WO2017132945A1 - miRancer分子的设计与应用 - Google Patents

miRancer分子的设计与应用 Download PDF

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WO2017132945A1
WO2017132945A1 PCT/CN2016/073517 CN2016073517W WO2017132945A1 WO 2017132945 A1 WO2017132945 A1 WO 2017132945A1 CN 2016073517 W CN2016073517 W CN 2016073517W WO 2017132945 A1 WO2017132945 A1 WO 2017132945A1
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microrna
sequence
target
target microrna
mirancer
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French (fr)
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朱涛
李高朋
洛比E·彼得
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University of Science and Technology of China USTC
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University of Science and Technology of China USTC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the invention relates to RNA molecules and uses thereof.
  • the present invention relates to oligomeric RNA molecules capable of specifically binding and enhancing the expression and activity of microRNAs and uses thereof.
  • miRNA A mature microRNA (miRNA) is a small RNA of about 22 nucleotides (nt) in length (e.g., about 18-25 nucleotides) that has a variety of important regulatory roles in the cell. miRNAs are widely found in eukaryotes, and they function in conjunction with mRNA molecules that are complementary to their sequences (eg, leading to gene silencing), an important strategy for regulating gene expression. The results show that miRNAs participate in the regulation of life activities such as development, apoptosis, proliferation and differentiation, and can play a role in the diagnosis and treatment of diseases, especially cancer.
  • RNA sequence that binds to a target microRNA, preferably the single-stranded RNA sequence enhances the activity of a target microRNA (referred to herein as a miRancer molecule) having 5 An 'end arm> and a 3' end arm, the 5' end arm binding to a target microRNA by base complementary pairing, the 3' end arm binding to the 5' end arm by base complementary pairing,
  • a target microRNA referred to herein as a miRancer molecule
  • the single-stranded RNA sequence has the following formula 1 from the 5' end to the 3' end:
  • n is a contiguous nucleotide or an analog thereof, and the number thereafter is the number of nucleotides or the like, and each n may be independently selected from the following nucleotides: A, U, G, and C. Or a nucleotide analog thereof, wherein n 1 is complementary to the nucleotide at the first position of the 5' end of the target microRNA, and the corresponding n 6-7 corresponds to the second to seventh of the target microRNA from the 5' end to the 3' end Bit or position 2-8, such that (n 5-11 )(n 0-3 )(n 6-7 ) is the 5' end arm and (n 11-21 )(n 0-2 ) is the 3' end arm Where n 0-3 is the inserted C or G to form a GC pair between the 5' end arm and the 3' end arm.
  • the single-stranded RNA molecules provided herein are capable of binding to a target microRNA and enhancing the activity of the microRNA.
  • the inventors referred to the RNA molecule capable of enhancing microRNA activity as a microRNA enhancer, referred to as miRancer.
  • the enhanced microRNA activity can include, for example, one or more of stabilizing the microRNA, extending the half-life of the microRNA, altering expression of a target gene regulated by the microRNA, and the like. Methods for measuring the expression of target genes regulated by microRNAs and microRNAs are known in the art.
  • the 5' end arm and the target microRNA are from the 5' end to the 3' end in addition to the inserted ( n0-3 ) as a gap in the RNA molecule described herein.
  • the complementary strand of nucleotides 2-7 or 2-8 of the 3' end has at least 80%, 90%, or 100% identity, preferably (n 11-21 ) and (n 5-11 ) (
  • the number of unpaired nucleotides in n 0-3 )(n 6-7 ) is less than 6, 5, 4, 3, 2, or 1 pair, preferably all unpaired nucleotides are around (n 1 ).
  • (n 5-11 )(n 0-3 )(n 6-7 ) and the target microRNA are at least 50%, 60 from nucleotides 2-18 of the 5' end to the 3' end. %, 70%, 80%, 90%, 95% or 100% complementary. In some embodiments, (n6-7) and the target microRNA are at least 80%, 90%, 95%, 99% from positions 5-7 or 2-8 of the 5' end to the 3' end. Or 100% complementary. In some embodiments, (n 11-21 ) and (n 5-11 )(n 0-3 )(n 6-7 ) are at least 50%, 60%, 70%, 80%, 90%, 95% or 100% complementary.
  • a vector such as a plasmid, comprising the single-stranded RNA sequence.
  • a vector such as a plasmid, comprising a polynucleotide sequence of the single-stranded RNA sequence.
  • RNA molecule of the single-stranded RNA sequence is provided herein.
  • provided herein is a cell comprising the vector.
  • the single-stranded RNA sequence, the vector, or the synthetic RNA molecule is used to specifically increase expression of an endogenous and/or exogenous target microRNA or other small RNA and/ Or the method of use and/or use of the activity.
  • the single-stranded RNA sequence, the vector, or the synthetic RNA molecule is used to specifically modulate (eg, increase) expression of a gene targeted by the target microRNA in vitro and/or in vivo. Use and / or use.
  • the single-stranded RNA sequence can be specifically regulated (eg, increased) by integration into a region of the genome that regulates microRNA-targeted gene expression, including, for example, the 5'UTR region of the gene. Expression of a gene targeted by the target microRNA.
  • RNA sequence for sensing the target microRNA or other small RNA.
  • the single-stranded RNA sequence, the vector, or the synthetic RNA molecule is used to sense the target microRNA or other small RNA to modulate the target microRNA or other small Use of expression of RNA-targeted genes.
  • a method comprising a method for specifically increasing the expression and/or activity of an endogenous and/or exogenous target microRNA or other small RNA for use in vitro and/or Or a method of specifically modulating expression of a gene targeted by said target microRNA in vivo, for use as a sensor for sensing said target microRNA or other small RNA, and for sensing said target microRNA or other small RNA
  • the method comprising providing the single-stranded RNA sequence, the vector, or the synthesis provided herein
  • the single-stranded RNA sequence, the vector, or the synthetic RNA molecule is introduced into a cell of interest.
  • the target microRNA or other small RNA is sensed by interacting with a single-stranded RNA sequence, the vector, or the synthetic RNA molecule, with a target microRNA released after cell lysis, and can be used The presence of the microRNA in the sample is detected.
  • RNA sequence that binds to a target microRNA
  • the method comprising selecting a target microRNA (eg, selecting a corresponding microRNA according to a gene of interest desired to be modulated), and then generating the single sheet provided herein A stranded RNA sequence, the vector, or the synthetic RNA molecule.
  • kits comprising the single-stranded RNA sequence, the vector, and/or the synthetic RNA molecule, and instructions for use, preferably the kit can be used herein The purpose and method of description.
  • the kit includes various reagents such as buffers and the like suitable for storing the single-stranded RNA sequence, the vector, and/or the synthetic RNA molecule.
  • the kit includes various reagents, such as enzymes, transformation or transfection reagents, suitable for performing the single-stranded RNA sequence, the vector, and/or the synthetic RNA molecule to react with a target microRNA, .
  • the kit comprises, by the single-stranded RNA sequence, the vector, and/or the synthetic RNA molecule, modulating expression of a gene of interest by reacting with a target microRNA (eg, enhancing expression of a gene of interest) ) Reagents.
  • polynucleotides that are capable of being transcribed into the single-stranded RNA sequence, wherein the polynucleotide comprises the single-stranded RNA expression vector, wherein the expression vector comprises the encoding DNA sequence of single-stranded RNA.
  • a method of binding to a target microRNA and enhancing the activity of the microRNA comprising introducing the single-stranded RNA sequence, the vector, or the synthetic RNA molecule into a target cell step.
  • the enhanced microRNA activity can include, for example, one or more of stabilizing the microRNA, extending the half-life of the microRNA, altering expression of a target gene regulated by the microRNA, and the like.
  • FIG. 1 Different forms of the miR-7 binding sequence.
  • FIG. 1 Different forms of the miR-9 binding sequence.
  • FIG. 3 Schematic representation of the miR-7 reporter and miR-9 reporter.
  • FIG. 4 Luciferase activity of miR-7 reporter or miR-9 reporter after overexpression of miRancers. *, p ⁇ 0.05, **, p ⁇ 0.01 by Student's t-test.
  • Figure 5 Schematic representation of the miR-7 binding sequence for different stem lengths.
  • FIG. 6 Luciferase activity of miR-7 reporter after overexpression of miRancers with different stem lengths **, p ⁇ 0.01 by Student's t-test.
  • FIG. 7 Luciferase activity of miR-7 reporter or miR-9 reporter after overexpression of miRancer. **, p ⁇ 0.01 by Student's t-test.
  • Figure 8 Expression levels of miR-7 or miR-9 after overexpression of miRancer. ***, p ⁇ 0.001 by Student's t test.
  • Figure 9 Schematic representation of a luciferase reporter vector containing a miRancer or sponge sequence.
  • Figure 10 Effect of overexpression of miR-7 or miR-9 on different luciferase activities. *, p ⁇ 0.05, **, p ⁇ 0.01, ***, p > 0.001 by Student's t test.
  • the single-stranded RNA molecule provided herein is capable of binding to a target microRNA and enhancing the activity of the microRNA, such an RNA molecule that enhances microRNA activity is referred to herein as a microRNA enhancer, referred to as a microRNA enhancer, miRancer.
  • enhancing microRNA activity can include, for example, stabilizing the microRNA, extending the half-life of the microRNA, altering (eg, increasing or decreasing) one or more of aspects of target gene expression regulated by the microRNA. Methods for measuring the half-life of microRNAs and expression of target genes are known in the art, see, for example, the methods exemplified in the Examples herein.
  • the miRancer is a single-stranded RNA sequence capable of binding to a target microRNA, the single-stranded RNA sequence having a 5'-end and a 3'-arm, the 5'-arm binding to the target microRNA via base-pair pairing
  • the 3' end arm binds to the 5' end arm by a base complementary pairing.
  • the order of nucleotide sequences refers to the order from the 5' end to the 3' end.
  • a single-stranded RNA sequence such as a miRancer molecule, comprises or consists of a molecule of Formula 1 below from the 5' end to the 3' end:
  • n is a contiguous nucleotide or analog thereof, and the number thereafter is the number of nucleotides or analogs thereof, wherein n 1 is the nucleotide of the first position at the 5' end of the target microRNA (usually U)
  • the corresponding n 6-7 corresponds to position 2-7 or position 2-8 of the target microRNA from the 5' end to the 3' end (referred to as "seed sequence"), thereby (n 5-11 ) (n 0 -3 ) (n 6-7 ) is a 5' end arm, (n 11-21 ) (n 0-2 ) is a 3' end arm, where n 0-3 is the inserted C or G to the 5' A GC pair is formed between the end arm and the 3' end arm, and (n 0-2 ) may be a 3' end overhang, which may be, for example, a UU.
  • (n 1 ) is A.
  • the 5' end arm and the target microRNA are from the 5' end to the 3' end of the 2-18th nucleus, except that the inserted ( n0-3 ) is not calculated as a gap
  • At least 50%, 60%, 70%, 80%, 90%, or 100% of the nucleotides are complementary, for example, the 5' end arm of the miRancer molecule and the 2-18 of the microRNA molecule from 5' to 3'
  • the bit sequence (or, for example, the 2-17th, 2-16th, 2-15th, 2-14th sequence) is 100%, about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92 %, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% complementary.
  • n 6-7 preferably wherein (n 6-7 ) is 100%, about 99%, 98%, 97% from the 2-7th or 2-8 sequence of the 5' to 3' of the target microRNA molecule. , 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80 %, 75%, 70%, 65%, 60%, 55%, or 50% complementary.
  • the number of unpaired nucleotides in (n 11-21 ) and (n 5-11 )(n 0-3 )(n 6-7 ) is less than 6, 5, 4, 3, 2, or 1 pair, preferably all unpaired nucleotides form a ring structure around (n 1 ).
  • the stem structure is 10-18 pairs of paired nucleotides in length, including, for example, 10 pairs, 11 pairs, 12 pairs, 13 pairs, 14 pairs, 15 pairs, 16 pairs, 17 pairs, 18 pairs. Paired nucleotides.
  • the loop structure comprises 1-13 nucleotides, including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleosides acid.
  • (n 0-2 ) can be a 3' end overhang, which can be, for example, a UU.
  • the acid forms a loop; for example, each of the 5' end and the 3' end of (n 1 ) has 1, 2, 3, 4, 5 or 6 nucleotides forming a 5' end and a 3' end arm, said 5' The ends and the 3' end arms are not complementary to form a loop.
  • the (n 1) of the 5 'and 3' ends each having 5 or 6 nucleotides to form a 5 'end and 3' end of the arm, the 5 ' The ends and the 3' end arms are not complementary to form a loop, and the remaining nucleotides that do not form a loop are 100% complementary to form a stem.
  • each n of the nucleotide sequences described herein are independently of one another selected from the group consisting of: A, U, T, G, and C, or nucleotide analogs thereof, eg, if The sequences are RNA sequences, each n being independently of one another selected from the group consisting of: A, U, G and C, or nucleotide analogs thereof.
  • (n 1 ) is A.
  • (n 6-7 ) is fully complementary to positions 2-7 or 2-8 of the microRNA, and (n 0-3 ) is inserted cytidine (C) or guanosine (G).
  • the corresponding position in the corresponding (n 11-21 ) sequence is a complementary guanosine (G) or cytidine (C) analog thereof to the 5' end arm and the 3' end arm
  • the GC pairing is formed, and the number of the pairings may be 0, 1, 2, and 3, and the inserted C is not counted as a gap when calculating the complementarity between the microRNA and the miRancer.
  • (n 5-11 ) is 5, 6, 7, 8, 9, 10, or 11 contiguous nucleotides. In some embodiments, (n 6-7 ) is 6 or 7 contiguous nucleotides. In some embodiments, (n 11-21 ) is 16, 17, or 18 contiguous nucleotides, wherein the number of unpaired nucleotides is less than 6, 5, 4, 3, 2, or 1 pair, preferably all Unpaired nucleotides form a loop structure around (n 1 ); in some embodiments, the stem structure is 10-18 pairs of paired nucleotides in length, including, for example, 10 pairs, 11 pairs, 12 pairs, 13 pairs, 14 pairs, 15 pairs, 16 pairs, 17 pairs, 18 pairs of paired nucleotides; the loop structure comprises 1-13 nucleotides including, for example, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13 nucleotides.
  • (n 0-2 ) can be a 3' end overhang, which can be, for example, a UU.
  • the 3 'end overhang can, for example, facilitate binding of Ago2.
  • the miRancer molecule can further comprise a sequence that promotes binding of Ago2 at the 3' end, such as an HDV ribozyme, which is capable of promoting the removal of additional sequences of the 3' segment of the RNA molecule, resulting in a short 3' end overhang, thereby The binding of RNA molecules to Ago2 is facilitated (see, for example, Renfu Shang et al, NATURE COMMUNICATIONS, 6: 8430, which is incorporated herein by reference in its entirety).
  • a single-stranded RNA sequence such as a miRancer molecule, comprises or consists of a molecule of Formula 2 below from the 5' end to the 3' end:
  • n is a contiguous nucleotide or analog thereof, and the number thereafter is the number of nucleotides or analogs thereof, wherein n 1 is the nucleotide of the first position at the 5' end of the target microRNA (usually U)
  • corresponding n 7 corresponds to position 2-8 of the target microRNA from the 5' end to the 3' end
  • n 2 is an inserted C or G to form a GC between the 5' end arm and the 3' end arm pair.
  • (n 7 ) is at least 50%, 60%, 70%, 80%, 90%, or 100% complementary to the 2nd to 8th positions of the target microRNA from the 5' end to the 3' end.
  • the acid forms a loop; for example, each of the 5' end and the 3' end of (n 1 ) has 1, 2, 3, 4, 5 or 6 nucleotides forming a 5' end and a 3' end arm, said 5' The ends and the 3' end arms are not complementary to form a loop.
  • the (n 1) of the 5 'and 3' ends each having 5 or 6 nucleotides to form a 5 'end and 3' end of the arm, the 5 ' The ends and the 3' end arms are not complementary to form a loop, and the remaining nucleotides that do not form a loop are 100% complementary to form a stem.
  • the miRancer provided herein can be an oligonucleotide.
  • oligonucleotide refers to a molecule formed by covalently linking two or more nucleotides.
  • the term oligonucleotide typically includes oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations of these.
  • a nucleotide or monomer sequence it may be a base sequence such as, for example, the sequence of A, T (or U), G or C or an analog thereof.
  • nucleotide refers to a moiety comprising a sugar moiety, a base moiety, and a glycoside of a covalently linked group (such as a phosphate or phosphorothioate internucleotide linking group) and includes a naturally occurring nucleotide (such as DNA or RNA) and a modified sugar and/or base moiety
  • Non-naturally occurring nucleotides which are also referred to herein as "nucleotide analogs.”
  • Non-naturally occurring nucleotides include nucleotides having a modified sugar moiety (e.g., a bicyclic nucleotide or a 2' modified nucleotide, such as a 2' substituted nucleotide).
  • nucleotide analog is a variant produced by a naturally occurring nucleotide (such as a DNA or RNA nucleotide) using modifications in the sugar and/or base moiety. Analogs may have no functional or functional impact on the oligonucleotide. For example, by increasing the binding affinity for the target and/or increased resistance to intracellular nucleases and/or increased ease of transport into the cell.
  • the miRancer molecule comprises 1, 2, 3 or more nucleotide analogs.
  • the oligomer comprises 3-8 nucleotide analogs, such as 6 or 7 nucleotide analogs.
  • the nucleotide analog comprises a locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • a modified RNA molecule as defined herein may contain a nucleotide analog/modification, such as a backbone modification, a sugar modification, or a base modification.
  • the backbone modification may be the chemistry of the phosphate ester of the nucleotide backbone contained in the nucleic acid molecule.
  • the phosphate group of the modified modified backbone can be modified by replacing one or more oxygen atoms with different substituents, examples of which include, for example, phosphorothioates.
  • the sugar modification can be a chemical modification of the sugar of the nucleotide of the nucleic acid molecule, for example, the 2' hydroxyl (OH) can be modified or replaced by a number of different "oxy" or "deoxy" substituents.
  • the base modification can be a chemical modification of the base portion of the nucleotide of the nucleic acid molecule.
  • the nucleotide analog or modification may be selected from nucleotide analogs suitable for transcription and/or translation. Nucleotide analogs/modifications may include 2-amino-6-chloropurine nucleoside-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2'-fluorothymidine- 5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 7-deazacyosine-5'-triphosphate, and Pseudouridine-5'-triphosphate and the like.
  • Modified nucleosides may include pyridin-4-one ribonucleoside, 5-aza-uridine, dihydro pseudouridine, 2-thio-dihydrouridine, 2,6-diaminopurine, N2, N2 -Dimethylguanosine, N1-methyl-pseudouridine, 5,6-dihydrouridine, 4-thio-uridine, 5-hydroxy-uridine, deoxy-thymidine, inosine, ⁇ - Thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino -6-chloro-indole, pseudo-cytidine, 6-chloro-indole, and the like.
  • the miRancer molecules provided herein may be included or consist of a total length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 consecutive nucleotide sequences composition.
  • the oligonucleotide comprises or consists of a total of about 30-45, such as about 33-40, such as about 33-37, such as about 33-35 contiguous nucleotide sequences in length. .
  • the oligonucleotides of the invention consist of no more than 40 nucleotides, such as no more than 38 nucleotides, such as no more than 34 nucleotides, such as 33, 35 or 37 Nucleotide.
  • miRancer expression vector refers to a vector comprising a miRancer molecule having a polynucleotide sequence encoding miRancer.
  • the miRancer expression vector is designed to produce a miRancer molecule from the vector.
  • MicroRNA refers to an oligoribonucleic acid, generally ranging from about 18 to about 25 nucleotides in length, which regulates the expression of a polynucleotide comprising a target sequence.
  • MicroRNAs are non-protein-encoding RNAs that have been identified in animals and plants. The microRNA is initially transcribed into long polyadenylated RNA which is then processed to form a shorter sequence with the ability to form a stable hairpin. Most microRNA genes synthesize pri-miRNAs under the action of RNA polymerase II.
  • the pri-miRNA is cleaved by the Drosha enzyme to form a stem-loop structure of about 70 nt, that is, a pre-miRNA. Subsequently, the pre-miRNA is cleaved by the Dicer enzyme to produce a microRNA single-stranded structure that forms a mature microRNA.
  • the miRancer expression vector is provided herein.
  • the miRancer expression vector expression vector comprises a polynucleotide that can be transcribed into a miRancer sequence that enhances microRNA activity.
  • the miRancer expression vector provides for formation with a similar hair
  • the miRancer molecule can stabilize a microRNA molecule to modulate the expression activity of the microRNA targeting gene.
  • the miRNA can be derived from any animal or plant.
  • the 5' end arm sequence of miRancer and the contiguous nucleotide sequence of the miRNA sequence starting from position 2 can be 100%, at least 99%, 98%, 97%, 96%, 95%, 90% , 85%, 80% or less complementary sequences.
  • the 5' end-arm sequence of miRancer comprises a sequence that has 1, 2, 3, 4, 5 or more mismatches with a contiguous nucleotide sequence starting from position 2 of the miRNA sequence, and is still sufficient The complement forms a double-stranded structure with the miRNA sequence, generates a miRNA binding sequence and enhances the activity of the microRNA.
  • the vector miRancer molecule is expressed by miRancer and has a sequence that is sufficiently complementary to the microRNA.
  • a "sufficiently complementary sequence" to a microRNA target sequence means that its complementarity is sufficient to allow the miRancer molecule to bind to the microRNA and increase the activity of the microRNA.
  • the miRancer having sufficient complementarity to the target sequence is 100% complementary to the microRNA target sequence or may be less than 100% complementary to the target sequence (ie, at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70% or less complementary sequences).
  • the miRancer and the target sequence can have 1, 2, 3, 4, 5 or up to 6 mismatches as long as the miRancer is sufficiently complementary to the target sequence to increase the activity of the target sequence, such as half-life.
  • the miRNA sequence can have a "U" at the 5' end.
  • a pair of base pair changes can be added within the 5' end of the miRNA such that the sequence differs from the target sequence by one nucleotide.
  • Target sequence refers to the sequence of the microRNA targeted by the miRancer.
  • the target sequence may be an endogenous sequence or may be an introduced heterologous sequence.
  • the miRancer produced by the miRancer expression vector is capable of increasing the activity of the microRNA.
  • Methods for determining the activity of a microRNA include measuring changes in the expression of a gene/protein that it targets.
  • a single miRNA can silence a plurality of proteins/genes or whole proteins and/or gene families in a protein and/or gene family.
  • “Increase” means normal levels of microRNA relative to wild-type organisms (or Increase in microRNA-targeted gene/protein levels). By “increasing miRancer activity” it can be an increase in expression activity to any statistically significant amount, for example an increase of at least 10%, 15%, 20%, 25%, 30%, 35%, 40 relative to wild-type expression activity. %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • the miRancer molecules described herein can be delivered to one or more of an animal such as a mammal, including a human or a plant cell.
  • isolated or recombinant polynucleotides encoding various components of a miRancer expression vector, a miRancer expression vector, together with a different product of a miRancer expression vector processed into a miRancer.
  • Polynucleotides can be polymers of RNA or DNA, which can be single or double stranded, optionally comprising synthetic, non-natural or modified nucleotide bases.
  • a polynucleotide in the form of a DNA polymer can be composed of one or more fragments of cDNA, genomic DNA, synthetic DNA, or a mixture thereof.
  • Polynucleotides can include ribonucleotides as well as combinations of ribonucleotides and deoxyribonucleotides. The deoxynucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs.
  • Polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpin structures, stem-loop structures, and the like.
  • compositions provided herein can comprise isolated or substantially purified polynucleotides.
  • An "isolated” or “purified” polynucleotide is substantially or essentially free of those free components that normally accompany or interact with a polynucleotide in a naturally occurring environment.
  • an isolated or purified polynucleotide is substantially free of other cellular material, or a culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the "isolated" polynucleotide is naturally ubiquitously present on the genomic DNA of the organism from which the polynucleotide is derived (ie, located 5' of the polynucleotide and The sequence of the 3' end).
  • a recombinant vector comprises a combination of artificial or heterologous nucleic acid sequences, such as non-naturally coexisting regulatory and coding sequences.
  • the recombinant vector may comprise regulatory sequences derived from different sources And coding sequences, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from the naturally occurring manner.
  • the carrier can be used alone or in combination with a carrier. If a vector is used, the choice of vector will depend on the method used to transform the host cell. For example, a plasmid vector can be used.
  • a genetic element that can be included on the vector In order to successfully transform, screen and propagate a host cell comprising any of the isolated nucleic acid fragments of the invention, a genetic element that can be included on the vector. Therefore, in order to obtain a cell line showing a desired expression level and pattern, screening can be performed by Southern blot analysis, Northern blot analysis, immunoblot analysis of protein expression, phenotypic analysis, and the like.
  • one or more miRancer expression vectors described herein can be expressed in different cell types in an expression cassette format.
  • the cassette can include 5' and 3' regulatory sequences operably linked to the polynucleotides provided herein.
  • the expression cassette can include a transcriptional and translational initiation region (ie, a promoter), a recombinant polynucleotide provided herein, and a transcriptional and translational termination region (ie, a termination region).
  • the miRancer expression vector may contain a promoter regulatory region (eg, a regulatory region that confers inducible, constitutive, environmental or developmental regulation, or cell or tissue specific/selective expression), a transcriptional initiation site, ribose A body binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • a promoter regulatory region eg, a regulatory region that confers inducible, constitutive, environmental or developmental regulation, or cell or tissue specific/selective expression
  • the methods provided herein comprise introducing a miRancer expression vector into a cell to increase the activity of the target microRNA.
  • the methods provided herein are limited to a particular method as long as the polynucleotide is introduced into the interior of at least one cell of the host.
  • Methods for introducing polynucleotides into host cells are known in the art and include, but are not limited to, virus-mediated methods.
  • Introduction includes the integration of a nucleic acid into a eukaryotic or prokaryotic cell in which the nucleic acid can be integrated into the genome of the cell and includes providing the nucleic acid or protein to the cell. Transformation protocols and protocols for introducing polynucleotide sequences into plants can vary depending on the type of cell being transformed.
  • a method of increasing microRNA activity by introducing a miRancer expression vector into a cell comprises introducing a miRancer expression vector into a cell, wherein the miRancer molecule produced by the miRancer expression vector increases microRNA and/or alters (eg, increases or decreases) the activity of the microRNA-targeted gene.
  • a miRancer molecule can be integrated into a regulatory region of a gene targeted by a target microRNA in the genome (including, for example, 5'UTR and/or 3'UTR and/or regions upstream, downstream, intron, etc.) Regulate the expression of genes.
  • the miRancer molecule provided herein is capable of increasing gene expression of a microRNA-targeted gene, which is very surprising, as the role of the microRNA is generally believed to reduce gene expression.
  • the miRancer molecules provided herein are capable of altering the effect of a target microRNA (eg, the target microRNA reduces expression of some of the genes, and then, by introducing a miRancer molecule or vector provided herein into the regulatory region of the gene, such that microRNA increases gene expression).
  • the expression of a gene of interest desired to alter its expression can be modulated by the corresponding microRNA encoding the corresponding microRNA of the gene of interest desired to be regulated.
  • variants of polynucleotides for use in the compositions and methods.
  • "Variant” refers to a substantially similar sequence.
  • a variant includes deletion and/or insertion of one or more nucleotides at one or more internal sites of the polynucleotide, and/or one or more positions in the polynucleotide.
  • a dot has one or more nucleotide substitutions.
  • Variant polynucleotides can include polynucleotides of synthetic origin, such as those produced, for example, by site-directed mutagenesis.
  • the miRancer expression vector, single-stranded RNA molecule, miRancer molecule disclosed herein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 with the prototype polynucleotide. Sequence identity of %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Sequence alignment methods for alignment are well known in the art. Procedures for aligning sequences and determining sequence identity include CLUSTAL, GAP, BESTFIT, BLAST, FASTA, and TFASTA. You can use the default parameters to make an alignment using these programs.
  • the target microRNA to which miRancer is capable of acting is not specifically limited.
  • a target microRNA molecule that is desired to be modulated can be selected from an existing database.
  • the corresponding microRNA molecule can be selected based on the gene that is desired to be modulated.
  • genes targeted by microRNAs can be searched for by bioinformatics methods (eg, using miRanda, TargetScan, RNAhybrid, etc.).
  • the miRancer molecules herein can increase the activity of the microRNA by binding to a target microRNA molecule.
  • the oligonucleotide when hybridized to a target microRNA molecule, can tolerate 1, 2, 3 or 4 (or more) mismatches and still fully bind to the target, Shows the desired effect (eg, increases the activity of the microRNA).
  • a mismatch can be compensated for by an increased length oligonucleotide sequence and/or an increased number of nucleotide analogs (such as locked nucleic acids (LNA)) present within the nucleotide sequence.
  • LNA locked nucleic acids
  • the contiguous nucleotide sequence comprises no more than 3 mismatches (eg, no more than 1 or no more than 2 mismatches) when hybridized to a target microRNA molecule. In some embodiments, the contiguous nucleotide sequence comprises no more than one mismatch when hybridized to a target microRNA molecule.
  • the miRancer molecule of the invention preferably is at least 80% complementary to the microRNA molecule in the complementary region, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, At least 97%, at least 98%, at least 99%, such as at least 100% complementary.
  • the miRancer may comprise additional 5' or 3' nucleotides or modifications, such as, independently, 1, 2, 3, 4 or 5 additional nucleotides 5' and/or 3' It is not complementary to the target sequence.
  • an oligonucleotide of the invention may comprise a contiguous nucleotide sequence joined to another nucleotide at the 5' and or 3' side.
  • the additional 5' or 3' nucleotide is a naturally occurring nucleotide, such as DNA or RNA.
  • the additional 5' or 3' nucleotide can be a nucleotide analog.
  • the complementary region may include positions 2-12, 2-13, 2-14, 2-15, 2-16, 2-17 of the microRNA molecule, 2-18 digits.
  • the miRancer molecules, vectors, and cells of the present invention can be used in pharmaceutical preparations and compositions, and can also be prepared into kits for convenient use.
  • the composition or kit comprises a pharmaceutically acceptable solvent such as water or saline, a diluent, a carrier, a salt or an adjuvant.
  • the invention also encompasses pharmaceutical compositions and formulations comprising the oligonucleotides of the invention.
  • the pharmaceutical compositions of the invention may be administered in a variety of ways, depending on whether local or systemic treatment is desired, and depending on the area to be treated.
  • the oligonucleotides of the invention can be used as research reagents, for example, for diagnosis, treatment, and prevention.
  • the oligonucleotides can be used to specifically bind to a target microRNA, which can increase its activity, thereby facilitating functional analysis of the target or its assessment as a target for therapeutic intervention.
  • the oligonucleotides can be used to detect and quantify target microRNA levels in tissues in a cell by Northern blotting, in situ hybridization, or similar techniques.
  • an animal or human suspected of having a disease or condition that can be treated by modulation of the expression of a gene targeted by the target microRNA can be treated by administering an oligonucleotide of the invention.
  • a method of treating a mammal (eg, a human) suspected of having or prone to have a disease or condition associated with expression of a gene targeted by a target microRNA by administering a therapeutically or prophylactically effective amount of one or A variety of oligonucleotides or compositions of the invention.
  • Oligonucleotides or pharmaceutical compositions of the invention are typically administered in an effective amount.
  • the invention also provides a method for treating a disease, such as a tumor, comprising administering to a patient in need thereof an oligonucleotide molecule described herein or a pharmaceutical composition comprising the molecule.
  • the cell line used in the following experiments was the MCF-7 human breast cancer cell line, purchased from ATCC, the medium was RPMI-1640, purchased from Gibco, and the fetal bovine serum FBS was purchased from Gibco.
  • the transfection reagent was lipofectamine 2000, purchased from invitrogen.
  • Plasmid pSilencer 4.1CMV was purchased from Ambion, and plasmid psiCheck2 and dual luciferase assay kits were purchased from promega. Primer synthesis was completed by Shanghai Biotech, and mimics synthesis was completed by Shanghai Jima.
  • the recombinant plasmid cloning method is a conventional method, and the sequencing is completed by Shanghai Biotech, and the details are explained below.
  • the dual luciferase reporter assay was performed according to the kit (Promega E1910) instructions.
  • the binding sequence of the microRNA is designed to have a single-stranded RNA with a similar hairpin structure.
  • the 5' end arm of the hairpin structure binds to the microRNA through base complementary pairing, while the 3' end arm binds to the 5' end arm through base complementary pairing.
  • the preferred double strands are within 18 nucleotides in length.
  • a 7 nt or 13 nt ring structure is introduced at the corner of the hairpin structure to determine the potential functions that different structures may bring.
  • RNA secondary structure Thereafter, two pairs of GCs are inserted in the middle of the hairpin loop structure to stabilize the RNA secondary structure.
  • FIG. 1 shows the different forms of the miR-7 binding sequence
  • Figure 2 shows the different forms of the miR-9 binding sequence.
  • the pSilencer 4.1CMV plasmid was used as an expression vector for small hairpin RNA. Plasmid passage After double digestion with BamHI and HindIII, they were separated by 1% agarose gel, then recovered by Axyprep DNA extraction kit (AP-GX-50) and stored at -20 °C.
  • miR-7 binding sequence B forward
  • the annealed DNA sequence was ligated between BamHI and HindIII of the pSilencer 4.1 CMV plasmid, and the kit used was a TAKARA ligation kit (D6022).
  • the recombinant plasmid was sequenced to confirm that the sequence was correct.
  • FIG. 3 shows a schematic of the miR-7 reporter and miR-9 reporter.
  • the miR-7 binding sequence and the miR-7 reporter were co-transfected into MCF-7 cells, and then a dual luciferase reporter assay (Promega E1910) was performed as described.
  • Figure 4 shows luciferase activity of miR-7 reporter or miR-9 reporter after overexpression of miRancers. *, p ⁇ 0.05, **, p ⁇ 0.01 by Student's t-test.
  • Type F is selected as a preferred microRNA binding sequence that enhances microRNA activity. All sequences that enhance microRNA activity are called microRNA enhancers, referred to as miRancers.
  • FIG. 5 shows a schematic representation of the miR-7 binding sequence for different stem lengths.
  • Figure 6 shows luciferase activity of miR-7 reporter after overexpression of miRancers of different stem lengths, p ⁇ 0.01 by Student's t-test.
  • miRancer (Shanghai Jima) was synthesized by chemical method according to F type.
  • the miRancer designed for the bantam miRNA in Drosophila was used as a control.
  • the inventors have found that chemically synthesized miRancers can also specifically affect the activity and level of microRNAs.
  • Figure 7 shows luciferase activity of miR-7 reporter or miR-9 reporter after overexpression of miRancer. **, p ⁇ 0.01 by Student's t-test.
  • Figure 8 shows the expression levels of miR-7 or miR-9 after overexpression of miRancer. ***, p ⁇ 0.001 by Student's t test.
  • MiRancer-7, miR-7 sponge, miRancer-9, miR-9 sponge were inserted upstream of luciferase, and then co-transfected with miR-7 mimics, miR-9 mimics (Shanghai Jima), respectively, and luciferase containing miRancer was found. It can be stabilized by the corresponding miRNA, while the luciferase containing the sponge is inhibited by the corresponding miRNA. This suggests that miRancer can be used to enhance gene expression.
  • Figure 9 shows a schematic representation of a luciferase reporter vector containing a miRancer or sponge sequence.
  • Figure 10 shows the effect of overexpression of miR-7 or miR-9 on different luciferase activities. *, p ⁇ 0.05, **, p ⁇ 0.01, ***, p > 0.001 by Student's t test.

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Abstract

提供一种结合目标microRNA的单链RNA序列，该单链RNA序列具有5'端臂和3'端臂，所述5'端臂通过碱基互补配对与目标microRNA结合，所述3'端臂通过碱基互补配对与所述5'端臂结合。

Description

miRancer分子的设计与应用 技术领域
本发明涉及RNA分子及其用途。具体而言，本发明涉及能够特异性结合和增强microRNA的表达及活性的寡聚RNA分子及其用途。
背景技术
成熟的microRNA(miRNA)是一类长度约22个核苷酸(nt)(例如约18-25个核苷酸)的小RNA，在细胞内具有多种重要的调节作用。miRNA广泛存在于真核生物中，它们能够与那些和它的序列互补的mRNA分子相结合发挥功能(例如导致基因的沉默)，这是调节基因表达的一个重要策略。研究结果表明，miRNA参与调控个体发育、细胞凋亡、增殖及分化等生命活动，可以在疾病特别是癌症的诊断和治疗中发挥作用。
发明内容
在一些实施方案中，本文提供一种结合目标microRNA的单链RNA序列，优选的所述单链RNA序列增强目标microRNA的活性(在本文中称为miRancer分子)，所述单链RNA序列具有5'端臂和3'端臂，所述5'端臂通过碱基互补配对与目标microRNA结合，所述3'端臂通过碱基互补配对与所述5'端臂结合，
所述单链RNA序列从5'端到3'端具有以下通式1：
(n5-11)(n0-3)(n6-7)(n1)(n11-21)(n0-2)，
其中n为连续的核苷酸或其类似物，其后数字为核苷酸或其类似物的个数，每个n可以彼此独立地选自下述核苷酸：A，U，G和C，或其核苷酸类似物，其中n1与目标microRNA的5'端第一位的核苷酸互补，相应的n6-7对应于目标microRNA从5'端到3'端的第2-7位或第2-8位，从而(n5-11)(n0-3)(n6-7)为5'端臂，(n11-21)(n0-2)为3'端臂，其中n0-3为插入的C或G 以在所述5'端臂和3'端臂之间形成GC配对。
在一些实施方案中，本文提供的所述单链RNA分子能够结合目标microRNA和增强所述microRNA的活性。发明人将所述可以增强microRNA活性的RNA分子称为microRNA增强子(microRNA enhancer)，简称miRancer。所述增强microRNA活性可以包括例如稳定所述microRNA，延长所述microRNA的半衰期，改变所述microRNA所调节的靶基因表达等方面中的一种或多种。测量microRNA和microRNA所调节的靶基因表达的方法是本领域已知的。
在一些实施方案中，本文描述的RNA分子中除了插入的(n0-3)作为缺口不计算同一性之外，所述5'端臂与所述目标microRNA从5'端到3'端的第2-18位核苷酸的互补链至少50％、60％、70％、80％、90％、或100％的同一性，优选其中(n6-7)与所述目标microRNA从5'端到3'端的第2-7位或第2-8位核苷酸的互补链具有至少80％、90％、或100％的同一性，优选(n11-21)与(n5-11)(n0-3)(n6-7)中不配对核苷酸的个数小于6、5、4、3、2、或1对，优选全部不配对的核苷酸在(n1)周围。在一些实施方案中，(n5-11)(n0-3)(n6-7)与所述目标microRNA从5'端到3'端的第2-18位核苷酸至少50％、60％、70％、80％、90％、95％或100％互补。在一些实施方案中，(n6-7)与所述目标microRNA从5'端到3'端的第2-7位或第2-8位核苷酸至少80％、90％、95％、99％或100％互补。在一些实施方案中，(n11-21)与(n5-11)(n0-3)(n6-7)至少50％、60％、70％、80％、90％、95％或100％互补。
在一些实施方案中，本文提供一种包含所述单链RNA序列的载体，如质粒。
在一些实施方案中，本文提供一种包含所述单链RNA序列的多核苷酸序列的载体，如质粒。
在一些实施方案中，本文提供一种所述单链RNA序列的合成的或重组的RNA分子。
在一些实施方案中，本文提供一种包含所述载体的细胞。
在一些实施方案中，本文提供所述单链RNA序列、所述载体、或所述合成的RNA分子用于特异性增加内源性和/或外源性目标microRNA或其它小RNA的表达和/或活性的使用方法和/或用途。
在一些实施方案中，本文提供所述单链RNA序列、所述载体、或所述合成的RNA分子用于体外和/或体内特异性调节(例如增加)所述目标microRNA靶向的基因的表达的使用方法和/或用途。在一些实施方案中，本文提供所述单链RNA序列可通过整合到基因组中调节microRNA靶向的基因表达的区域(包括例如所述基因的5'UTR区)来特异性调节(例如增加)所述目标microRNA靶向的基因的表达。
在一些实施方案中，本文提供所述单链RNA序列、所述载体、或所述合成的RNA分子用于感测所述目标microRNA或其它小RNA的使用方法和/或用途。
在一些实施方案中，本文提供所述单链RNA序列、所述载体、或所述合成的RNA分子用于感测所述目标microRNA或其它小RNA的从而调节所述所述目标microRNA或其它小RNA靶向的基因的表达的用途。
在一些实施方案中，本文提供一种方法，所述方法包括用于特异性增加内源性和/或外源性目标microRNA或其它小RNA的表达和/或活性的方法，用于体外和/或体内特异性调节所述目标microRNA靶向的基因的表达的方法，用作感受器用于感测所述目标microRNA或其它小RNA的方法，和用于通过感测所述目标microRNA或其它小RNA从而调节所述所述目标microRNA或其它小RNA靶向的基因的表达方法中的任意一种或多种，所述方法包括将本文提供所述单链RNA序列、所述载体、或所述合成的RNA分子与包含目标microRNA或其它小RNA的样品(例如细胞、血液、组织等样品)接触的步骤。在一些实施方案中，通过将所述单链RNA序列、所述载体、或所述合成的RNA分子引入到目的细胞中发挥作用。在一些实施方案中，通过将单链RNA序列、所述载体、或所述合成的RNA分子与细胞裂解后释放的目标microRNA相互作用，从而感测所述目标microRNA或其它小RNA，并可以用于检测所述microRNA的在样品中的存在。
在一些实施方案中，本文提供一种产生结合目标microRNA的单链RNA序列的方法，所述方法包括选择目标microRNA(例如根据希望调节的目的基因选择相应的microRNA)，然后产生本文提供所述单链RNA序列、所述载体、或所述合成的RNA分子。
在一些实施方案中，本文提供一种组合物，其包含所述单链RNA序列、所述载体、和/或所述合成的RNA分子，优选的所述组合物可以用于本文描述的用途和方法。在一些实施方案中，本文提供一种试剂盒，其包含所述单链RNA序列、所述载体、和/或所述合成的RNA分子，以及使用说明书，优选的所述试剂盒可以用于本文描述的用途和方法。在一些实施方案中，所述试剂盒来包括适合用于储存所述单链RNA序列、所述载体、和/或所述合成的RNA分子的各种试剂如缓冲液等。在一些实施方案中，所述试剂盒包括适合进行所述单链RNA序列、所述载体、和/或所述合成的RNA分子与目标microRNA反应的各种试剂包括酶、转化或转染试剂等。在一些实施方案中，所述试剂盒包括适合通过所述单链RNA序列、所述载体、和/或所述合成的RNA分子通过与目标microRNA反应而调节目的基因表达(如增强目的基因表达的)试剂。
在一些实施方案中，本文提供能够被转录为所述单链RNA序列的分离或重组多核苷酸，其中所述多核苷酸包含所述单链RNA表达载体，其中所述表达载体包含编码所述单链RNA的DNA序列。
在一些实施方案中，本文提供一种结合目标microRNA和增强所述microRNA的活性的方法，所述方法包括将所述单链RNA序列、所述载体、或所述合成的RNA分子引入靶细胞的步骤。所述增强microRNA活性可以包括例如稳定所述microRNA，延长所述microRNA的半衰期，改变所述microRNA所调节的靶基因表达等方面中的一种或多种。
附图说明
图1：miR-7结合序列的不同形式。
图2：miR-9结合序列的不同形式。
图3：miR-7 reporter和miR-9 reporter的示意图。
图4：过表达miRancers后的miR-7 reporter或miR-9 reporter的荧光素酶活性。*,p<0.05,**,p<0.01 by Student's t-test.。
图5：不同茎长的miR-7结合序列示意图。
图6：过表达不同茎长的miRancers后miR-7 reporter的荧光素酶活性 **，p<0.01 by Student's t-test。
图7:过表达miRancer后miR-7 reporter或miR-9 reporter的荧光素酶活性。**,p<0.01 by Student's t-test。
图8：过表达miRancer后miR-7或miR-9的表达水平。***，p<0.001by Student's t test。
图9：含有miRancer或sponge序列的荧光素酶报告载体示意图。
图10：过表达miR-7或miR-9对不同荧光素酶活性的影响。*，p<0.05,**,p<0.01,***,p>0.001 by Student’s t test。
具体实施方式
现将结合附图更全面地描述本发明，其中描述了本发明的一部分而非全部实施方案。实际上，这些发明可以许多不同的形式来体现，并且不应理解为受本文所示实施方案的限制，其修改形式和其他实施方案也包括在所附权利要求的范围内。
1.microRNA增强子(microRNA enhancer)
在一些实施方案中，本文提供的所述单链RNA分子能够结合目标microRNA并增强所述microRNA的活性，这种可以增强microRNA活性的RNA分子在本文中称为microRNA增强子(microRNA enhancer)，简称miRancer。在一些实施方案中，增强microRNA活性可以包括例如稳定所述microRNA，延长所述microRNA的半衰期，改变(例如增加或降低)所述microRNA所调节的靶基因表达等方面中的一种或多种。测量microRNA的半衰期和靶基因表达的方法是本领域已知的，参见例如本文实施例中例示的方法。
在一些实施方案中，miRancer为能够结合目标microRNA的单链RNA序列，所述单链RNA序列具有5'端臂和3'端臂，所述5'端臂通过碱基互补配对与目标microRNA结合，所述3'端臂通过碱基互补配对与所述5'端臂结合。
在本文中，除另有明确说明外，核苷酸序列的顺序均是指从5'端到3'端的顺序。
在一些实施方案中，单链RNA序列如miRancer分子从5'端到3'端包含以下通式1的分子或由所述通式1的分子组成：
(n5-11)(n0-3)(n6-7)(n1)(n11-21)(n0-2)，
其中n为连续的核苷酸或其类似物，其后数字为核苷酸或其类似物的个数，其中n1与目标microRNA的5'端第一位的核苷酸(通常是U)互补，相应的n6-7对应于目标microRNA从5'端到3'端的第2-7位或第2-8位(称为“种子序列”)，从而(n5-11)(n0-3)(n6-7)为5'端臂，(n11-21)(n0-2)为3'端臂，其中n0-3为插入的C或G以在所述5'端臂和3'端臂之间形成GC配对，(n0-2)可以为3'端悬突(overhang)，其可以是例如UU。
在一些实施方案中，(n1)为A。在一些实施方案中，其中除了插入的(n0-3)作为缺口不计算同一性之外，所述5'端臂与所述目标microRNA从5'端到3'端的第2-18位核苷酸至少50％、60％、70％、80％、90％、或100％互补，例如，所述miRancer分子的5'端臂与所述microRNA分子从5'至3'的第2-18位序列(或例如第2-17、2-16、2-15、2-14位序列)100％，约99％，98％，97％，96％，95％，94％，93％，92％，91％，90％，89％，88％，87％，86％，85％，84％，83％，82％，81％，80％，75％，70％，65％，60％，55％，或50％互补。在一些实施方案中，优选其中(n6-7)与所述目标microRNA分子从5'至3'的第2-7位或第2-8序列100％，约99％，98％，97％，96％，95％，94％，93％，92％，91％，90％，89％，88％，87％，86％，85％，84％，83％，82％，81％，80％，75％，70％，65％，60％，55％，或50％互补。在一些实施方案中，(n5-11)(n0-3)(n6-7)与(n11-21)之间100％，约99％，98％，97％，96％，95％，94％，93％，92％，91％，90％，89％，88％，87％，86％，85％，84％，83％，82％，81％，80％，75％，70％，65％，60％，55％，或50％互补，形成茎结构。在一些实施方案中，优选(n11-21)与(n5-11)(n0-3)(n6-7)中不配对核苷酸的个数小于6、5、4、3、2、或1对，优选全部不配对的核苷酸在(n1)周围，形成环结构。在一些实施方案中，所述茎结构的长度为10-18对配对核苷酸，包括例如10对，11对，12对，13对，14对，15对，16对，17对，18对配对核苷酸。在一些实施方案中，所述环结构包括1-13个核苷酸，包括例如1，2，3，4，5，6，7，8，9，10，11，12，或13个核苷酸。在一些实施方案中，(n0-2)可以为3'端悬突(overhang)， 其可以是例如UU。在一些实施方案中，紧邻(n1)周围(包括(n1)在内)的1，2，3，4，5，6，7，8，9，10，11，12或13个核苷酸形成环；例如，在(n1)的5'端和3'端各具有1，2，3，4，5或6个核苷酸形成5'端和3'端臂，所述5'端和3'端臂之间不互补，从而形成环。在一些实施方案中，在(n1)的5'端和3'端各具有1，2，3，4，5或6个核苷酸形成5'端和3'端臂，所述5'端和3'端臂之间不互补，从而形成环，而不形成环的剩余核苷酸之间至少50％、60％、70％、80％、90％、或100％互补，形成茎。在一些实施方案中，在(n1)的5'端和3'端各具有1，2，3，4，5或6个核苷酸形成5'端和3'端臂，所述5'端和3'端臂之间不互补，从而形成环，而不形成环的剩余核苷酸之间100％互补，形成茎。
在一些实施方案中，本文描述的核苷酸序列的每个n彼此独立地选自下述核苷酸：A，U，T，G和C，或其核苷酸类似物，例如如果所述序列为RNA序列，每个n可以彼此独立地选自下述核苷酸：A，U，G和C，或其核苷酸类似物。
在一些实施方案中，(n1)为A。在一些实施方案中，(n6-7)与microRNA的第2-7或第2-8位完全互补，(n0-3)为插入的胞苷(C)或鸟苷(G)其类似物，从而其相对应的(n11-21)序列中的相应位置为互补的鸟苷(G)或胞苷(C)其类似物，以在所述5'端臂和3'端臂之间形成GC配对，所述配对的个数可以是0，1，2，3个，所述插入的C在计算所述microRNA与所述miRancer之间的互补性时作为缺口不计算在内。在一些实施方案中，(n5-11)为5，6，7，8，9，10，或11个连续核苷酸。在一些实施方案中，(n6-7)为6或7个连续核苷酸。在一些实施方案中，(n11-21)为16、17或18个连续核苷酸，其中不配对核苷酸的个数小于6、5、4、3、2、或1对，优选全部不配对的核苷酸在(n1)周围，形成环结构；在一些实施方案中，所述茎结构的长度为10-18对配对核苷酸，包括例如10对，11对，12对，13对，14对，15对，16对，17对，18对配对核苷酸；所述环结构包括1-13个核苷酸，包括例如1，2，3，4，5，6，7，8，9，10，11，12，13个核苷酸。在一些实施方案中，(n0-2)可以为3'端悬突(overhang)，其可以是例如UU。在一些实施方案中，所述3'端悬突可以例如促进Ago2的结合。在一些实施方案中，miRancer分子可以在3'端另外包含促进Ago2的结合 的序列，例如HDV核酶，其能够促进RNA分子3'段额外序列的去除，产生短的3'端悬突，从而促进RNA分子与Ago2的结合(参见例如Renfu Shang等人，NATURE COMMUNICATIONS，6:8430，其全文通过引用并入本文)。
在一些实施方案中，单链RNA序列如miRancer分子从5'端到3'端包含以下通式2的分子或由所述通式2的分子组成：
(n7-9)(n2)(n7)(n1)(n16-18)，
其中n为连续的核苷酸或其类似物，其后数字为核苷酸或其类似物的个数，其中n1与目标microRNA的5'端第一位的核苷酸(通常是U)互补，相应的n7对应于目标microRNA从5'端到3'端的第2-8位，其中n2为插入的C或G以在所述5'端臂和3'端臂之间形成GC配对。在一些实施方案中，(n7)与目标microRNA从5'端到3'端的第2-8位至少50％、60％、70％、80％、90％、或100％互补。在一些实施方案中，(n7)与目标microRNA从5'端到3'端的第2-8位完全互补。在一些实施方案中，紧邻(n1)周围(包括(n1)在内)的1，2，3，4，5，6，7，8，9，10，11，12或13个核苷酸形成环；例如，在(n1)的5'端和3'端各具有1，2，3，4，5或6个核苷酸形成5'端和3'端臂，所述5'端和3'端臂之间不互补，从而形成环。在一些实施方案中，在(n1)的5'端和3'端各具有1，2，3，4，5或6个核苷酸形成5'端和3'端臂，所述5'端和3'端臂之间不互补，从而形成环，而不形成环的剩余核苷酸之间至少50％、60％、70％、80％、90％、或100％互补，形成茎。在一些实施方案中，在(n1)的5'端和3'端各具有1，2，3，4，5或6个核苷酸形成5'端和3'端臂，所述5'端和3'端臂之间不互补，从而形成环，而不形成环的剩余核苷酸之间100％互补，形成茎。
本文提供的miRancer可以是一种寡核苷酸。术语“寡核苷酸”是指通过共价连接两种以上的核苷酸形成的分子。术语寡核苷酸通常包括寡核苷、寡核苷酸类似物、寡核苷酸模拟物和这些的嵌合组合。当指核苷酸或单体序列时，其可以碱基序列，诸如，例如，A，T(或U)，G或C或其类似物的序列。
本文中“核苷酸”用在本文中是指包含糖结构部分、碱基结构部分和 共价连接的基团(如磷酸或硫代磷酸核苷酸间连接基团)的糖苷，并且包括天然存在的核苷酸(如DNA或RNA)和包含修饰的糖和/或碱基结构部分的非天然存在的核苷酸，其在本文中还称为“核苷酸类似物”。非天然存在的核苷酸包括具有修饰的糖结构部分(如二环核苷酸或2’修饰的核苷酸，如2’取代的核苷酸)的核苷酸。“核苷酸类似物”是天然存在的核苷酸(如DNA或RNA核苷酸)利用在糖和/或碱基结构部分中的修饰产生的变体。类似物可以对所述寡核苷酸没有功能性影响或具有功能性影响。例如，通过产生针对靶标的增加的结合亲和力和/或增加的对细胞内核酸酶的抗性和/或增加的转运到细胞内的容易性。在一些实施方案中，所述miRancer分子包含1个、2个、3个或更多核苷酸类似物。在一些实施方案中，所述寡聚体包含3-8个核苷酸类似物，例如6或7个核苷酸类似物。在一些实施方案中，所述核苷酸类似物包括锁定核酸(LNA)。例如，所述核苷酸类似物中1个、2个、3个或更多核苷酸类似物可以是LNA。
本文定义的修饰的RNA分子可以含有核苷酸类似物/修饰，例如骨架修饰，糖修饰或碱基修饰。骨架修饰可以是核酸分子中包含的核苷酸骨架的磷酸酯被化学。修饰的修饰骨架的磷酸酯基团可以通过用不同取代基替代一个或多个氧原子来修饰，其实例包括例如硫代磷酸酯。糖修饰可以是核酸分子的核苷酸的糖的化学修饰，例如，2'羟基(OH)可以被很多不同"氧基"或"脱氧"取代基修饰或替代。碱基修饰可以是核酸分子的核苷酸的碱基部分的化学修饰。核苷酸类似物或修饰可以选自适于转录和/或翻译的核苷酸类似物。核苷酸类似物/修饰可以包括2-氨基-6-氯嘌呤核苷-5'-三磷酸酯，2'-氨基-2'-脱氧胞苷-三磷酸酯，2'-氟胸苷-5'-三磷酸酯，5-甲基胞苷-5'-三磷酸酯，5-溴胞苷-5'-三磷酸酯，7-去氮杂鸟苷-5'-三磷酸酯，和假尿苷-5'-三磷酸酯等。修饰的核苷可以包括吡啶-4-酮核糖核苷，5-氮杂-尿苷，二氢假尿苷，2-硫代-二氢尿苷，2，6-二氨基嘌呤，N2,N2-二甲基鸟苷，N1-甲基-假尿苷，5,6-二氢尿苷，4-硫代-尿苷，5-羟基-尿苷，脱氧-胸苷，肌苷，α-硫代-鸟苷，6-甲基-鸟苷，5-甲基-胞苷，8-氧代-鸟苷，7-去氮杂-鸟苷，N1-甲基-腺苷，2-氨基-6-氯-嘌呤，假异-胞苷，6-氯-嘌呤等。
本文提供的miRancer分子可以包含或由长度总共为25，26，27，28， 29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50个连续的核苷酸序列组成。在一些实施方案中，所述寡核苷酸包含或由长度总共约为30–45个，诸如约33–40个，诸如约33-37个，诸如约33-35个连续核苷酸序列组成。在一些实施方案中，本发明所述的寡核苷酸由不超过40个核苷酸组成，诸如不超过38个核苷酸，诸如不超过34个核苷酸，诸如33、35或37个核苷酸。
2.组合物
本文提供包含miRancer分子的方法和组合物，所述miRancer当在细胞中表达时能增强microRNA活性。此类方法和组合物包含miRancer表达载体。“miRancer表达载体”是指包含miRancer分子的载体，其具有编码miRancer的多核苷酸序列。miRancer表达载体被设计成从所述载体产生miRancer分子。
“microRNA”或“miRNA”是指寡核糖核酸，一般来讲长度为约18至约25个核苷酸，其调控包含靶序列的多核苷酸的表达。microRNA为非蛋白编码RNA，在动物和植物中已被鉴定。microRNA被初始转录为长多腺苷酸化的RNA，然后被加工形成具有形成稳定发夹能力的较短序列。大多数microRNA基因在RNA聚合酶Ⅱ的作用下合成pri-miRNA。在细胞核内pri-miRNA经Drosha酶剪切，形成约70nt的茎环结构，即pre-miRNA。随后，pre-miRNA在Dicer酶的作用下剪切产生microRNA单链结构，形成成熟microRNA。
3.miRancer表达载体
本文提供miRancer表达载体。miRancer表达载体表达载体包含能被转录为miRancer序列的多核苷酸，所述miRancer序列能增强microRNA活性。
在一些实施方案中，所述miRancer表达载体提供了形成具有类似发 夹RNA结构的miRancer分子。在一些实施方案中，所述miRancer分子可以稳定microRNA分子，从而调节所述microRNA靶向基因的表达活性。所述miRNA可来源于任何动物或植物。
在一些实施方案中，miRancer的5'端臂序列与miRNA序列从第2位开始的连续核苷酸序列可以是100％、至少99％、98％、97％、96％、95％、90％、85％、80％或更低互补的序列。在实施方案中，miRancer的5'端臂序列包含与miRNA序列从第2位开始的连续核苷酸序列具有1、2、3、4、5或更多处错配的序列，并仍具有足够的互补与miRNA序列形成双链结构，生成miRNA结合序列并增强所述microRNA的活性。
由miRancer表达载体miRancer分子，并具有与microRNA足够互补的序列。与microRNA靶序列的“足够互补的序列”是指其互补性足够使得miRancer分子与microRNA结合并增加所述microRNA的活性。在具体的实施方案中，具有与靶序列足够互补的miRancer可与microRNA靶序列共有100％互补的序列或可与靶序列共有小于100％互补的序列(即至少99％、98％、97％、96％、95％、90％、85％、80％、75％、70％或更低互补的序列)。在其它实施方案中，miRancer与靶序列可具有1、2、3、4、5或至多6处错配，只要miRancer与靶序列具有足够的互补以增加靶序列的活性如半衰期。
在一些实施方案中，miRNA序列可具有位于5'端的“U”。任选地，在miRNA的5'端内可加入一对碱基对变化，以使所述序列与靶序列相差一个核苷酸。
“靶序列”是指设计miRancer所靶向的microRNA的序列。靶序列可以是内源性序列，或者可以是导入的异源性序列。
由miRancer表达载体产生的miRancer能够增加microRNA的活性。用于测定microRNA的活性的方法包括测量其靶向的基因/蛋白的表达的改变。在一些实施方案中，单个miRNA可沉默蛋白和/或基因家族中的多个蛋白/基因或整个蛋白和/或基因家族。
“增加”表示相对于野生型生物体中microRNA正常水平(或 microRNA靶向的基因/蛋白水平)的增加。通过“增加miRancer活性”可以是增加表达活性至任一统计意义上的显著性数量，例如相对于野生型表达活性增加至少10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、或100％。
本文所述的miRancer分子可以被递送到动物如哺乳动物包括人或植物细胞中的一种或多种中。
4.编码miRancer表达载体的多核苷酸和制备方法
本文提供分离或重组体多核苷酸，其编码miRancer表达载体、miRancer表达载体的各种组分、连同被加工成miRancer的miRancer表达载体的不同产物。
多核苷酸可以是RNA或DNA的聚合物，它们可以是单链或双链，任选地包含合成的、非天然的或改性的核苷酸碱基。DNA聚合物形式的多核苷酸可由cDNA、基因组DNA、合成DNA或它们的混合物的一个或多个片段构成。多核苷酸可包括核糖核苷酸以及核糖核苷酸和脱氧核糖核苷酸的组合。所述脱氧核苷酸和核糖核苷酸包括天然存在的分子和合成类似物。本发明所述多核苷酸也涵盖所有形式的序列，包括但不限于单链形式、双链形式、发夹结构、茎环结构等。
本文提供的组合物可包含分离或基本上经纯化的多核苷酸。“分离”或“纯化的”多核苷酸，是基本上或本质上不含那些在天然存在环境中正常伴随多核苷酸或与其相互作用的游离组分。因此，分离或经纯化的多核苷酸基本上不含其他细胞物质、或当通过重组体技术生产时的培养基、或基本上不含当通过化学合成时的化学前体或其他化学物质。在一些实施方案中，“分离”多核苷酸不含多核苷酸所来源于的生物的基因组DNA中天然地存在于所述多核苷酸两侧(即，位于所述多核苷酸的5'和3'端)的序列。
还提供了包含miRancer表达载体和其不同组分的重组多核苷酸。重组载体包含人工的或异源的核酸序列的组合例如非天然共存的调控和编码序列。在其它实施方案中，重组载体可包含来源于不同来源的调控序列 和编码序列，或来源于相同来源但以不同于天然存在的方式排列的调控序列和编码序列。所述载体可独自使用或与载体组合使用。如果使用载体，则载体的选择取决于用以转化宿主细胞的方法。例如可以使用质粒载体。为了成功地转化、筛选和繁殖包括本发明任何分离核酸片段的宿主细胞，载体上可以包含的遗传元件。因此为了获得显示期望的表达水平和模式的细胞系，可通过DNA印迹分析、RNA印迹分析、蛋白表达的免疫印迹分析或表型分析等进行筛选。
在具体的实施方案中，本文所述的一个或多个miRancer表达载体可以表达盒形式提供在不同细胞类型中表达。所述盒可包括5'和3'调控序列，其被可操作地与本文提供的多核苷酸连接。在转录的5'-3'方向中，所述表达盒可包括转录和翻译起始区(即启动子)、本文所提供的重组多核苷酸、以及转录和翻译终止区(即终止区)。
许多启动子可用于本文提供的miRancer表达载体。通过在miRancer表达载体中使用不同启动子，可以调节miRancer表达的时间、位置和/或水平。如果需要，miRancer表达载体可含有启动子调节区(例如赋予诱导型、组成型、环境或发育调节的、或细胞或组织特异性/选择性表达的调节区)，转录起始开始位点、核糖体结合位点、RNA加工信号、转录终止位点和/或聚腺苷酸化信号。
5.导入的方法
本文提供的方法包括将miRancer表达载体导入细胞，以提高目标microRNA的活性。本文提供的方法限于特定方法，只要使多核苷酸进入宿主的至少一个细胞的内部即可。将多核苷酸导入宿主细胞中的方法是本领域已知的，包括但不限于病毒介导的方法。导入包括指将核酸整合进真核或原核细胞中，在该细胞中核酸可整合进细胞的基因组内，并且包括指将核酸或蛋白提供给细胞。转化方案以及用于将多核苷酸序列引入植物内的方案可以根据被转化细胞的类型而改变。
6.使用方法
提供了通过将miRancer表达载体导入细胞增加microRNA活性的方法。本文提供的方法包括将miRancer表达载体导入细胞，其中由miRancer表达载体产生的miRancer分子，其能增加microRNA和/或改变(如增加或降低)所述microRNA靶向的基因的活性。在一些实施方案中，可以将miRancer分子整合到基因组中目标microRNA靶向的基因的调节区(包括例如5'UTR和/或3'UTR和/或基因上游、下游、内含子等区域)以调节基因的表达。在一些实施方案中，本文提供的miRancer分子能够增加microRNA靶向的基因的基因表达，这是非常令人吃惊的，因为通常认为microRNA的作用是降低基因的表达。在一些实施方案中，本文提供的miRancer分子能够改变目标microRNA的作用(例如，目标microRNA降低一些基因的表达，然后，通过将本文提供的miRancer分子或载体引入到所述基因的调节区，使得目标microRNA增加基因的表达)。在一些实施方案中，可以通过针对期望调节的目的基因的相应的microRNA涉及相应的miRancer分子，从而调节期望改变其表达的目的基因的表达。
7.活性变体
本文还提供在组合物和方法中应用的多核苷酸的活性变体。“变体”是指基本上类似的序列。就多核苷酸而言，变体包括在多核苷酸的一个或多个内部位点有一个或多个核苷酸的缺失和/或插入，和/或在多核苷酸的一个或多个位点有一个或多个核苷酸的置换。变体多核苷酸可包括合成来源的多核苷酸，诸如那些例如通过定点诱变生成的。一般来讲，本文所公开的miRancer表达载体、单链RNA分子、miRancer分子，与原型多核苷酸具有至少约60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同一性。用于比对的序列比对方法是本领域熟知的。比对序列、测定序列同一性的程序包括CLUSTAL、GAP、BESTFIT、BLAST、FASTA和TFASTA等。可以使用默认参数进行使用这些程序的比对。
8.目标microRNA分子
在一些实施方案中，本文的miRancer能够作用的目标microRNA不受特定限制。例如，可以从现有数据库中选择希望被调节的目标microRNA分子。在一些实施方案中，可以根据希望调节的基因选择所对应的microRNA分子。另外，可以通过生物信息学方法(例如应用miRanda、TargetScan、RNAhybrid等程序)寻找microRNA所靶向的基因。
本文的miRancer分子可通过与目标microRNA分子结合提高所述microRNA的活性。在一些实施方案中，当与目标microRNA分子杂交时，所述寡核苷酸可以耐受1，2，3或4个(或更多个)错配，并且仍然与所述靶标充分结合，以显示出需要的作用(例如，提高所述microRNA的活性)。例如，错配可以由所述核苷酸序列内存在的增加长度的寡核苷酸序列和/或增加数量的核苷酸类似物(如锁定核酸(LNA))而得到补偿。在一些实施方案中，当与目标microRNA分子杂交时，所述连续的核苷酸序列包含不超过3个错配(例如，不超过1个或不超过2个错配)。在一些实施方案中，当与目标microRNA分子杂交时，所述连续的核苷酸序列包含不超过一个错配。
本发明的miRancer分子优选地与microRNA分子在互补区至少80％互补，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，诸如至少100％互补。
在一些实施方案中，所述miRancer可以包含另外的5’或3’核苷酸或修饰，诸如，独立地1，2，3，4或5个另外的核苷酸5’和/或3’，其与靶序列不互补。在这一方面，在一些实施方案中，本发明的寡核苷酸可以包含在5’和或3’侧连另外的核苷酸的连续的核苷酸序列。在一些实施方案中，所述另外的5’或3’核苷酸是天然存在的核苷酸，诸如DNA或RNA。在一些实施方案中，所述另外的5’或3’核苷酸可以是核苷酸类似物。互补区可以包括microRNA分子的第2-12、2-13位，2-14、2-15、2-16、2-17、 2-18位。
9.药物组合物
本发明的miRancer分子、载体、细胞可以用在药物制剂和组合物中，也可制备成方便应用的试剂盒。适当地，所述组合物或试剂盒包含药用溶剂，诸如水或盐水，稀释剂，载体，盐或辅药。
本发明还包括含有本发明的寡核苷酸的药物组合物和制剂。本发明的药物组合物可以以多种方式施用，取决于是需要局部治疗还是系统治疗，以及取决于待治疗的区域。
10.应用
本发明的寡核苷酸可以用作研究试剂，例如，用于诊断、治疗和预防。在研究中，所述寡核苷酸可以用于特异性结合目标microRNA，可以增加其活性，由此促进对靶标的功能性分析或对其作为治疗干预的靶标的评估。
在诊断剂中，所述寡核苷酸可以用于通过RNA印迹、原位杂交或相似的技术来检测并定量细胞中组织中的目标microRNA水平。
对于治疗剂，怀疑患有可以通过调节目标microRNA靶向的基因的表达进行治疗的疾病或病症的动物或人可以通过施用本发明所述的寡核苷酸进行治疗。进一步提供治疗怀疑患有或倾向于患有与目标microRNA靶向的基因的表达相关的疾病或病症的哺乳动物(如治疗人)的方法，所述通过通过施用治疗或预防有效量的一种或多种本发明的寡核苷酸或组合物。本发明所述的寡核苷酸或药物组合物典型地以有效量施用。
本发明还提供用于治疗疾病如肿瘤的方法，所述方法包括给有此需要的患者施用本文所述的寡核苷酸分子或包含所述分子的药物组合物。
11.实验结果
试剂和材料
以下实验中用到的细胞系为MCF-7人乳腺癌细胞系,购于ATCC,培养基为RPMI-1640,购于Gibco,胎牛血清FBS购于Gibco。转染试剂为lipofectamine 2000,购于invitrogen.质粒pSilencer 4.1CMV购于Ambion公司，质粒psiCheck2及双荧光素酶检测试剂盒购于promega公司。引物合成由上海生工完成，mimics合成由上海吉玛完成。
重组质粒克隆方法为常规方法，测序由上海生工完成，细节在下文中阐述。
双荧光素酶报告实验按照试剂盒(Promega E1910)说明书进行。
实验过程
1.设计有高级结构的microRNA结合序列。
首先，microRNA的结合序列被设计为有类似发卡结构的单链RNA，发卡结构5'端臂通过碱基互补配对与microRNA结合，而3'端臂则通过碱基互补配对与5’端臂结合。为了防止Dicer1的加工和类似siRNA效应发发生，优选的双链的长度在18个核苷酸以内。
然后，在发卡结构的拐角处分别引入7nt或13nt的环结构，用以确定不同结构可能带来的潜在功能。
再后，在发卡环结构的中部插入两对GC以稳定RNA二级结构。
最后，也设计了一个线性的microRNA结合序列作为阴性对照。
这样，就得到了8种不同形式的示例性microRNA结合序列(以miR-7和miR-9为例)。图1显示了miR-7结合序列的不同形式；图2显示了miR-9结合序列的不同形式。
2.制备表达载体。
选用pSilencer 4.1CMV质粒作为小发卡RNA的表达载体。质粒经过 BamHI和HindIII双酶切后，用1％的琼脂糖胶分离，然后用Axyprep DNA extraction kit(AP-GX-50)回收，存放于-20℃。
3.制备插入序列。
合成了如下DNA序列：
miR-7结合序列A正向:
GATCCAAAATCACTAGTCTTCCAGGAAGACTAGTGATTTTA
miR-7结合序列A反向:
AGCTTAAAATCACTAGTCTTCCTGGAAGACTAGTGATTTTG
miR-7结合序列B正向:
GATCCAAAATCACTAGTCTTCCACCTAGACTAGTGATTTA
miR-7结合序列B反向:
AGCTTAAAATCACTAGTCTAGGTGGAAGACTAGTGATTTTG
miR-7结合序列C正向:
GATCCAAAATCACTAGTCTTCCACCTTCTCTAGTGATTTTA
miR-7结合序列C反向:
AGCTTAAAATCACTAGAGAAGGTGGAAGACTAGTGATTTTG
miR-7结合序列D正向:
GATCCAAAATCACTAGTCTTCCAA
miR-7结合序列D反向:
AGCTTTGGAAGACTAGTGATTTTG
miR-7结合序列E正向:
GATCCAATCACTACCGTCTTCCAGGAAGACGGTAGTGATTA
miR-7结合序列E反向:
AGCTTAATCACTACCGTCTTCCTGGAAGACGGTAGTGATTG
miR-7结合序列F正向:
GATCCAATCACTACCGTCTTCCACCTAGACGGTAGTGATTA
miR-7结合序列F反向:
AGCTTAATCACTACCGTCTAGGTGGAAGACGGTAGTGATTG
miR-7结合序列G正向:
GATCCAATCACTACCGTCTTCCACCTTCTCGGTAGTGATTA
miR-7结合序列G反向:
AGCTTAATCACTACCGAGAAGGTGGAAGACGGTAGTGATTG
miR-7结合序列H正向:
GATCCAATCACTACCGTCTTCCAA
miR-7结合序列H反向:
AGCTTTGGAAGACGGTAGTGATTG
miR-9结合序列A正向:
GATCCCAGCTAGATAACCAAAGACTTTGGTTATCTAGCTGA
miR-9结合序列A反向:
AGCTTCAGCTAGATAACCAAAGTCTTTGGTTATCTAGCTGG
miR-9结合序列B正向:
GATCCCAGCTAGATAACCAAAGAGAATGGTTATCTAGCTGA
miR-9结合序列B反向:
AGCTTCAGCTAGATAACCATTCTCTTTGGTTATCTAGCTGG
miR-9结合序列C正向:
GATCCCAGCTAGATAACCAAAGAGAAACCTTATCTAGCTGA
miR-9结合序列C反向:
AGCTTCAGCTAGATAAGGTTTCTCTTTGGTTATCTAGCTGG
miR-9结合序列D正向:
GATCCCAGCTAGATAACCAAAGAA
miR-9结合序列D反向:
AGCTTTCTTTGGTTATCTAGCTGG
miR-9结合序列E正向:
GATCCGCTAGATACCACCAAAGACTTTGGTGGTATCTAGCA
miR-9结合序列E反向:
AGCTTGCTAGATACCACCAAAGTCTTTGGTGGTATCTAGCG
miR-9结合序列F正向:
GATCCGCTAGATACCACCAAAGAGAATGGTGGTATCTAGCA
miR-9结合序列F反向:
AGCTTGCTAGATACCACCATTCTCTTTGGTGGTATCTAGCG
miR-9结合序列G正向:
GATCCGCTAGATACCACCAAAGAGAAACCTGGTATCTAGCA
miR-9结合序列G反向:
AGCTTGCTAGATACCAGGTTTCTCTTTGGTGGTATCTAGCG
miR-9结合序列H正向:
GATCCGCTAGATACCACCAAAGAA
miR-9结合序列H反向:
AGCTTTCTTTGGTGGTATCTAGCG
这些DNA序列两两成对在1×TE buffer中按如下程序退火：
95℃ 2min
touch down at 0.1℃every 8s
4℃  30min
退火后的DNA序列连接入pSilencer 4.1CMV质粒的BamHI和HindIII之间，使用的试剂盒是TAKARA ligation kit(D6022)。重组质粒通过测序确认序列正确。
4.制备microRNA报告载体。
在psi-CHECK2质粒的XhoI和NotI之间插入microRNA的结合位点以获得microRNA报告载体。图3显示了miR-7 reporter和miR-9 reporter的示意图。
5.荧光素酶报告实验.
向MCF-7细胞中共转染了miR-7结合序列和miR-7 reporter,然后按照说明做了双荧光素酶报告实验(Promega E1910)。图4显示了过表达miRancers后的miR-7 reporter或miR-9 reporter的荧光素酶活性。*,p<0.05,**,p<0.01 by Student's t-test.
选择F型作为优选的可以增强microRNA活性的microRNA结合序列。将所有的可以增强microRNA活性的序列称为microRNA增强子，简称 miRancers.
6.优化miRancer结构
以F型为原型，继续摸索合适的miRancer结构，通过缩短或延长miRancer的Stem区结构，得到了含有15bp(miR-7结合序列F1)或13bp(miR-7结合序列F2)的发卡结构，结果显示，二者均仍然可以起作用。图5显示不同茎长的miR-7结合序列示意图。图6显示过表达不同茎长的miRancers后miR-7 reporter的荧光素酶活性**，p<0.01 by Student's t-test。
7.化学合成miRancers.
进而根据F型用化学方法合成了miRancer(上海吉玛)。针对果蝇中的bantam miRNA设计的miRancer作为对照。发明人发现化学合成的miRancers也可以特异地影响microRNA的活性和水平。图7显示过表达miRancer后miR-7 reporter或miR-9 reporter的荧光素酶活性。**,p<0.01by Student's t-test.图8显示过表达miRancer后miR-7或miR-9的表达水平。***，p<0.001 by Student's t test。
8.miRancer motif增强基因表达.
在luciferase的上游分别插入miRancer-7,miR-7 sponge,miRancer-9,miR-9 sponge，然后分别与miR-7 mimics,miR-9 mimics(上海吉玛)共转染，发现含有miRancer的luciferase可以被相应的miRNA稳定，而含有sponge的luciferase则被相应的miRNA抑制。这说明miRancer可以用来增强基因的表达。图9显示含有miRancer或sponge序列的荧光素酶报告载体示意图。图10显示过表达miR-7或miR-9对不同荧光素酶活性的影响。*，p<0.05,**,p<0.01,***,p>0.001 by Student’s t test。

Claims (10)

  1. 一种结合目标microRNA的单链RNA序列，所述单链RNA序列具有5'端臂和3'端臂，所述5'端臂通过碱基互补配对与目标microRNA结合，所述3'端臂通过碱基互补配对与所述5'端臂结合，
    所述单链RNA序列从5'端到3'端具有以下通式1：
    (n5-11)(n0-3)(n6-7)(n1)(n11-21)(n0-2)，
    其中n为连续的核苷酸或其类似物，其后数字为核苷酸或其类似物的个数，其中n1与目标microRNA的5'端第一位的核苷酸互补，相应的n6-7对应于目标microRNA从5'端到3'端的第2-7位或第2-8位，从而(n5-11)(n0-3)(n6-7)为5'端臂，(n11-21)(n0-2)为3'端臂，其中n0-3为插入的C或G以在所述5'端臂和3'端臂之间形成GC配对。
  2. 权利要求1所述的单链RNA序列，其中除了插入的(n0-3)作为缺口不计算同一性之外，所述5'端臂与所述目标microRNA从5'端到3'端的第2-18位核苷酸的互补链至少50％、60％、70％、80％、90％、或100％的同一性，优选其中(n6-7)与所述目标microRNA从5'端到3'端的第2-7位或第2-8位核苷酸的互补链具有至少80％、90％、或100％的同一性，优选(n11-21)与(n5-11)(n0-3)(n6-7)中不配对核苷酸的个数小于6、5、4、3、2、或1对，优选全部不配对的核苷酸在(n1)周围。
  3. 一种包含权利要求1或2所述单链RNA序列的载体，如质粒。
  4. 一种包含编码权利要求1或2所述单链RNA序列的多核苷酸序列的载体，如质粒。
  5. 一种包含权利要求1或2所述单链RNA序列的合成的RNA分子。
  6. 一种包含权利要求3或4的载体的细胞。
  7. 权利要求1或2的单链RNA序列、权利要求3或4的载体、或权利要求5的合成的RNA分子的用途，所述用途包括用于特异性增加内源性和/或外源性目标microRNA或其它小RNA的表达和/或活性，用于体外和/或体内特异性调节所述目标microRNA靶向的基因的表达，用作感受器用于感测所述目标microRNA或其它小RNA，和用于通过感测所述目标 microRNA或其它小RNA从而调节所述所述目标microRNA或其它小RNA靶向的基因的表达。
  8. 一种方法，所述方法包括用于特异性增加内源性和/或外源性目标microRNA或其它小RNA的表达和/或活性的方法，用于体外和/或体内特异性调节所述目标microRNA靶向的基因的表达的方法，用作感受器用于感测所述目标microRNA或其它小RNA的方法，和用于通过感测所述目标microRNA或其它小RNA从而调节所述所述目标microRNA或其它小RNA靶向的基因的表达方法，所述方法包括将权利要求1或2的单链RNA序列、权利要求3或4的载体、或权利要求5的合成的RNA分子与包含目标microRNA或其它小RNA的样品接触的步骤。
  9. 一种产生结合目标microRNA的单链RNA序列的方法，所述方法包括产生权利要求1或2所述的结合目标microRNA的单链RNA序列。
  10. 一种组合物或试剂盒，其包含权利要求1或2的单链RNA序列、权利要求3或4的载体、和/或权利要求5的合成的RNA分子，优选的所述组合物或试剂盒用于权利要求7所述的用途或权利要求8所述的方法。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101437942A (zh) * 2006-04-03 2009-05-20 桑塔里斯制药公司 包含抗微小rna反义寡核苷酸的药物组合物
CN102264898A (zh) * 2008-10-23 2011-11-30 国立大学法人东京大学 微小rna的功能抑制方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101437942A (zh) * 2006-04-03 2009-05-20 桑塔里斯制药公司 包含抗微小rna反义寡核苷酸的药物组合物
CN102264898A (zh) * 2008-10-23 2011-11-30 国立大学法人东京大学 微小rna的功能抑制方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BOUTLA, A. ET AL.: "Developmental defects by antisense-mediated inactivation of micro-RNAs 2 and 13 in Drosophila and the identification of putative target genes", NUCLEIC ACIDS RESEARCH, vol. 31, no. 17, 1 September 2003 (2003-09-01), pages 4973 - 4980, XP002993014, ISSN: 0305-1048 *

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