus-related miRNA and uses thereof - Google Patents

Abstract

  Described herein are novel polynucleotides associated with viral infections. This polynucleotide is a miRNA and a miRNA precursor. Related methods and compositions that can be used for diagnosis, prognosis and treatment of medical conditions are disclosed. Also described herein are methods that can be used to identify modulators of viral infection.

Description

  The present invention relates generally to viral microRNA molecules and groups of human microRNA molecules associated with viral infections, as well as various nucleic acid molecules associated therewith or derived therefrom.

  MicroRNA (miRNA) is a short RNA oligonucleotide of about 22 nucleotides that is involved in gene regulation. MicroRNAs control gene expression by targeting mRNA for cleavage or translational repression. miRNAs are present in a wide range of species, including C. elegans, Drosophila and humans, but these have only been recently identified. More importantly, the role of miRNAs in disease development and progression has only recently been recognized.

  Due to their small size, miRNA has been difficult to identify using standard methodologies. A limited number of miRNAs have been identified by extracting large amounts of RNA. Moreover, it has been confirmed that miRNA contributes to presentation of a phenotype that can be visually discerned. Expression array data indicates that miRNAs are expressed at different developmental stages or in different tissues. The fact that miRNAs are limited to certain tissues or limited developmental stages indicates that miRNAs identified to date are likely to be a very small part of the total miRNA.

  Recently, computational approaches have been developed to identify the remaining miRNAs in the genome. Tools such as MiRscan and MiRseeker identified miRNAs that were later confirmed experimentally. Based on these computer tools, it is estimated that the human genome contains 200 to 255 miRNA genes. However, these estimates are based on the hypothesis that the remaining miRNAs to be identified will have the same properties as the already identified miRNAs. In light of the fundamental importance of miRNAs in mammalian biology and disease, techniques are needed to identify unknown miRNAs. The present invention fulfills this need and provides a large number of miRNAs and uses thereof. To date, no viral miRNA has been detected.

  The present invention aims to provide nucleic acid sequences of virus miRNAs and virus-related miRNAs, precursors thereof, target sequences and related sequences thereof that may be useful for diagnostic purposes and for modification of target gene expression. To do.

The present invention relates to an isolated nucleic acid comprising a sequence of a pri-miRNA, pre-miRNA, miRNA, miRNA ^* , anti-miRNA, or miRNA binding site, or a variant thereof. The nucleic acid is SEQ ID NO: 4097721-420493; the sequence of the precursor referred to in Table 1, 11-12, or 21-23; SEQ ID NO: 1-1142416 or 4204914-4204915; Table 1, 13-14 or 21 The sequence of the miRNA referenced to -23; SEQ ID NO: 11424417-4097720; the sequence of the target gene binding site referenced in Table 4, 10 or 15-16; its complement; or at least 70% identical to it, at least 12 It may contain a sequence comprising consecutive nucleotides. Isolated nucleic acids can be from 5 to 250 nucleotides in length.

  The present invention also relates to a probe containing the nucleic acid. The probe comprises at least 8-22 consecutive nucleotides complementary to the miRNAs referenced in SEQ ID NOs: 1-1142416 or 4204914-4204915, Table 1, 13-14 or 21-23, or variants thereof. May be included. The probe may also comprise at least 8-22 consecutive nucleotides complementary to a human miRNA that is differentially expressed upon viral infection, or a variant thereof.

  The present invention also relates to a plurality of the probes. The plurality of probes may include at least 10 of the probes. The plurality of probes may include at least 100 probes. The present invention also relates to a composition comprising one probe or a plurality of probes. The present invention also relates to a biochip including a solid substrate, and the substrate includes a plurality of the probes. Each probe can be attached to the substrate at a spatially defined address. The biochip can include a probe that is complementary to the viral miRNA. The biochip can also include a probe that is complementary to a human miRNA characterized by expression during viral infection.

  The present invention also relates to a method for detecting differential expression of miRNA associated with a disease. Prepare a biological sample and measure the level of nucleic acid that is at least 70% identical to SEQ ID NO: 1-1214416 or 4204914-4204915; sequences referenced in Tables 1, 13-14, and 21-23; or variants thereof . Differences in nucleic acid levels compared to controls indicate differential expression.

  The invention also relates to methods for identifying compounds that modulate pathological conditions. A cell capable of expressing a nucleic acid that is at least 70% identical to a sequence of the miRNA referenced in Tables 1, 13-14 and 21-23; or variants thereof is provided. After contacting the cell with a candidate modulator, the expression level of the nucleic acid is measured. A difference in the level of nucleic acid compared to a control identifies the compound as a modulator of a pathological condition associated with the nucleic acid.

  The present invention also relates to a method for inhibiting the expression of a target gene in a cell. The nucleic acid is introduced into the cell in an amount sufficient to inhibit expression of the target gene. The target gene may comprise a binding site substantially identical to a binding site referenced in Tables 4, 10 or 15-16, or variants thereof. The nucleic acid can comprise a portion of SEQ ID NOs: 1-1142416 or 4204914-4204915; the sequences of miRNAs referenced in Tables 1, 13-14, and 21-23; or variants thereof. Target gene expression can be inhibited both in vitro and in vivo.

  The present invention also relates to a method for increasing the expression of a target gene in a cell. The nucleic acid is introduced into the cell in an amount sufficient to increase expression of the target gene. The target gene may comprise a binding site substantially identical to a binding site referenced in Tables 4, 10 or 15-16, or variants thereof. A portion of the nucleic acid can be substantially complementary to SEQ ID NOs: 1-1142416 or 4204914-4204915; the sequences of miRNAs referenced in Tables 1, 13-14, and 21-23; or variants thereof. Target gene expression can be inhibited both in vitro and in vivo. Target gene expression can be increased both in vitro and in vivo.

  The present invention also includes the step of administering to a patient in need thereof a nucleic acid comprising the sequence of SEQ ID NO: 1 to 760616; the sequence shown in Table 10; the sequence shown in Table 17; or a variant thereof. It also relates to a method of treating patients having the diseases shown in Table 6.

  The present invention also provides that a portion of the nucleic acid is substantially complementary to SEQ ID NOs: 1-1142416 or 4204914-4420915; the sequences of miRNAs referenced in Tables 1, 13-14, and 21-23; or variants thereof It also relates to a method of treating a patient with a viral infection or a condition associated with a viral infection comprising administering to a patient in need thereof a nucleic acid that is of interest.

  The present invention provides nucleic acid sequences of virus miRNAs and virus-related miRNAs, precursors thereof, targets thereof and related sequences. Such nucleic acids are useful for diagnostic applications and for modifying target gene expression. Other aspects of the invention will be apparent to those skilled in the art from the following description of the invention.

1. Definitions Prior to disclosing and describing the compounds, products and compositions and methods of the present invention, the terminology used herein is for the purpose of describing particular embodiments only and is intended for purposes of limitation. Please understand that it does not. As used herein and in the appended claims, the singular forms “a”, “an”, and “the” do not expressly indicate otherwise in the context. It should be noted that as long as multiple instructions are included. Furthermore, it should be noted that the terms “and” and “or” may encompass both connected and disconnected meanings unless the context clearly indicates otherwise. .

  As used herein, “animal” may mean fish, amphibians, reptiles, birds, and mammals such as mice, rats, rabbits, goats, cats, dogs, cows, monkeys and humans.

  As used herein, “attached” or “immobilized” with reference to a probe and a solid support refers to the binding, washing, analysis, and removal of the bond between the probe and the solid support. It is sufficient to be stable under conditions. The bond may be a covalent bond or a non-covalent bond. Covalent bonds are formed directly between the probe and the solid support, or by cross-linking agents, or by inclusion of specific reactive groups on either the solid support or the probe or both molecules. Can be done. The non-covalent bond may be one or more of electrostatic interaction, hydrophilic interaction, and hydrophobic interaction. Included in the non-covalent bond is a covalent bond of a molecule such as streptavidin to a support and a non-covalent bond of a biotinylated probe with streptavidin. Immobilization can also include a combination of covalent and non-covalent interactions.

  As used herein, “biological sample” can mean a sample of biological tissue or biological fluid containing nucleic acids. Such samples include, but are not limited to, tissue isolated from animals. Biological samples also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, and skin. It is done. Biological samples also include explants derived from patient tissue and primary cell culture and / or transformed cell culture. A biological sample can be prepared by removing a sample of cells from an animal, but has already been isolated (eg, isolated from another person, isolated at another time, and / or It can also be achieved by using cells isolated for another purpose) or by carrying out the method of the invention in vivo. Storage tissues such as those having treatment or outcome history may be used.

  “Complement” or “complementary” as used herein may mean Watson-Crick base pairing or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

  “Differential expression” can mean a qualitative or quantitative difference in temporal and / or cellular gene expression patterns within and between cells and between tissues and tissues. Thus, differentially expressed genes can qualitatively change their expression, including, for example, activation or inactivation in normal or diseased tissues. Since a gene can be switched on and off in a particular state compared to another state, two or more states can be compared. Qualitatively controlled genes present a pattern of expression that can be detected by standard techniques within a state or cell type. Some genes are expressed in one state or cell type, but not both. Alternatively, differential expression is quantified in that, for example, when expression is regulated, it is upregulated resulting in an increase in the amount of transcript or downregulated resulting in a decrease in the amount of transcript. Can be the target. Different degrees of expression need only be large enough to be quantified via standard characterization techniques such as expression arrays, quantitative reverse transcriptase PCR, Northern analysis, and RNase protection.

  As used herein, “gene” refers to a genomic gene comprising transcriptional and / or translational control sequences and / or coding regions and / or untranslated sequences (eg, introns, 5′- and 3′-untranslated sequences). It can be. The coding region of a gene can be an amino acid sequence or a nucleic acid sequence that encodes a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA and antisense RNA. A gene can also be mRNA or cDNA corresponding to a coding region (eg, exons and miRNAs) that includes a 5'- or 3'-untranslated sequence optionally associated with it. The gene may also amplify in vitro generated nucleic acid molecules that contain all or part of the coding region and / or the 5'- or 3'-untranslated sequence associated therewith.

  As used herein, a “host cell” can be a naturally occurring cell or a transformed cell that contains a vector and supports the replication of the vector. Host cells can be cultured cells, explants, in vivo cells, etc. The host cell can be a prokaryotic cell such as E. coli or a eukaryotic cell such as a yeast, insect, amphibian, or mammalian cell, such as CHO, HeLa, and the like.

  As used herein in the context of two or more nucleic acid or polypeptide sequences, “identical” or “identity” refers to nucleotides or amino acids in which these sequences are the same for a particular region in a particular percentage. It means having. This ratio can be calculated by optimally aligning and comparing two sequences, comparing two sequences in a specific region, and measuring the number of positions where the same residue appears in both sequences. The number of matched positions is obtained, the number of matched positions is divided by the total number of positions in the specific region, and the result is multiplied by 100 to obtain the percent sequence identity. If the two sequences are of different lengths or have alignment-twisted ends and only a single sequence is included in a particular comparison region, then the single sequence residues are included in the denominator of the numerator Not included. When comparing DNA and RNA, thymine (T) and uracil (U) are considered equivalent. Confirmation of identity may be done manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

  As used herein, “inhibit” can mean prevent, suppress, reduce or eliminate.

As used herein, “label” means a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. sell. For example, useful labels include ³² P, fluorescent dyes, high electron density reagents, enzymes (such as those commonly used in ELISAs), biotin, digoxigenin, or haptens and detectable Other entities that can be mentioned. The label may be incorporated at any position in the nucleic acid and protein.

  As used herein, “nucleic acid” or “oligonucleotide” or “polynucleotide” may mean at least two nucleotides covalently linked together. As will be apparent to those skilled in the art, the depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the depicted single-stranded complementary strand. Also, as will be apparent to those skilled in the art, many variants of a nucleic acid can be used as a given nucleic acid for the same purpose. Thus, nucleic acids also include substantially identical nucleic acids and their complements. Also, as will be apparent to those skilled in the art, a single strand provides a probe for a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, nucleic acids also include probes that hybridize under stringent hybridization conditions.

  Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. Nucleic acids may be genomic DNA and cDNA, DNA, RNA, or hybrids, where the nucleic acid is a combination of deoxyribonucleotides and ribonucleotides, as well as uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypopo Combinations of bases including xanthine, isocytosine and isoguanine may be included. These nucleic acids can be obtained by chemical synthesis or recombinant methods.

Nucleic acids generally contain a phosphodiester bond, but at least one different bond, such as a phosphoramidate bond, a phosphorothioate bond, a phosphorodithioate bond, or an O-methyl phosphoramidite bond, and a peptide nucleic acid backbone and bond Nucleic acid analogs that may have Other nucleic acid analogs include the positive backbone; the nonionic backbone, and those described in US Pat. Nos. 5,235,033 and 5,034,506, incorporated herein by reference. Examples thereof include those having a skeleton that does not contain ribose. Nucleic acids containing one or more non-naturally occurring nucleotides or modified nucleotides are also included in one definition of nucleic acid. Modified nucleotide analogues can be located, for example, at the 5 ′ end and / or the 3 ′ end of the nucleic acid molecule. Representative examples of nucleotide analogs can be selected from sugar-modified or backbone-modified ribonucleotides. However, it should be noted that nucleobases are modified ribonucleotides, ie uridine or cytidine modified at position 5, eg 5- (2-amino) propyluridine, 5-bromouridine; modified at position 8 Natural instead of naturally occurring nucleobases such as adenosine and guanosine, eg 8-bromoguanosine; diazanucleotides, eg 7-diaza-adenosine; O-alkylated and N-alkylated nucleotides, eg N6-methyladenosine Also suitable are ribonucleotides containing nucleobases not present in 2'-OH @ - group, H, OR, R, _{halo, SH, SR, NH 2,} NHR, NR 2 or CN (where, R is C ₁ -C ₆ alkyl, alkenyl or alkynyl, halo represents F , Cl, Br or I). Modification of the ribose-phosphate backbone can be made for a variety of reasons, for example, to increase the stability and half-life of such molecules in a physiological environment, or as a probe on a biochip. Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs and mixtures of naturally occurring nucleic acids and analogs may be made.

  As used herein, “operably linked” can mean that expression of a gene is under the control of a promoter spatially linked to the gene. The promoter can be located 5 '(upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene that the promoter controls in the gene from which the promoter is derived. As is known in the art, variations in this distance can be accommodated without compromising promoter function.

  As used herein, a “probe” means an oligonucleotide that can bind to a target nucleic acid of a complementary sequence by one or more chemical bonds, usually by complementary base pairing, usually by hydrogen bond formation. Depending on the stringency of the hybridization conditions, the probe may bind to a target sequence that lacks complete complementarity with the probe sequence. There can be any number of base pair mismatches that can interfere with hybridization between the target sequence and the single stranded nucleic acid of the invention. However, if the number of mutations is so high that hybridization does not occur even under low stringency hybridization conditions, the sequence is not a complementary target sequence. The probe may be single stranded, or may be partially single stranded and partially double stranded. The number of probe strands is affected by the structure, composition, and properties of the target sequence. The probe may be labeled directly or indirectly with biotin or the like that later binds to the streptavidin complex.

  As used herein, “promoter” can refer to a synthetic or naturally-derived molecule that can cause, activate, or enhance the expression of a nucleic acid in a cell. A promoter can include one or more specific regulatory elements to further enhance its expression and / or to alter its spatial and / or temporal expression. A promoter can also include a terminal enhancer element or repressor element, which can be located as much as several thousand base pairs from the transcription start site. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter constitutively controls the expression of genetic components, or differentially controls with respect to the cell, tissue or organ where expression occurs, or with respect to the stage of development where expression occurs, or physiological stress, pathogen, It can be controlled in response to external stimuli such as metal ions or inducers. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter Or SV40 late promoter and CMV IE promoter.

As used herein, a “selectable marker” is any that confers a phenotype to the cell in which it is expressed to facilitate the identification and / or selection of cells transfected or transformed with the gene construct. Can mean a gene. Representative examples of selectable markers include ampicillin resistance gene (Amp ^r ), tetracycline resistance gene (Tc ^r ), bacterial kanamycin resistance gene (Kan ^r ), zeocin resistance gene, and resistance to the antibiotic aureobasidin A. AURI-C gene, phosphinotricin resistance gene, neomycin phosphotransferase gene (nptII), hygromycin resistance gene, β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein coding gene And the luciferase gene.

As used herein, “stringent hybridization conditions” refers to hybridization of a first nucleic acid sequence (eg, a probe) with a second nucleic acid sequence (eg, a target), such as in a complex mixture of nucleic acids. May mean conditions that do not hybridize to other sequences. Stringent conditions are sequence-dependent and will vary with different circumstances. Generally, stringent conditions are selected to be about 5-10 ° C. lower than the thermal melting temperature (T _m ) for the specific sequence at a defined ionic strength pH. T _m can be the temperature (under defined ionic strength, pH, and nuclear concentration) at which 50% of the probe complementary to the target hybridizes in equilibrium with the target sequence (the target sequence is present in excess). Therefore, at T _m , 50% of the probe is occupied in equilibrium). Stringent conditions are a salt concentration of pH 7.0-8.3 and less than about 1.0M sodium ion, generally about 0.01-1.0M sodium ion concentration (or other salt), and the temperature is short It may be at least about 30 ° C. for probes (eg, about 10-50 nucleotides) and at least about 60 ° C. for long probes (eg, longer than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2 to 10 times the hybridization background. Exemplary stringent hybridization conditions include the following conditions: 50% formamide, 5 × SSC, and 1% SDS, incubated at 42 ° C., or 5 × SSC, 1% SDS, 65 ° C. Incubate, wash in 0.2 × SSC, and 0.1% SDS at 65 ° C.

  As used herein, “substantially complementary” means that the first sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, At least 60%, 65%, 70%, 75%, 80%, 85 with the complement of the second sequence with respect to a region of 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides %, 90%, 95%, 97%, 98% or 99% identical or may mean that the two sequences hybridize under stringent hybridization conditions.

  As used herein, “substantially identical” means that the first and second sequences are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, At least 60%, 65%, 70%, 75%, 80%, 85%, 90% for 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotide or amino acid regions, It can mean 95%, 97%, 98% or 99% identical, or in terms of nucleic acids, where the first sequence is substantially complementary to the complement of the second sequence.

  As used herein, “target” can mean a polynucleotide that can be bound by one or more probes under stringent hybridization conditions.

  As used herein, “terminator” may mean a sequence at the end of a transcription unit that signals termination of transcription. The terminator may be a 3 'untranslated DNA sequence that contains a polyadenylation signal, which may facilitate the addition of polyadenylic acid sequences to the 3' end of the primary transcript. Terminators can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Representative examples of terminators include SV40 polyadenylation signal, HSV TK polyadenylation signal, CYC1 terminator, ADH terminator, SPA terminator, Agrobacterium tumefaciens nopaline synthase (NOS) gene terminator Viral (CaMV) 35S gene terminator, maize-derived zein gene terminator, Rubisco small subunit gene (SSU) gene terminator sequence, subclover atrophy virus (SCSV) gene sequence terminator, low factor-independent E. coli terminator, and lacZ α Examples include terminators.

  “Treatment” or “treating” as used herein in reference to protecting an animal from a condition refers to preventing, suppressing, suppressing, or eliminating the condition. means. Preventing a condition involves administering the composition of the invention to an animal prior to the occurrence of the condition. Suppressing a condition involves administering the composition of the invention to an animal after induction of the condition but before its clinical appearance. Suppressing a condition involves administering the composition of the present invention to an animal so that the condition is alleviated or prevented from aggravating after the condition appears clinically. Elimination of the condition involves administering to the animal the composition of the present invention so that the animal does not suffer any further after the condition has clinically appeared.

  As used herein, “vector” can mean a nucleic acid sequence containing an origin of replication. The vector may be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. The vector can be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector or a vector that integrates into the host genome.

2. MicroRNA
The current model for mammalian miRNA maturation is shown in FIG. 1, but is not bound by theory. The gene encoding miRNA is transcribed resulting in the production of a miRNA precursor known as pri-miRNA. The pri-miRNA can be part of a polycistronic RNA that includes multiple pri-miRNAs. pri-miRNA can form hairpins with stems and loops. As shown in FIG. 1, the stem may contain mismatched bases.

  The hairpin structure of pri-miRNA can be recognized by Drosha, an RNase III endonuclease. Drosha recognizes the end loop of the pri-miRNA and enters the stem to cut approximately two helical turns to produce a 60-70 nt precursor known as pre-miRNA. Drosha cleaves pri-miRNA in a twisted state common to RNase III endonucleases, resulting in a pre-miRNA stem loop with a 5 'phosphate and a 3' overhang of about 2 nucleotides. Approximately one helical turn (about 10 nucleotides) of the stem extending from Drosha's cleavage site may be essential for effective processing. The pre-miRNA is then actively transported from the nucleus to the cytoplasm by Ran-GTP and the transport receptor Exportin-5.

The pre-miRNA can be recognized by Dicer, which is also an RNase III endonuclease. Dicer can recognize the double-stranded stem of pre-miRNA. Dicer can also recognize 5 'phosphate and 3' overhangs at the base of stem loops. Dicer cuts the loop from the base of the stem loop through two helical turns leaving an additional 5 ′ phosphate and a 3 ′ overhang of about 2 nucleotides. The resulting siRNA-like duplex may contain mismatches, but contains fragments of similar size known as mature miRNA and miRNA ^* . miRNA and miRNA ^* can be derived from the opposite arm of pri-miRNA and pre-miRNA. miRNA ^* sequences can be found in a library of cloned miRNAs, but are generally less frequent than miRNAs.

Although initially present as a double-stranded species containing miRNA ^* , the miRNA is eventually incorporated as a single-stranded RNA into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Various proteins can form RISCs, and these proteins can bring variability in specificity for miRNA / miRNA ^* duplexes, target gene binding sites, miRNA activity (suppression or activation), and miRNA / MiRNA ^* Both strands of the duplex are incorporated into the RISC.

miRNA: miRNA ^{* If} a double miRNA strand is incorporated into a RISC, the miRNA ^* can be removed and degraded. MiRNA incorporated into RISC: miRNA ^* A duplex strand can be a strand whose 5 'end is not closely paired. miRNA: Both miRNA and miRNA ^* can have gene silencing activity if both ends of the miRNA: miRNA ^* form an approximately equivalent 5 'pair.

  RISC can identify target nucleic acids based on high levels of complementarity between miRNA and mRNA, particularly by nucleotides 2-8 of the miRNA. Only one example has been reported in which the interaction between miRNA and its target has been along the entire length of the miRNA in animals. This was shown for mir-196 and Hox B8, and it was further shown that mir-196 mediates cleavage of Hox B8 mRNA (Yekta et al 2004, Science 304-594). Otherwise, such interactions are known only in plants (Bartel & Bartel 2003, Plant Physiol 132-709).

  Numerous studies have looked at the requirements for base pairing between miRNA and its mRNA target to achieve efficient translational inhibition (reviewed in Bartel 2004, Cell 116-281. ) In mammalian cells, the first 8 nucleotides of the miRNA may be important (Doench & Sharp 2004 Genes Dev 2004-504). However, other parts of the microRNA may also be involved in mRNA binding. Furthermore, sufficient base pairing at 3 'can compensate for insufficient pairing at 5' (Brennecke at al, 2005 PLoS 3-e85). Evaluation studies that analyze miRNA binding in whole genomes suggest a specific role for miRNAs 5 ′ bases 2-7 in target binding, but also the role of the first nucleotide normally found to be “A” Recognized (Lewis et at 2005 Cell 120-15). Similarly, nucleotides 1-7 or 2-8 were used by Krek et al. For target identification and confirmation (2005, Nat Genet 37-495).

  The target site in the mRNA can be in the 5'UTR, in the 3'UTR or in the coding region. Interestingly, multiple miRNAs can control the same mRNA target by recognizing the same or multiple sites. The presence of multiple miRNA complementary sites in most genetically identified targets can indicate that the cooperative action of multiple RISCs results in the most efficient translational inhibition.

  miRNAs can direct RISC to down-regulate gene expression by either of two mechanisms: mRNA cleavage or translational repression. If the mRNA has some degree of complementarity to the miRNA, the miRNA can direct the cleavage of the mRNA. If the miRNA leads to cleavage, the cleavage can be between the nucleotides paired with residues 10 and 11 of the miRNA. Alternatively, if the miRNA does not have the necessary degree of complementarity to the miRNA, the miRNA can suppress translation. Translational suppression will be more prevalent in animals because animals may have a lower degree of complementarity.

It should be noted that there may be variability at the 5 ′ and 3 ′ ends of any pair of miRNA and miRNA ^* . This variability may be due to processing variability by Drosha and Dicer enzymes with respect to the site of cleavage. Also, the variability of the 5 ′ and 3 ′ ends of miRNA and miRNA ^* may be due to mismatches in the stem structures of pri-miRNA and pre-miRNA. Stem strand mismatches can result in a population of different hairpin structures. Variability in the stem structure also leads to variability in products of cleavage by Drosha and Dicer.

3. Nucleic acids The present invention relates to isolated nucleic acids comprising the nucleic acid sequences referenced in SEQ ID NOs: 1-4420915, the sequences referenced in Tables 1, 4, 10-14 and 21-23, and variants thereof. The variant may be a complement of the referenced nucleic acid sequence. The variant may also be a nucleic acid sequence that is substantially identical to the referenced nucleic acid sequence or its complement. The variant may also be a nucleic acid sequence that hybridizes under stringent conditions to a referenced nucleic acid sequence, its complement, or a nucleic acid sequence substantially identical thereto.

  The nucleic acid can be 10-100 nucleotides in length. The nucleic acid is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, It can be 45, 50, 60, 70, 80 or 90 nucleotides in length. Nucleic acids are synthesized or expressed intracellularly (in vitro or in vivo) using the synthetic genes described below. Nucleic acids can be synthesized as single stranded molecules and hybridized with substantially complementary nucleic acids to form duplexes, which are considered nucleic acids of the present invention. Nucleic acids are used in methods known to those skilled in the art, including those described in US Pat. No. 6,506,559, in single stranded or double stranded form, or incorporated herein by reference. And can be introduced into cells, tissues or organs in a form that can be expressed by a synthetic gene.

a. Pri-miRNA
The nucleic acid of the present invention may comprise a pri-miRNA sequence or a variant thereof. The pri-miRNA sequence may comprise 45-250, 55-200, 70-150 or 80-100 nucleotides. The sequence of the pri-miRNA may include pre-miRNA, miRNA and miRNA ^* as described below. The pri-miRNA can also include miRNA or miRNA ^* and its complement, and variants thereof. The pri-miRNA can comprise at least 19% adenosine nucleotides, at least 16% cytosine nucleotides, at least 23% thymine nucleotides and at least 19% guanine nucleotides.

  The pri-miRNA can form a hairpin structure. The hairpin structure can include a first nucleic acid sequence and a second nucleic acid sequence that are substantially complementary. The first and second nucleic acid sequences can be 37-50 nucleotides. The first and second nucleic acid sequences can be separated by a third sequence of 8-12 nucleotides. This hairpin structure is described in Hofacker et al., The contents of which are incorporated herein. , Monatshefte f. As described in Chemie 125: 167-188 (1994), it may have a free energy of less than −25 Kcal / mol calculated by the Vienna algorithm using the initial value parameters. The hairpin can have a terminal loop of 4-20, 8-12 or 10 nucleotides.

  The sequence of the pri-miRNA may comprise SEQ ID NOs: 4097721 to 4209913, the precursors referenced in Table 1, sequences of the sequences referenced in Tables 11-12 and 21-23, or variants thereof.

b. Pre-miRNA
The nucleic acids of the invention can also include a pre-miRNA sequence or a variant thereof. The pre-miRNA sequence can comprise 45-90, 60-80, or 60-70 nucleotides. The sequence of Pre-miRNA can include miRNA and miRNA ^* as described below. The pre-miRNA can also include miRNA or miRNA ^* and its complement, and variants thereof. Further, the sequence of the Pre-miRNA may be the sequence of the pri-miRNA excluding 0 to 160 nucleotides from the 5 ′ end and 3 ′ end of the pri-miRNA.

  The sequence of the pre-miRNA may comprise SEQ ID NOs: 4097721 to 4249913, the precursors referenced in Table 1, sequences of sequences referenced in Tables 11-12 and 21-23, or variants thereof.

c. miRNA
The nucleic acids of the invention can also include the sequence of miRNA, miRNA ^* or a variant thereof. The miRNA sequence can comprise 13-33, 18-24 or 21-23 nucleotides. The sequence of the miRNA can be the first 13-33 nucleotides of the pre-miRNA. The sequence of the miRNA can be the last 13-33 nucleotides of the pre-miRNA.

  The sequence of the miRNA may comprise SEQ ID NOs: 1-1142416 or 4204914-4204915, the miRNA referenced in Table 1, the sequences of the sequences referenced in Tables 11-12 and 21-23, or variants thereof.

d. Anti-miRNA
The nucleic acids of the invention can also include anti-miRNA sequences capable of inhibiting the activity of miRNA or miRNA ^* . The anti-miRNA can comprise a total of 5-100 or 10-60 nucleotides. In addition, the anti-miRNA is at least a total of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or It can contain 26 nucleotides. The sequence of the anti-miRNA is (a) at least 5 nucleotides substantially identical to the 5 ′ of the miRNA, and at least 5 to 5 substantially complementary to the adjacent region of the target site from the 5 ′ end of the miRNA. 12 nucleotides, or (b) at least 5-12 nucleotides that are substantially identical to the 3 ′ of the miRNA, and at least 5 nucleotides that are substantially complementary to the adjacent region of the target site from the 3 ′ end of the miRNA. Can be included.

  The sequence of the anti-miRNA can comprise the complement of SEQ ID NOs: 1-1214416 or 4204914-4204915, the sequence of miRNA referenced in Tables 1, 13-14, or 21-23, or variants thereof.

e. Target Binding Site The nucleic acid of the invention can also include a sequence of a target miRNA binding site, or a variant thereof. The target site sequence can comprise a total of 5-100 or 10-60 nucleotides. The target site sequence may comprise at least 5 nucleotides of SEQ ID NOs: 1142417-4097720, the sequence of the target gene binding site referenced in Tables 4, 10 or 15-16, or variants thereof.

4). Synthetic genes The present invention also relates to synthetic genes comprising a nucleic acid of the invention operably linked to transcriptional and / or translational control sequences. A synthetic gene may be capable of modifying the expression of a target gene that includes a binding site for a nucleic acid of the invention. The expression of the target gene can be modified in cells, tissues or organs. Synthetic genes can be synthesized or obtained from naturally occurring genes by standard recombinant techniques. The synthetic gene can also include a terminator at the 3 ′ end of the transcription unit of the synthetic gene sequence. A synthetic gene can also include a selectable marker.

5. Vector The present invention also relates to a vector comprising the synthetic gene of the present invention. The vector can be an expression vector. The expression vector can include additional elements. For example, this expression vector allows it to be maintained in two organisms, for example in mammalian or insect cells for expression and in prokaryotic hosts for cloning and amplification. There can be one replication system. In order to integrate multiple expression vectors, the expression vector can include at least one sequence homologous to the host cell genome, and preferably two homologous sequences flanking the expression construct. An integrative vector can be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. The vector can also include a selectable marker gene that allows for selection of transformed host cells.

6). Host cells The present invention also relates to host cells comprising the vectors of the present invention. The cell may be a bacterial, fungal, plant, insect or animal cell.

7). Probe The present invention also relates to a probe comprising the nucleic acid of the present invention. Probes can be used in screening and diagnostic methods, as outlined below. The probe can be attached or immobilized to a solid substrate, such as a biochip.

  Probes can be 8 to 500, 10 to 100, or 20 to 60 nucleotides in length. The probe is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, It can be 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 or 300 nucleotides in length. The probe may further comprise a 10-60 nucleotide linker sequence.

8). Biochip The present invention also relates to a biochip. The biochip may include a solid substrate including the probe of the present invention or a plurality of probes attached thereto. The probe may be capable of hybridizing to the target sequence under stringent hybridization conditions. The probe can be deposited on the substrate with a spatially defined address. One or more probes can be used per target sequence, which can be overlapping probes or probes for different sections of a particular target sequence. The probe may be capable of hybridizing to a target sequence associated with a single disease.

  The probe can be attached to the biochip in a variety of ways, as will be appreciated by those skilled in the art. The probe may be synthesized on the biochip after it is first synthesized or synthesized directly on the biochip.

  The solid substrate may be a material that can be modified to include separate and distinct sites suitable for probe attachment or association and is sensitive to at least one detection method. Typical examples of the substrate include glass and modified glass or functional glass, plastics (acrylic, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon (registered trademark) J, Others), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silica and modified silicon, carbon, metals, inorganic glasses and inorganic plastics. The substrate can allow optical detection without appreciable fluorescence.

  The substrate may be planar, but other substrate arrangements may be used as well. For example, for analysis of a flowing sample, a probe may be placed on the inner surface of the tube to minimize the sample volume. Similarly, the substrate may be flexible, such as a flexible foam that contains special plastic closed cells.

  The biochip and probe can be derivatized with a chemical functional group and then attached to both. For example, biochips can be derivatized with chemical functional groups including but not limited to amino groups, carboxyl groups, oxo groups or thiol groups. These functional groups can be used to attach a probe directly or indirectly using a functional group on the probe using a linker. The probe can be attached to the solid support either via the 5 'end, 3' end, or via an internal nucleotide.

  Alternatively, the probe can be non-covalently attached to the solid support. For example, a biotinylated oligonucleotide can be created and attached that can bind it to a surface covalently coated with streptavidin. Alternatively, the probe can be synthesized on the surface using techniques such as photopolymerization and photolithography.

9. In addition, the present invention relates to a method for identifying miRNA associated with a disease or a pathological state such as viral infection, the method comprising contacting a biological sample with the probe or biochip of the present invention, and hybridization Detecting the amount of. It is also possible to use PCR that amplifies the nucleic acid in the sample, which can lead to high sensitivity.

  The ability to identify miRNAs that are more overexpressed or less expressed in pathological cells compared to controls is in the fields of diagnostics, therapeutics, drug development, pharmacogenetics, biosensor development , And other related fields, can result in high resolution, high sensitivity data sets. The expression profile generated by this method can be a “fingerprint” of the state of the sample with respect to the number of miRNAs. Even if there are no specific miRNAs similarly expressed in the two states, the assessment of the number of miRNAs simultaneously generates a gene expression profile specific to the state of the cell. That is, normal tissue can be distinguished from diseased tissue. By comparing tissue expression profiles in different known disease states, information can be obtained regarding which miRNAs are associated with these conditions. A diagnosis can then be made or confirmed to determine whether the expression profile of the tissue sample is that of a normal tissue or a diseased tissue. This can provide molecular diagnosis of relevant conditions.

10. Determination of Expression Level The present invention also relates to a method for determining the expression level of a disease-related miRNA comprising the steps of contacting a biological sample with a probe or biochip of the present invention and measuring the amount of hybridization. The expression level of disease-related miRNA is useful information in many respects. For example, differential expression of a disease-related miRNA compared to a control can be used as a diagnosis that a patient is afflicted with the disease. The expression level of disease-related miRNA can also be used to monitor patient treatment and disease status. Furthermore, the expression level of disease-related miRNAs may allow screening of candidate agents to alter a specific expression profile or to suppress a disease-related expression profile.

  The target nucleic acid can be detected by contacting a sample containing the target nucleic acid with a biochip containing an attachment probe that is sufficiently complementary to the target nucleic acid and detecting hybridization with a probe that exceeds the control level. it can.

  The target nucleic acid can be detected by immobilizing the nucleic acid to be tested on a solid support such as a nylon membrane and hybridizing a labeled probe with the sample. Similarly, the target nucleic acid can be detected by immobilizing the labeled probe to a solid support and hybridizing a sample containing the labeled target nucleic acid. After washing to remove non-specific hybridization, the label may be detected.

  The target nucleic acid can also be detected in situ by contacting permeabilized cells or tissue samples with a labeled probe to allow hybridization with the target nucleic acid. The label may be detected after washing to remove the non-specifically bound probe.

  These assays can be direct hybridization assays, or generally U.S. Pat. Nos. 5,681,702; 5,597,909; 5,545,730; 5,594, No. 117; No. 5,591,584; No. 5,571,670; No. 5,580,731; No. 5,571,670; No. 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124,246; and 5,681, As outlined in US Pat. No. 697, a sandwich assay involving the use of multiple probes can be included. Each of the above references is incorporated herein by reference.

  As outlined above, a variety of hybridization conditions can be used, including high, medium and low stringency conditions. The assay may be performed under stringency conditions that allow probe hybridization only with the target. Stringency can be controlled by varying step parameters that are thermodynamic variables including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, or organic solvent concentration. .

  Hybridization reactions can be accomplished in a variety of ways. The components of the reaction can be added simultaneously or sequentially in a different order. In addition, the reaction can include a variety of other reagents, including salts, buffers, neutral proteins (eg, albumin), surfactants, etc., that facilitate optimal hybridization and detection. And / or can be used to reduce non-specific or background interactions. Reagents that improve the efficiency of other assays, such as protease inhibitors, nuclease inhibitors and antibacterial agents, can also be used appropriately depending on the sample preparation method and target purity.

a. Diagnosis The present invention also relates to a method of diagnosis comprising detecting a differential expression level of a disease-related miRNA or an infection-related miRNA in a biological sample. The miRNA can be a viral miRNA that can be expressed in an infected subject. The miRNA may be derived from a subject whose expression level has been modified due to viral infection. The sample may be derived from a patient. Diagnosis of a disease state in a patient allows for prognosis and treatment strategy selection. In addition, the stage of cell development can be classified by determining the temporarily expressed miRNA molecules.

  In situ hybridization of the labeled probe with the tissue array can be performed. If fingerprints are compared between an individual and a specimen, those skilled in the art can perform diagnosis, prognosis, or prediction based on the knowledge. Furthermore, it is understood that genes that indicate diagnosis may differ from genes that indicate prognosis, and that molecular profiling of the cellular state can lead to differentiation of reactive or refractory states or can predict outcome .

b. Drug screening The present invention also provides a therapeutic agent comprising the steps of contacting a pathological cell capable of expressing a disease-related miRNA with a candidate therapeutic agent, and evaluating the effect of the candidate drug on the expression profile of the disease-related miRNA. It also relates to the method of screening. After identifying differentially expressed miRNA, various assays can be performed. Test compounds can be screened for the ability to modulate gene expression of disease-related miRNA. Regulation includes both reducing and increasing gene expression.

  The test compound or candidate drug can be any molecule that is tested for the ability to directly or indirectly alter the disease phenotype or expression of a disease-related miRNA, such as a protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc. It can be. Candidate agents include a number of chemical classes, such as small organic molecules having a molecular weight greater than 100 and less than about 500, 1,000, 1,500, 2,000, or 2,500 daltons. Candidate compounds contain structural structural interactions with proteins, in particular functional groups required for hydrogen bonding, and generally at least amine, carbonyl, hydroxyl or carboxyl groups, preferably at least of these functional chemical groups Two can be included. Candidate agents can include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found in biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

  Combinatorial libraries of potential modulators can be screened for the ability to bind or modulate the activity of disease-related miRNAs. A combinatorial library can be a collection of various chemicals created by either chemical synthesis or biological synthesis by combining multiple chemical building blocks such as reagents. The preparation and screening of chemical combinatorial libraries is well known to those skilled in the art. Such chemical combinatorial libraries include, but are not limited to, peptide libraries encoded with peptides, diversomers such as benzodiazepines, hydantoins, benzodiazepines and dipeptides, vinyl polypeptides, small compounds Similar organic compounds of the library, oligocarbamic acid and / or peptidyl phosphonates, nucleic acid libraries, peptide nucleic acid libraries, antibody libraries, carbohydrate libraries, and small organic molecule libraries.

11. Gene Silencing The present invention also relates to a method for reducing the expression of a target gene in a cell, tissue or organ using the nucleic acid of the present invention. Target gene expression can be reduced by expressing a nucleic acid of the invention comprising a sequence that is substantially complementary to one or more binding sites of the target mRNA. The nucleic acid can be miRNA or a variant thereof. The nucleic acid can also be a pri-miRNA, a pre-miRNA, or a variant thereof that can be processed to produce a miRNA. The expressed miRNA can hybridize to a substantially complementary binding site of the target mRNA, which can cause activation of gene silencing mediated by RISC. An example of a study using miRNA overexpression is Yekta et al 2004, Science 304-594, incorporated herein by reference. One skilled in the art will recognize the nucleic acids of the invention in antisense methods well known in the art, as well as in US Pat. Nos. 6,506,559 and 6,573,099, which are incorporated herein by reference. It will be appreciated that the RNAi method described can be used to inhibit target gene expression.

  The target gene can be a viral gene that can be reduced by expressing viral miRNA or human miRNA. Alternatively, the target gene can be a human gene that is expressed upon viral infection and can be reduced by expressing viral miRNA or human miRNA. The target of gene silencing can be a protein that causes silencing of a second protein. By suppressing the expression of the target gene, the expression of the second protein can be increased. Examples of efficient suppression of miRNA expression are described in Esau et al 2004 JBC 275-52361; and Cheng et al 2005 Nucleic Acids Res. 33-1290.

12 Gene Enhancement The present invention also relates to a method for increasing the expression of a target gene in a cell, tissue or organ using the nucleic acid of the present invention. Target gene expression may be increased by expressing a nucleic acid of the invention comprising a sequence that is substantially complementary to a pri-miRNA, pre-miRNA, miRNA or variant thereof. The nucleic acid can be an anti-miRNA. Anti-miRNA can hybridize with pri-miRNA, pre-miRNA or miRNA, and as a result can reduce gene repression activity. In addition, the expression of the target gene is performed by using the nucleic acid of the present invention that is substantially complementary to a part of the target gene binding site so that miRNA binding can be avoided by the binding of the nucleic acid of the present invention and the target gene binding site. There is a possibility to increase by expressing.

  The target gene may be a viral gene, which when expressed can reduce the infectivity of the virus. The target gene may also be a human gene, which when expressed can reduce the infectivity of the virus or increase resistance or immunity to viral infection.

13. Treatment The invention also relates to methods of using the nucleic acids of the invention as modulators or targets for diseases or disorders such as those associated with viral infections. In general, the nucleic acid molecule of the present invention can be used as a regulator of the expression of a gene that is at least partially complementary to the nucleic acid. Furthermore, miRNA molecules may serve as targets for therapeutic screening procedures, for example, inhibition or activation of miRNA molecules may modulate the differentiation process of cells, such as apoptosis.

  In addition, existing miRNA molecules can be used as starting materials for the production of miRNA molecules (eg, oncogenes, multidrug resistance genes or other therapeutic target genes) whose sequences have been modified to modify their target specificity. it can. Furthermore, after the miRNA molecule is processed, the miRNA molecule can be modified so that it is generated as a double stranded siRNA that is redirected to a therapeutically relevant target. In addition, miRNA molecules can be used in tissue reprogramming procedures. For example, expression of miRNA molecules may transform differentiated cell lines into different cell types or stem cells.

14 Compositions The present invention also relates to pharmaceutical compositions comprising the nucleic acids of the invention and optionally containing a pharmaceutically acceptable carrier. The composition can be used for diagnostic or therapeutic applications. Administration of the pharmaceutical composition may be accomplished by known methods that introduce nucleic acids into the desired target cells in vitro or in vivo. Commonly used gene transfer techniques include calcium phosphate, DEAE-dextran, electroporation, microinjection, viral methods and cationic liposomes.

15. Kits The invention also includes any or all of the following with the nucleic acids of the invention: assay reagents, buffers, probes and / or primers, and sterile saline or other pharmaceutically acceptable emulsions and suspending bases. It also relates to a kit containing In addition, the kit can include instructions including usage (eg, a protocol) for performing the methods of the invention.

Prediction of miRNAs We predict US Patent Application Nos. 60 / 522,459, 10 / 709,577 and 10/10, the contents of which are incorporated herein by reference to predict miRNAs. A number of viral genomes were investigated for the potential of genes encoding miRNAs using three computational approaches similar to those described in 709,572. Predicted hairpins and promising miRNAs were evaluated by thermodynamic stability and structural and contextual characteristics. The algorithm was modified with the miRNA in the Sanger database that was valid.

1.1 First and Second Screening Tables 11 and 12 show the sequence ("PRECURSOR SEQUENCE"), sequence identifier ("PRECURR SEQ-ID") and derived organism (predicted from the first computer screening) “GAM ORGANISM”) is shown along with the predicted miRNA (“GAM NAME”). Tables 13 and 14 show the sequence ("GAM RNA SEQUENCE") and sequence identifier ("GAM SEQ-ID") for each miRNA ("GAM NAME"), the origin organism ("GAM ORGANISM") and the Dicer cleavage position ( "GAM POS").

2. Third round of selection Table 1 lists the sequence numbers for each predicted hairpin (“HID”) of a third round of computer selection of a particular viral genome (“V”; see also Table 10). Table 1 lists the positions of hairpins in the genome (“Hairpin Location (Hairpin Loc.)”). The format of the genome position is a continuous form of <strand><startposition>. The location of the gene is based on the NCBI-Entrez nucleotide database. The Entrez nucleotide database is a collection of sequences from several sources, including GenBank, RefSeq, and PDB. Table 10 shows the accession number and construct (version) for each genome used in this selection.

Table 1 also lists the sequence numbers (“MID”) for each predicted miRNA and miRNA ^* . Table 1 also shows Hofcker et al. , Monatshefte f. Chemie 125: 167-188, 1994 lists the 0-1 grade (1 most reliable hairpin) prediction score grade ("P") for each hairpin. Table 1 also lists the p-values (“Pval”) calculated from the background hairpin for each P-score value. All p values are significantly lower than 0.05. As shown in Table 1, there are a few cases where Pval is 0.0. In each of these cases, this value is less than 0.0001. The p-value was calculated by comparing the pal grades of the tested hairpins with the pal grades of other sequences without pre-selection of the hair pins.

  Table 1 also lists whether miRNA was effective by expression analysis (“E”), as detailed in Table 2 (Y = effective, N = not). Table 1 also lists whether miRNAs were effective by sequencing (“S”), as detailed in Table 3 (Y = effective, N = not). If there is a difference in sequence between the predicted miRNA and the sequenced miRNA, the sequenced sequence is presented. It should be noted that failure to sequence or detect expression of miRNA does not necessarily mean that the miRNA is not present. Such undetected miRNA may be expressed in tissues other than the test tissue. Furthermore, such undetected miRNAs are expressed in the test tissue, but may be expressed at a different stage or under different conditions than the experimental cells.

  Table 1 also lists whether miRNAs showed differential expression (“D”) in at least one disease, as detailed in Table 2 (Y = shown, N = No). Table 1 also shows miRNAs in Sanger DB Release 6.0 (April 2005) (http://nar.upjournals.org/) as detected or predicted in humans or mice. (“F”) is listed (Y = present, N = not). As discussed above, miRNAs listed in the Sanger database are a component of the prediction algorithm and are a contrast of results.

  Table 1 also lists gene location clusters ("LC") for hairpins within 1,000 nucleotides of each other for a particular virus. Each miRNA with the same LC shares the same gene cluster. Overlapping hairpins are not clustered. Table 1 also lists seed clusters ("SC") for grouping miRNAs based on their 2-7 seeds based on perfect match, regardless of source virus. Each miRNA with the same SC has the same seed. For a discussion on 6 nucleotide long seeds, see Lewis et al. , Cell, 120; 15-20 (2005).

Example 2
Target gene prediction Next, using miRNA predicted from the three rounds of computer selection of Example 1, U.S. Patent Application No. 60 / 522,459, the contents of which are incorporated herein by reference. Using two computational approaches for predicting miRNA, similar to those described in 10 / 709,577 and 10 / 709,572, human and viral target genes and their binding The site was predicted.

1.1 First and Second Screening Tables 15 and 16 show the predicted target gene (“TARGET”) and binding site sequence (“TARGET BINDING SITE SEQUENCE”) and binding site sequences obtained from the first computer screening. The identifier (“TARGET BINDING SITE SEQ-ID”), as well as the target source organism (“TARGET ORGANISM”) are listed.

2. Third selection a. Human target genes Table 4 lists the human target genes predicted for each miRNA (MID) from a particular virus (V) and its hairpin (HID), obtained from the third round of computer selection. The name of the target gene is NCBI Reference Sequence Release 9 (http://www.ncbi.nlm.nih.gov; Pruit et al., Nucleic Acids Res, 33 (1): D501-D504, 2005; Pruit et al. , Trends Genet., 16 (1): 44-47, 2000; and Tatusova et al., Bioinformatics, 15 (7-8): 536-43, 1999). The target gene was identified by a perfect complementary match of the 7 nucleotide miRNA seed (positions 2-8) and A on the UTR (total 8 nucleotides). For considerations in identifying target genes, see Lewis et al. , Cell, 120: 15-20, (2005). For a discussion of seeds sufficient for binding miRNA to UTR, see Lim Lau et al. , (Nature 2005) and Brenneck et al, (PLoS Biol 2005).

  Selection of binding sites took into account only the first 4000 nucleotides per UTR, taking into account the longest transcript when there were several transcripts per gene. Filtering reduced the total number of transcripts from 23626 to 14239. Table 4 lists the predicted binding site sequence numbers for each target gene. Since the binding site sequence is located on the spliced mRNA, it contains the 5 'and 3' 20 nucleotides of the binding site. Where the binding site consists of two exons, 20 nucleotides are included from both the 5 'and 3' ends of both exons.

  Table 5 shows the relationship between miRNA (MID) / hairpin (HID) of specific virus (V) and disease for each human target gene. The name of the disease was obtained from OMIM. For a discussion of the rationale linking the host gene where the hairpin is located to the disease, see Baskerville and Bartel, RNA, 11: 241-247 (2005) and Rodriguez et al. Genome Res. 14: 1902-1910 (2004). Table 5 shows the number of miRNA target genes (“N”) associated with the disease. Table 5 also shows the total number of genes associated with disease (“T”) obtained from genes predicted to have miRNA binding sites. Table 5 shows the ratio of N in T and the p-value (“Pval”) of hypergeometric analysis. When pval is described as 0.0, it indicates that the value is less than 0.0001. For a reference to hypergeometric analysis, see Schaum's Outline of Elements of Statistics II: Informal Statistics. Table 7 shows the disease codes of Tables 5 and 6.

b. Viral Target Genes Similar to those described for human target genes in Table 4 above to date, Table 10 shows the same specific virus (V) and its hairpin (from its third round of computer selection) The viral target genes predicted for each miRNA (MID) from HID) are listed. For the prediction of the virus binding site, the complete gene was used instead of UTR as shown in Table 4 according to the method for human target genes shown in Table 10 above. If the candidate target gene is known to have a role in the viral life cycle, the candidate target gene was included in the selection. MiRNAs that have binding sites on viral genes that play a role in the viral life cycle can affect diseases that may be associated with viruses.

  Human herpesviruses 1 and 2 are associated with any of a number of inflammatory diseases caused by herpesviruses, in some cases prominent skin or mucosal blisters (such as those in the mouth and lips) above the waist. In other instances, such blisters are prominent in the genitals. Human herpesvirus 4 (Epstein-Barr virus) causes infectious mononucleosis and is associated with Burkitt lymphoma and nasopharyngeal carcinoma. HIV strains are involved in acquired immune deficiency syndrome (AIDS). Hepatitis B and hepatitis C viruses cause inflammation of the liver.

Example 3
Validation of miRNAs To confirm the hairpins and miRNAs predicted in Example 1, we have made US patent applications 60 / 522,459, 10/709, the contents of which are incorporated herein by reference. , 577 and 10 / 709,572 were used to detect expression in various tissues using a high-throughput microarray similar to that described in US Pat. For each predicted precursor miRNA, mature miRNAs from both stems of the hairpin were tested.

1. Expression analysis-set 1 and set 2
Tables 17-19 list the results of microarray expression analysis to detect miRNA sequences (“GAM RNA SEQUENCE”).

2. Expression analysis-set 3
Table 2 shows the third predicted set of hairpins ("HID"), as well as expression, confirmed to be effective by detecting the expression of the related miRNA ("MID") from a particular virus ("V") The code of the detected tissue (“Tissue”) is shown. If a score of 1 or more is obtained from the same miRNA in the same tissue, only the higher score is shown.

  Tissue and disease codes are listed in Table 6 and Table 7, respectively. Table 8 shows the relationship between genes and diseases. This allows all miRNAs to be linked to disease. In Table 4, at least one target gene is assigned to each miRNA. Table 5 shows the significant relationship between miRNA and disease by showing the results of statistical analysis in Table 4 and OMIM. Table 8 is basically a reduced version of OMIM. Table 8 lists all numeric codes for the diseases associated with each gene for each gene.

  All disclosed tissues provide an indication of viral disease. The fact that significant expression of the virus was measured means that in this tissue the tissue can be involved in viral disease. For example, if an HIV mir is expressed in a T cell line, it may have an effect on AIDS. Of course, a cell line represents only a subset of tissue characteristics when the tissue functions in an organ, but can be inferred from the expression measured in the cell line.

  Table 2 also shows the grade of expression score of the chip (range of 500-65000) (“S”). A threshold of 500 was used to remove insignificant signals and scores were normalized with the MiraChip probe signal from different experiments. Variations in the intensity of the fluorescent material between experiments can be attributed to variability in RNA preparation or labeling efficiency. We standardized based on the hypothesis that the total amount of miRNA in each sample is relatively constant. Initially we subtracted the background signal from the raw signal of each probe (background signal was defined as 400). The inventors then divided each miRNA probe signal by the average signal of all miRNAs and multiplied the result by 10,000 to get back the background signal of 400. Thus, by definition, the sum of all miRNA probe signals for each experiment is 10400.

  Table 2 also shows a statistical analysis of the standardized signal calculated based on the standardized score (“Spval”). For each miRNA, we used the relevant control group from the complete predicted miRNA list. Each miRNA has an internal probe control that contains a mismatch. Related control groups included probes with similar C and G ratios (absolute difference <5%) to have similar Tm. The P value of the probe signal is the ratio relative to the related control probe with the same or higher signal. The result is a p-value ≦ 0.05 and a score greater than 500. When SPVAL is described as 0.0, this value is less than 0.0001.

3. Sequencing-set 3
In order to further validate the second predicted hairpin (“HID”), a number of miRNAs can be obtained from US Patent Application Nos. 60 / 522,459, 10/709, the contents of which are incorporated herein by reference. , 577 and 10 / 709,572 have been validated by sequencing methods similar to those described. Table 3 shows hairpins (“HID”) that have been validated by sequencing the viral (“V”) miRNA (“MID”) in the indicated tissue (“Tissue”).

4). Northern analysis Groups of miRNAs were verified by Northern analysis as shown in FIGS.

Example 4
Differential expression of miRNA Viral miRNA
Table 20 lists effective viral miRNAs that have been demonstrated to be differentially expressed in diseased tissues compared to healthy human tissues or human-derived cell lines. All miRNA sequences were confirmed to be valid using the miRNA microarray as described above. Alzheimer's disease affects mildly, moderately, or severely affected and healthy human amygdala, cingulate cortex, caudate nucleus, pallidum, occipital and occipital cortex, Alzheimer's symptoms GAM RNA expression in a mixture of tissues from all areas of the brain that have been shown to do was studied. In Parkinson, GAM RNA expression was studied in substantia nigra tissue from diseased and healthy human tissue. The MT2 cell line was infected with a T cell directed clinical isolate of Clade A human immunodeficiency virus (HIV), while the healthy control was not infected. The cMagi cell line was infected with macrophage-directed clade B HIV, while healthy controls were not infected. Human fibroblasts (TC) were infected with HSV1 or HSV2, or were not infected and served as controls. GAM RNASEQUENCE: Sequence of mature, “diced” GAM RNA (5 ′ to 3 ′). CHIP SEQUENCE is a sequence of oligonucleotides containing predicted GAM RNA placed on the microarray (not including non-genomic sequences used as separators from the microarray surface). DISEASE: A disease in which GAM RNA is differentially expressed—BAL refers to macrophage-directed HIV1 subtype B, lab strain, BLAI refers to T cell-directed HIV-1 (LAV-1), subtype B; SIGNAL (HEALTHY): signal on a microarray for GAM RNA in a sample consisting of human tissue or a human-derived cell line not suffering from the designated disease; SIGNAL (DISEASE): human tissue suffering from the designated disease Or a signal on a microarray for GAM RNA in a sample consisting of a human-derived cell line.

2. Human miRNA
Table 21 lists the following miRNA expression data. HID: hairpin sequence number, MID: miRNA sequence number, Tissue: test tissue, S: grade of chip expression score (range = 100-65000), Dis. Diff. Exp. : Disease-related differential expression and the tissue to be tested, R: ratio of disease-related expression (range = 0.01-99.99), and abbreviation: Brain Mix A-Brain tissue mixture affecting Alzheimer; Brain Mix B-a mixture of all tissues of the brain; and Brain SN-substantia nigra. Tables 22 and 23 provide the following details regarding differentially expressed miRNAs. HID: hairpin sequence number, Hairpin_Loc: <chr_id><strand> space <start position> position of the hairpin genome in a continuous form (for example, 19 + 13540000 means chr19 + chain, start position 13540000), C: during evolution Conservation (presence / absence, and “-” if data is not applicable; existence—conservation level exceeds threshold of 0.7), T: type of genome, intergenic region (G), intron (I), Exon (E), MID: miRNA SEQ ID NO; Target Gene, Disease: Target gene (HUGO database) and related diseases (OMIM database), P: Predictive score grade (range 0-9), E: Chip expression information-Valid / No (Y / N), S: Verification by sequencing-Valid / No (Y / N), HID : Hairpin sequence number. Table 24 lists the sequences referred to in Tables 21-23.

Example 5
Analysis of EBV miRNA Validation of Expression FIG. 4 shows the validation of miRNA expression predicted in EBV (Epstein-Barr virus) miRNA; expression validation. Three cell lines were tested. Two were normal B cells immediately infected (PBMC-1 / 2-EBV) and one was an EBV-transformed cell line (B-95-8). The three cell lines showed a similar degree of EBV infection (FIG. 4A). However, EBV-miR-RG-1 and EBV-miR-RG-2 were highly expressed in the B-95-8 cell line relative to B cells immediately infected (FIG. 4B).

2. Expression Knockout FIG. 5 shows EBV miRNA knockout. Addition of 2-O-methyl to the B-95-8 cell line obtained against EBV-miR-RG-1 dramatically reduces the number of cells expressing EBV antigen. Addition of 2-O-methyl to B-95-8 obtained against EBV-miR-RG-2 had a moderate effect of slightly increasing EBV expression.

CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of US patent application Ser. No. 10 / 709,739, filed May 26, 2004, which is hereby incorporated by reference. We claim the benefit of US Provisional Application No. 60 / 522,459 and US Provisional Application No. 60 / 665,094 filed May 25, 2005.

It is a figure which shows the model of maturation of miRNA. The HIV's 5'UTR (U5R) containing two predicted miRNAs (bold) is shown. The mature miRNA is underlined. Shown is the one near the 5 'end of the mature miRNA (underlined in Figure 2A) in the 5'UTR (U5R) of HIV1 containing two predicted miRNAs (bold). FIG. 3 shows another near the 3 ′ end of the mature miRNA (underlined in FIG. 2A) in the 5′UTR (U5R) of HIV1 containing two predicted miRNAs (bold). The most 5 'miRNA corresponds to a known HIV1 RNA structure called TAR (Nature 1987.330: 489-93) that binds to the TAT protein. A similar miRNA (GAM NAME 506033) also shined on the chip. This miRNA probe is designed based on the T cell-directed HIV-1 (LAV-1), subtype B sequence, and is one nucleotide different from the miRNA shown in FIG. 2B. FIGS. 2B and 2C represent Northern blot analysis of miRNA oligonucleotides present in U5R and hybridized with a predicted mature miRNA probe. The upper arrow indicates the molecular size of the entire 355 nt U5R transcript. The predicted molecular sizes of the two GAM RNAs are 22nt and 17nt, respectively. The lower arrow indicates the 22 nt cell marker. Lanes: 1- Hela lysate; 2- U5R transcript in HeLa lysate without incubation; and 3-5 U5R transcript incubated with Hela lysate for 24 hours. Shown is a partial transcript of HIV1 RNA reacted with a predicted mature HIV1 miRNA probe. Shown is a partial transcript of HIV1 RNA reacted with a predicted mature HIV1 miRNA probe. In each of FIGS. 2D and 2E, the experimental transcription sequence is shown and the predicted mature miRNA is underlined. Figure 6 shows Northern blot analysis of miRNA precursors. It is demonstrated that one miRNA precursor transcript is 163 nt and the other miRNA precursor transcript is 200 nt. The predicted molecular size of mature miRNAs is both 24 nt. The 22 nt cell marker is shown. Lane: 1-transcript in HeLa lysate without incubation, and 2-transcript incubated with HeLa lysate for 24 hours. The result of a Northern blot is shown. Figure 2 shows the expression profiles of GAM506333 and GAM506336 in EBV infected (B95 / 8 EBV) and uninfected (pBMC) cells. The expression of these miRNAs was demonstrated on miRNA microarrays hybridized with RNA of B-95 / 8 cell line infected with EBV. Probes for these valid miRNA predictors were hybridized with total RNA in a Northern blot. Northern blots confirmed high expression of these two miRNAs in infected cells on the microarray. Figure 3 shows validation of miRNA expressed by EBV. EBV miRNA knockout is shown.

Claims (16)

(A) SEQ ID NO: 4097721-4204913;
(B) sequence of precursors referenced in Tables 1, 11-12 and 21-23;
(C) SEQ ID NOs: 1-1142416 or 4204914-4204915;
(D) the sequence of the miRNA referenced in Tables 1, 13-14 and 21-23;
(E) SEQ ID NOs: 1142417-4097720;
(F) the sequence of the target gene binding site referenced in Tables 4, 10 and 15-16;
(G) the complement of (a)-(h);
(H) a nucleic acid sequence comprising at least 12 contiguous nucleotides that is at least 70% identical to (a)-(h);
An isolated nucleic acid comprising a portion of a sequence selected from the group consisting of 5 to 250 nucleotides in length.   A probe comprising the nucleic acid according to claim 1.   The nucleic acid comprises at least 8-22 consecutive nucleotides complementary to the miRNA referenced in SEQ ID NOs: 1-1214416 or 4204914-4204915, Table 1, 13-14 or 21-23, or variants thereof The probe according to claim 2.   The probe according to claim 2, wherein the nucleic acid comprises at least 8-22 consecutive nucleotides complementary to a human miRNA differentially expressed in viral infection.   A plurality of probes selected from the group consisting of the probes according to claim 3 or 4.   The plurality of probes of claim 5, comprising at least 10 probes.   6. The plurality of probes of claim 5, comprising at least 100 probes.   The composition containing the some probe of any one of Claims 5-7.   A biochip comprising a solid substrate, the substrate comprising a plurality of probes according to any one of claims 5 to 7, wherein each probe is attached to the substrate at a spatially defined address. The biochip. (A) preparing a biological sample; and (b) (i) SEQ ID NOs: 1-1142416 or 4204914-4420915, (ii) sequences of miRNAs referenced in Tables 1, 13-14, and 21-23, or ( iii) measuring the level of nucleic acid that is at least 70% identical to a variant of (i)-(ii);
A method for detecting differential expression of a viral infection-related miRNA, wherein the difference in the level of nucleic acid compared to the control indicates differential expression. (A) (i) SEQ ID NO: 1-1142416 or 4204914-4204915, (ii) miRNA sequences referenced in Tables 1, 13-14, and 21-23, or (iii) mutations in (i)-(ii) Providing a cell capable of expressing a nucleic acid that is at least 70% identical to the body;
(B) contacting the cell with a candidate modulator;
(C) measuring the level of expression of the nucleic acid;
A method for identifying a compound that modulates viral infection, wherein the compound is identified as a modulator of a pathological condition associated with the nucleic acid with a difference in the level of the nucleic acid compared to the control.   A method for inhibiting the expression of a target gene in a cell comprising introducing a nucleic acid into the cell in an amount sufficient to inhibit the expression of the target gene, wherein the target gene is selected from Table 4, 10 or 15 to 16 comprising a binding site substantially identical to the binding site referenced in FIG. 16, or a variant thereof, wherein the nucleic acid is (i) SEQ ID NO: 1-1214416 or 4204914-4204915, (ii) Table 1, 13-14 or 21 The method comprising a portion of the sequence of the miRNA referenced to -23, or a variant of (iii) (i)-(ii).   13. A method according to claim 12, wherein expression is inhibited in vitro or in vivo.   A method of increasing expression of a target gene in a cell comprising introducing a nucleic acid into the cell in an amount sufficient to increase expression of the target gene, wherein the target gene is Table 4, 10 or 15-16. Or a variant thereof, wherein a portion of the nucleic acid comprises (i) SEQ ID NOs: 1-1142416 or 4204914-4204915, (ii) Table 1, 13-14 or The method, wherein the method is substantially complementary to the sequence of miRNA referenced 21-21, or a variant of (iii) (i)-(ii).   15. The method of claim 14, wherein expression is inhibited in vitro or in vivo.   A method of treating a virally infected patient or a patient associated with a viral infection comprising the step of administering said nucleic acid to a patient in need of said nucleic acid, wherein a portion of said nucleic acid is (i) SEQ ID NO: 1-1142416 Or 4204914-4204915, (ii) the sequence of the miRNA referenced in Table 1, 13-14 or 21-23, or (iii) a variant of (i)-(ii) substantially complementary, Method.

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