WO2020014948A1 - 核酸单元及其聚合核酸与应用 - Google Patents

核酸单元及其聚合核酸与应用 Download PDF

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WO2020014948A1
WO2020014948A1 PCT/CN2018/096397 CN2018096397W WO2020014948A1 WO 2020014948 A1 WO2020014948 A1 WO 2020014948A1 CN 2018096397 W CN2018096397 W CN 2018096397W WO 2020014948 A1 WO2020014948 A1 WO 2020014948A1
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sequence
nucleic acid
acid molecule
polymerized
stranded rna
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PCT/CN2018/096397
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English (en)
French (fr)
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张必良
杨秀群
萨玛斯基·丹米其
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广州市锐博生物科技有限公司
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Priority to AU2018433363A priority Critical patent/AU2018433363B2/en
Priority to CN201880088412.1A priority patent/CN111712508B/zh
Priority to JP2020572590A priority patent/JP7213897B2/ja
Priority to US17/042,946 priority patent/US20210253623A1/en
Priority to EP18926927.7A priority patent/EP3825321A4/en
Priority to PCT/CN2018/096397 priority patent/WO2020014948A1/zh
Publication of WO2020014948A1 publication Critical patent/WO2020014948A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/51—Physical structure in polymeric form, e.g. multimers, concatemers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/52—Physical structure branched

Definitions

  • the invention belongs to the field of nucleic acid technology, and particularly relates to a nucleic acid unit with a novel structure and a self-assembling polymerized nucleic acid, which can interfere with the expression of one or more target genes.
  • CON Complementary Oligonucleotide
  • CON technology includes RNAi (RNA interference) technology and ASO (antisense oligonucleotide) technology. It has been widely used in functional genomics research and is expected to become the third largest treatment besides small molecule compounds and biological agents. means.
  • mipomersen which is used to treat homozygous familial hypercholesterolemia (FoFH), is a synthetic phosphorothioate oligonucleotide developed by Genzyme.
  • the coding regions of 100 protein mRNAs are complementary paired, inhibiting the translation and synthesis of Apo B-100 protein (the main apolipoprotein of LDL and VLDL), thereby effectively reducing the levels of LDL-C, TC, Non-HDL-C in FoFH patients.
  • Apo B-100 protein the main apolipoprotein of LDL and VLDL
  • CON technology has achieved some success, this technology still needs improvement.
  • RNAi reagents have the following disadvantages: 1) tedious synthesis steps and relatively high production costs; 2) highly sensitive to endonucleases and exonucleases, and low stability; 3) the efficiency of inhibition is not high enough to prevent It is guaranteed that a single molecule can inhibit the target gene expression; 4) side effects caused by non-specific activities, which are mainly derived from the sense chain; 5) difficult to introduce into cells, especially animals.
  • the technical problem to be solved by the present invention is how to inhibit or reduce or interfere with the expression of a target gene.
  • the present invention first provides a polymerized nucleic acid molecule for inhibiting or reducing or interfering with the expression of a target gene.
  • the polymerized nucleic acid molecule for inhibiting or interfering the expression of a target gene provided by the present invention consists of n X-type nucleic acid molecules;
  • Each X-type nucleic acid molecule is composed of a targeting fragment 1, a targeting fragment two, and a linker fragment three in sequence from the 5 'end.
  • a target fragment of each of the X-type nucleic acid molecules is three complementary pairs with a linker fragment of an adjacent X-type nucleic acid molecule.
  • n X-type nucleic acid molecules are named X1 unit, X2 unit, X3 unit, and so on, Xn-1 unit, Xn unit; the linker fragment three of the X1 unit and the targeting fragment of the X2 unit A complementary pairing (completely complementary), the linker fragment three of the X2 unit and the targeting fragment of the X3 unit are a complementary pairing (complete complementarity), and so on
  • the target fragment of the unit is complementary paired (fully complementary), and the linker fragment three of the Xn unit is complementary paired (fully complementary) to the target fragment of the X1 unit.
  • the targeting fragments of each of the X-type nucleic acid molecules are not complementary to other X-type nucleic acid molecule sequences.
  • Targeting fragment one and targeting fragment two of each of the polymerized nucleic acid molecules are complementary paired with the target gene; each of the polymerized nucleic acid molecules is combined with a specific sequence of the target gene through targeting fragment one and targeting fragment two to achieve Inhibit or reduce or interfere with the expression of target genes.
  • the region complementary to the target gene sequence can be extended to a partial sequence of the linker fragment three.
  • Targeting fragment one and targeting fragment two in the same X-type nucleic acid molecule may be the same or different.
  • the targeting fragment one or the targeting fragment two in two different X-type nucleic acid molecules may be the same or different.
  • Each of the X-type nucleic acid molecules has the same length (and the same structure).
  • the n is an integer greater than or equal to 3.
  • the n X-type nucleic acid molecules are sequentially connected end-to-end (through the complementary fragment of the former X-type nucleic acid molecule three and the target fragment of the next adjacent X-type nucleic acid molecule to be complementary) ) To finally form a polymerized nucleic acid molecule with a circular secondary structure.
  • the n X-type nucleic acid molecules may further form a polymerized nucleic acid molecule having a linear structure, and the polymerized nucleic acid molecule having a linear structure further includes an H-type nucleic acid molecule; the H-type nucleic acid molecule is of H1 type Nucleic acid molecules and Hn-type nucleic acid molecules.
  • X-type nucleic acid molecules are respectively named X1 unit, X2 unit, X3 unit, and so on, Xn-1 unit, Xn unit;
  • the linker fragment three of the X1 unit is complementary paired with the target fragment of the X2 unit
  • the linker fragment three of the X2 unit and the target fragment of the X3 unit are complementary paired, and so on, and the analogy of the linker fragment three of the Xn-1 unit and the target fragment of the Xn unit are complementary paired;
  • the H1 type nucleic acid molecule is complementary paired with the targeting fragment of the X1 unit;
  • the Hn type nucleic acid molecule is triple complementary paired with the linker fragment of the Xn unit.
  • the 5 'end or 3' end of the H-type nucleic acid molecule further includes a Hy fragment; the H-type nucleic acid molecule is composed of an Hx fragment and a Hy fragment.
  • the complementary region of the H1 type nucleic acid molecule and the X1 unit may extend to part or all of the sequence of the target fragment two of the X1 unit; the complementary region of the Hn type nucleic acid molecule and the Xn unit may extend to Said part or all of the target fragment two of the Xn unit.
  • the polymerized nucleic acid molecule having a circular structure also includes a C-type nucleic acid molecule; the C-type nucleic acid molecule is formed by sequentially connecting n fragments, and the n fragments are each of the X type nucleic acid molecules. Targeting fragment II of the reverse complement.
  • the C-type nucleic acid molecule is composed of a cap-end fragment 1, a cap-end fragment 2, a cap-end fragment 3, and so on, starting from the 3 ′ end, and so on, and a cap-end fragment Cn-1 and a cap-end fragment n;
  • X-type nucleic acid molecules are named X1 unit, X2 unit, X3 unit, and so on, Xn-1 unit, Xn unit;
  • the linker fragment three of the X1 unit is complementary paired with the target fragment of the X2 unit, the linker fragment three of the X2 unit is complementary paired with the target fragment of the X3 unit, and so on, and Xn-1
  • the linker fragment three of the unit is complementary paired with the targeting fragment one of the Xn unit, the linker fragment three of the Xn unit is complementary pairing with the targeting fragment one of the X1 unit; the cap-end fragment 1 and the X1 unit
  • the target segment two is complementary paired, the cap-end fragment 2 is complementary paired with the target fragment two of the X2 unit, and so on, the cap-end fragment Cn-1 and the target fragment two of the Xn-1 unit are complementary.
  • Complementary pairing, the cap-end fragment n and the targeting fragment of the Xn unit are two complementary pairs.
  • the nucleic acid molecule (X-type nucleic acid molecule, H-type nucleic acid molecule, or C-type nucleic acid molecule) may be DNA or RNA or an oligonucleotide composed of DNA and RNA.
  • nucleic acid molecule (X-type nucleic acid molecule, H-type nucleic acid molecule or C-type nucleic acid molecule) is a single-stranded RNA molecule.
  • the length of the X-type nucleic acid molecule is 15-50 nt, preferably 24-36 nt;
  • the length of the targeting fragment 1 is 5-24 nt;
  • the length of the second targeting fragment is 1-20 nt;
  • the length of the linker segment three is 5-24 nt;
  • the total length of the targeting fragment one and the targeting fragment two is at least 14-16 nt;
  • the length of the Hy fragment is 2-6 nt or longer.
  • the length of the X-type nucleic acid molecule is 24-36 nt.
  • the polymerized nucleic acid molecule includes at least one modified nucleotide.
  • the modification is a phosphate backbone modification, a base modification, and / or a ribose modification.
  • the ribose is modified such that the hydroxyl group at the 2-position of the ribose is substituted with a halogen group or an O-alkyl group.
  • the alkyl group is methyl, ethyl, propyl or methylethyl.
  • the linker fragment of the X-type nucleic acid molecule has three consecutive 5-9 nucleotide ribose groups from the first nucleotide at the 3 ′ end of the hydroxyl group at the 2-position to a halogen group or an O-alkyl group.
  • the Hx fragment of the H-type nucleic acid molecule is substituted by a halogen group or an O-alkyl group with a hydroxyl group at the 2 position of 5-9 nucleotides consecutively from the first nucleotide at the 3 'end;
  • the H-type nucleic acid molecule has a continuous 8-30 nucleotide ribose 2 hydroxyl group from the first nucleotide at the 3 ′ end to a halogen group or an O-alkyl group; preferably, all The H-type nucleic acid molecule has a continuous 14-18 nucleotide ribose 2 hydroxyl group from the first nucleotide at the 3 ′ end of the H-type nucleic acid molecule to be replaced by a halogen group or an O-alkyl group;
  • Each cap-end fragment of the C-type nucleic acid molecule has a continuous 2-6 nucleotide ribose group at the 5 ′ end from the first nucleotide at the 5 ′ end of the hydroxyl group at the 2-position by a halogen group or an O-alkyl group. To replace.
  • n is 3 or 4 or 5 or 6 or 7 or 8.
  • n 4 or 5 or 6.
  • the number of the target genes is one or two or more, and the number of the target genes does not exceed n; each X-type nucleic acid molecule corresponds to one target gene, or a plurality of X Type nucleic acid molecules correspond to different regions of the same target gene.
  • the number of the target genes is one or four or six.
  • the target gene is at least one of the following genes: PPIB gene, p65 gene, BIRC5 gene, CTNNB gene, COPS5 gene, CLU gene, EIF4E gene, HIF1A gene, TP53 gene, VEGFA gene, and SOD1 gene.
  • polymerized nucleic acid molecules used to interfere with the expression of the target gene PPIB are the following a1) -a5):
  • a1) consists of a single-stranded RNA molecule shown in sequence 1, sequence 2, sequence 3 and sequence 4;
  • a2) consists of single-stranded RNA molecules shown in sequence 1, sequence 2, sequence 3, sequence 4, sequence 20 and sequence 21;
  • a3) consists of a single-stranded RNA molecule shown in sequence 1, sequence 2, sequence 3, sequence 4 and sequence 8;
  • a4) consists of single-stranded RNA molecules shown in sequence 2, sequence 3, sequence 9, sequence 10, sequence 11 and sequence 12;
  • a5) consists of the single-stranded RNA molecule shown in sequence 2, sequence 3, sequence 6, sequence 9, sequence 10, sequence 11, sequence 13 and sequence 14;
  • the polymerized nucleic acid molecules used to interfere with the expression of the target gene P65 are as follows b1) -b5):
  • b1) consists of a single-stranded RNA molecule shown in sequence 15, sequence 16, 17 and 18;
  • b2) consists of single-stranded RNA molecules shown in sequence 6, sequence 15, sequence 16, sequence 17, 17 and 19;
  • b3) consists of single-stranded RNA molecules shown in sequence 15, sequence 16, sequence 18, sequence 18 and sequence 22;
  • b4) consists of a single-stranded RNA molecule shown in sequence 15, sequence 16, sequence 23, sequence 24, and sequence 25;
  • b5) consists of the shown single-stranded RNA molecules shown in sequence 15, sequence 16, sequence 20, sequence 23, sequence 24, sequence 26 and sequence 27;
  • the polymerized nucleic acid molecule used to simultaneously interfere with the expression of the target genes BIRC5, CTNNB, COPS5 and CLU is composed of single-stranded RNA molecules shown in sequence 28, sequence 29, sequence 30 and sequence 31;
  • the polymerized nucleic acid molecule used to simultaneously interfere with the expression of the target genes BIRC5, CTNNB, COPS5, CLU, EIF4E, and HIF1A is composed of single-stranded RNA molecules shown in sequence 28, sequence 29, sequence 30, sequence 32, sequence 33, and sequence 34;
  • the polymerized nucleic acid molecules used to simultaneously interfere with the expression of the target genes SOD1, PPIB, P65 and VEGFA are as follows c1) -c3):
  • c1) consists of a single-stranded RNA molecule shown in sequence 35, sequence 36, sequence 37 and sequence 38;
  • c2) consists of a single-stranded RNA molecule represented by sequence 35, sequence 36, sequence 37, sequence 38, sequence 39 and sequence 40;
  • c3) consists of a single-stranded RNA molecule represented by sequence 35, sequence 36, sequence 37, sequence 38 and sequence 41;
  • the polymerized nucleic acid molecules used to interfere with the expression of the target gene VEGFA are the following d1) -d11):
  • d1) consists of a single-stranded RNA molecule represented by sequence 42, sequence 43, sequence 44, sequence 45, sequence 46 and sequence 47;
  • d2) consists of a single-stranded RNA molecule represented by sequence 48, sequence 49, sequence 50, sequence 51, sequence 52 and sequence 53;
  • d3) consists of a single-stranded RNA molecule represented by sequence 54, sequence 55, sequence 56, sequence 57, sequence 58 and sequence 59;
  • d4) consists of a single-stranded RNA molecule represented by sequence 42, sequence 43, sequence 45, sequence 46 and sequence 47;
  • d5) consists of a single-stranded RNA molecule represented by sequence 48, sequence 49, sequence 51, sequence 52 and sequence 53;
  • d6 consists of a single-stranded RNA molecule represented by sequence 54, sequence 55, sequence 57, sequence 58 and sequence 59;
  • d7) consists of a single-stranded RNA molecule represented by sequence 42, sequence 45, sequence 46 and sequence 47;
  • d8) consists of a single-stranded RNA molecule represented by sequence 48, sequence 51, sequence 52 and sequence 53;
  • d9) consists of a single-stranded RNA molecule represented by sequence 54, sequence 57, sequence 58 and sequence 59;
  • d10) consists of a single-stranded RNA molecule represented by sequence 60, sequence 61, sequence 62, sequence 63, sequence 64, and sequence 65;
  • d11 consists of a single-stranded RNA molecule represented by sequence 66, sequence 67, sequence 68, sequence 69, sequence 70, and sequence 71;
  • the polymerized nucleic acid molecules used to interfere with the expression of the target gene TP53 are e1) -e11) as follows:
  • e1 consists of a single-stranded RNA molecule represented by sequence 72, sequence 73, sequence 74, sequence 75, sequence 76 and sequence 77;
  • e2 consists of a single-stranded RNA molecule represented by sequence 78, sequence 79, sequence 80, sequence 81, sequence 82, and sequence 83;
  • e3 consists of a single-stranded RNA molecule represented by sequence 84, sequence 85, sequence 86, sequence 87, sequence 88, and sequence 89;
  • e4 consists of a single-stranded RNA molecule represented by sequence 73, sequence 74, sequence 75, sequence 76 and sequence 77;
  • e5 consists of a single-stranded RNA molecule represented by sequence 79, sequence 80, sequence 81, sequence 82, and sequence 83;
  • e6 consists of a single-stranded RNA molecule represented by sequence 54, sequence 55, sequence 57, sequence 58 and sequence 59;
  • e7 consists of a single-stranded RNA molecule represented by sequence 74, sequence 75, sequence 76 and sequence 77;
  • e8 consists of a single-stranded RNA molecule represented by sequence 80, sequence 81, sequence 82, and sequence 83;
  • e9 consists of a single-stranded RNA molecule represented by sequence 86, sequence 87, sequence 88, and sequence 89;
  • e10) consists of a single-stranded RNA molecule represented by sequence 90, sequence 91, sequence 92, sequence 93, sequence 94, and sequence 95;
  • e11 consists of a single-stranded RNA molecule represented by sequence 96, sequence 97, sequence 98, sequence 99, sequence 100, and sequence 101.
  • the present invention further provides a derivative of the aforementioned polymerized nucleic acid molecule.
  • the derivative of the polymerized nucleic acid molecule provided by the present invention is any one of the following (m1)-(m5):
  • a signal molecule and / or an active molecule and / or a functional group are indirectly attached to one end or the middle of the polymer nucleic acid molecule to obtain a derivative of the polymer nucleic acid molecule having the same function as the polymer nucleic acid molecule.
  • the present invention also provides a method for preparing the aforementioned polymerized nucleic acid molecule.
  • the method for preparing the polymerized nucleic acid molecule provided by the present invention includes the following steps:
  • M1 synthesizing the X-type nucleic acid molecule and / or H-type nucleic acid molecule and / or C-type nucleic acid molecule;
  • the annealing reaction system is a system obtained by mixing an equimolar amount of each single-stranded RNA molecule, an RNA annealing buffer, and water.
  • the annealing reaction condition is 90 ° C in the PCR instrument, which is sufficiently denatured in 2 minutes, and then the temperature is lowered to 25 ° C in the PCR instrument to anneal.
  • the annealing system is as follows (total volume is 100 ⁇ L): 20 ⁇ L of each single-stranded RNA molecule solution (concentration 100 ⁇ M), 15 ⁇ L of RNA annealing buffer (5X), and 5 ⁇ L of DEPC water.
  • the annealing system is as follows (total volume is 150 ⁇ L): 20 ⁇ L of each single-stranded RNA molecule solution (concentration 150 ⁇ M), 30 ⁇ L of RNA annealing buffer (5X), and 20 ⁇ L of DEPC water.
  • the annealing system is as follows (total volume is 200 ⁇ L): 20 ⁇ L of each single-stranded RNA molecule solution (concentration 200 ⁇ M), 40 ⁇ L of RNA annealing buffer (5X), and 40 ⁇ L of DEPC water.
  • the present invention also provides new uses of the above-mentioned polymerized nucleic acid molecules or derivatives.
  • the present invention provides the application of the aforementioned polymerized nucleic acid molecule or derivative in the following A1) or A2);
  • A1 regulate the target gene expression level in the cell
  • the regulation is inhibition or reduction or interference.
  • the cells are tumor cells.
  • the target gene is a disease-related gene; the disease-related gene is specifically a tumor-related gene; the tumor-related gene is at least one of the following genes: PPIB gene, p65 gene, BIRC5 gene, CTNNB gene, COPS5 gene, CLU gene, EIF4E gene, HIF1A gene, TP53 gene, VEGFA gene and SOD1 gene.
  • the present invention also provides a reagent or a kit or a medicament for inhibiting or reducing or interfering with the expression level of a target gene in a cell.
  • the reagent or kit or medicament provided by the present invention for inhibiting or reducing or interfering the expression level of a target gene in a cell includes the aforementioned polymerized nucleic acid molecule or the aforementioned derivative.
  • the present invention finally provides a method for inhibiting or reducing or interfering the expression level of a target gene in a cell.
  • the method provided by the present invention for inhibiting or reducing or interfering the expression level of a target gene in a cell includes the steps of: introducing the above-mentioned polymerized nucleic acid molecule or derivative into the cell to achieve inhibition or reduction of the target gene expression level in the cell.
  • the introduction method is to mix the polymerized nucleic acid molecule, transfection reagent and buffer solution and add it to the cell culture medium to obtain a reaction system; the final concentration of the polymerized nucleic acid molecule in the reaction system It is 1-300nM.
  • the cells are tumor cells.
  • the target gene is a disease-related gene; the disease-related gene is specifically a tumor-related gene; the tumor-related gene is at least one of the following genes: PPIB gene, p65 gene, BIRC5 gene, CTNNB gene, COPS5 gene, CLU gene, EIF4E gene, HIF1A gene, TP53 gene, VEGFA gene and SOD1 gene.
  • RNAi Some structures of polymerized nucleic acids do not require the participation of Dicer enzymes to play the role of RNAi, so they are more resistant to chemical modifications and can be partially or completely modified.
  • the present invention combines CON technology with nanotechnology, and realizes a multi-target interference effect by constructing a nucleic acid structure that can be accurately designed and has self-assembly ability, and can be used to inhibit the expression of multiple genes in the signal pathway of disease occurrence or development Or, it can inhibit the expression of multiple disease target genes at the same time, which has broad application prospects in multiple disciplines such as biology and chemistry.
  • FIG. 1 is a schematic diagram of a targeting nucleic acid unit-X form.
  • FIG. 2 is a schematic diagram of a flanking nucleic acid unit-H type.
  • Fig. 3 is a schematic diagram of a cap-type nucleic acid unit-C.
  • FIG. 4 is a schematic diagram of the R structure polymerized nucleic acid plane and loop formation.
  • the figure above is a schematic plan view of R structure polymerized nucleic acid.
  • Fig. 5 is a schematic plan view of an L-structured polymerized nucleic acid.
  • FIG. 6 is a schematic plan view of a Cr structure polymerized nucleic acid.
  • Figure 7 shows the relative expression level of a single gene.
  • Figure 8 shows the relative expression levels of multiple genes.
  • the invention designs three types of nucleic acid units, which are named X-type targeted nucleic acid units, H-type flanking nucleic acid units, and C-type capped nucleic acid units, respectively. These nucleic acid units can be freely combined into polymer nucleic acids of various structures.
  • the structure of the X-type targeted nucleic acid unit is as follows: 5'-T1-T2-A3-3 '.
  • T1 is targeting fragment one
  • T2 is targeting fragment two
  • A3 is linker fragment three.
  • T1 and T2 form a sequence complementary to the target gene sequence.
  • the region complementary to the target gene sequence can be extended to a partial sequence of A3.
  • the type X targeting nucleic acid unit is 15-50 nt in length, preferably 24-36 nt.
  • T1 is 5-24 nt in length; T2 is 1-20 nt in length, and A3 is 5-24 nt in length.
  • a schematic diagram of the X-type targeted nucleic acid unit is shown in FIG. 1.
  • Flanking nucleic acid unit H type
  • H-type flanking nucleic acid unit is as follows: 3'-Hx-Hy-5 'or 3'-Hy-Hx-5'.
  • Hx is a flanking body segment and Hy is a flanking extension segment (Hy may not exist).
  • Hx and Hy are linked via a phosphodiester bond.
  • Hy is located at the 5 'or 3' end of Hx.
  • Hx is complementary paired to the T1 fragment or A3 fragment of the X unit;
  • Hy and T2 fragment of the X unit are complementary paired to one, two, or more consecutive nucleotides from the 5 'end or the 3' end.
  • Hy can be 2-6 nt or longer in length.
  • the schematic diagram of the H-type flanking nucleic acid unit is shown in FIG. 2.
  • Cap-end nucleic acid unit Type C
  • the structure of the C-type capped nucleic acid unit is as follows: 3'-C1-C2 -... Cn-5 '. Among them, C1 is the cap-end fragment 1 and is complementary to the T2 fragment of the X1 unit; C2 is the cap-end fragment 2 and is complementary to the T2 fragment of the X2 unit, and so on. The fragments are complementary in reverse.
  • a schematic diagram of the C-type capped nucleic acid unit is shown in FIG. 3.
  • polymerized nucleic acids are designed according to the three types of nucleic acid units in step 1.
  • the polymerized nucleic acids are named as R-structured polymerized nucleic acid, L-structured polymerized nucleic acid, and Cr-structured polymerized nucleic acid. These structures can target different sites of the same gene at the same time, and can also target different sites of different genes at the same time, so as to interfere with the expression of target genes.
  • Polymeric nucleic acid structure 1 R structure
  • the structure of the R structure polymerized nucleic acid is as follows: (Xi) n. Where Xi is the targeted nucleic acid unit. n is an integer greater than or equal to 3, preferably an integer of 3-8, and more preferably 4, 5, 6;
  • n X-type targeted nucleic acid units as X1 unit, X2 unit, X3 unit, and so on, Xn-1 unit, Xn unit;
  • each targeted nucleic acid unit forms a double-stranded region with two targeted nucleic acid units other than itself in the polymerized nucleic acid through sequence complementation, that is, adjacent X units pass the T1 fragment and A3
  • the fragments are complementary paired and connected. Specifically, the A3 fragment of the X1 unit is complementary paired with the T1 fragment of the X2 unit, the A3 fragment of the X2 unit is complementary paired with the T1 fragment of the X3 unit, and so on.
  • Complementary pairing (T1 fragment of each targeted nucleic acid unit is complementary paired with its adjacent targeted nucleic acid unit A3 fragment, and T1 fragment of each targeted nucleic acid unit is not complementary paired with other targeted nucleic acid unit sequences), thus Form a cyclic secondary structure.
  • the structure and length of each targeted nucleic acid unit are the same.
  • Each targeted nucleic acid unit can bind to a specific sequence of a target gene through its T1 and T2, thereby regulating the expression of the target gene.
  • FIG. 4 A schematic diagram of the planar and loop formation of the R-structure polymerized nucleic acid is shown in FIG. 4.
  • Polymeric nucleic acid structure 2 L structure
  • the structure of the L-structure polymerized nucleic acid is as follows: H1- (Xi) n-Hn. H1 and Hn are flanking nucleic acid units, and Xi is a targeted nucleic acid unit. n is an integer greater than or equal to 3, preferably an integer of 3-8, and more preferably 4, 5, 6;
  • X-type targeted nucleic acid units are named as X1 unit, X2 unit, X3 unit, and so on, Xn-1 unit, Xn unit.
  • the A3 fragment of the X1 unit is complementary paired with the T1 fragment of the X2 unit
  • the A3 fragment of the X2 unit is complementary paired with the T1 fragment of the X3 unit
  • the A3 fragment of the Xn-1 unit and the Xn unit The T1 fragment is complementary paired
  • the H1 unit is complementary paired with the T1 fragment of the X1 unit.
  • the complementary region of the H1 unit and the X1 unit can also extend to part or all of the T2 fragment of the X1 unit; the Hn unit and the A3 fragment of the Xn unit are complementary paired, and the complementary region of the Hn unit and the Xn unit can also extend to the T2 fragment of the Xn unit Part or all of it.
  • the 5 ′ end of the H1 unit or the 3 ′ end of the Hn unit also includes a Hy fragment, and the Hy fragment and the X1 unit or the targeting fragment of the Xn unit are two, one or two from the 5 ′ end or the 3 ′ end. Multiple consecutive nucleic acid molecules are complementary paired.
  • FIG. 5 A schematic plan view of the L-structure polymerized nucleic acid is shown in FIG. 5.
  • the structure of Cr polymerized nucleic acid is as follows: (Xi) n-C. Where Xi is a targeted nucleic acid unit and C is a capped nucleic acid unit. n is an integer greater than or equal to 3, preferably an integer of 3-8, and more preferably 4, 5, 6;
  • X-type targeted nucleic acid units are named as X1 unit, X2 unit, X3 unit, and so on, Xn-1 unit, Xn unit.
  • the A3 fragment of the X1 unit and the T1 fragment of the X2 unit are complementary paired, the A3 fragment of the X2 unit and the T1 fragment of the X3 unit are complementary paired, and so on.
  • T1 fragment is complementary paired;
  • cap-end nucleic acid unit C is composed of cap-end fragment 1, cap-end fragment 2, and so on, and cap-end fragment n.
  • Cap-end fragment 1 is complementary paired with T2 fragment of unit X1, and cap-end fragment 2 and X2.
  • the T2 fragments of the unit are complementary paired, and so on, and the cap-end fragment n is complementary to the T2 fragment of the Xn unit.
  • the cap-end fragment 1, the cap-end fragment 2, ..., and the cap-end fragment n are connected by a phosphodiester bond to form a single unit.
  • FIG. 6 A schematic plan view of the Cr structure polymerized nucleic acid is shown in FIG. 6.
  • the nucleic acid units of the present invention anneal to each other in a sequence-specific manner, and their complementarity promotes the self-assembly of this polymeric nucleic acid to form a polymeric nucleic acid molecule with a secondary structure.
  • the specific synthesis method is as follows:
  • the nucleic acid unit is placed in an annealing condition to anneal each other, and self-assembles to form a secondary structure.
  • the annealing reaction system is a system obtained by mixing equimolar amounts of each nucleic acid unit, RNA annealing buffer (5X) (Biyuntian Annealing Buffer for RNA oligos (5X), R0051) and water (DEPC water).
  • the annealing reaction conditions were 90 ° C in the PCR instrument, which was fully denatured in 2 minutes, and then the temperature was lowered to 25 ° C in the PCR instrument to anneal.
  • nucleic acid unit designed by the present invention includes the following modifications:
  • the A3 linker fragment in the targeted nucleic acid unit has 5-9 nucleotides of ribose 2 consecutive hydroxyl groups from the first nucleotide at the 3 'end replaced by a halogen group or an O-alkyl group .
  • flanking body fragment of the flanking nucleic acid unit has 5-9 nucleotides of ribose 2 hydroxyl group consecutive from the first nucleotide at the 3 'end to a halogen group or an O-alkyl group; or
  • the flanking nucleic acid unit has a continuous 8-30 nucleotide ribose hydroxyl group from the first nucleotide at the 3 'end to a halogen group or an O-alkyl group.
  • the flanking nucleic acid unit has a consecutive 14-18 nucleotide ribose 2 hydroxyl group from the first nucleotide at the 3 'end to a halogen group or an O-alkyl group.
  • Each capped nucleic acid fragment of the capped nucleic acid unit is substituted with a 2-6 nucleotide ribose 2 hydroxyl group from the 5 'end to the 3' end by a halogen group or an O-alkyl group.
  • R structure polymerized nucleic acid inhibition experiment targeting different regions of the same gene
  • a polymerized nucleic acid of the following structure is prepared, and the targeted nucleic acid unit of each structure targets four, five, or six different regions of the VEGFA and TP53 genes, respectively.
  • A indicates that the T1 and A3 fragments of the targeted nucleic acid unit are 12 nt and the T2 fragment is 6 nt.
  • T1 and A3 fragments of the targeted nucleic acid unit are 12 nt and the T2 fragment is 5 nt.
  • T1 and A3 fragments of the targeted nucleic acid unit are 12 nt, and the T2 fragment is 3 nt.
  • D indicates that the T1 and A3 fragments of the targeted nucleic acid unit are 11 nt, and the T2 fragment is 6 nt.
  • T1 and A3 fragments of the targeted nucleic acid unit are 10 nt, and the T2 fragment is 6 nt.
  • the annealing system is as follows (total volume is 100 ⁇ L): 20 ⁇ L of each nucleic acid unit solution (concentration 100 ⁇ M), 15 ⁇ L of RNA annealing buffer (5X), and 5 ⁇ L of DEPC water.
  • the annealing system is as follows (total volume is 150 ⁇ L): 20 ⁇ L of each nucleic acid unit solution (concentration 150 ⁇ M), 30 ⁇ L of RNA annealing buffer (5X), and 20 ⁇ L of DEPC water.
  • the annealing system is as follows (200 ⁇ L in total volume): 20 ⁇ L of each nucleic acid unit solution (200 ⁇ M concentration), 40 ⁇ L of RNA annealing buffer (5X), and 40 ⁇ L of DEPC water.
  • the polymerized nucleic acid whose target gene is VEGFA is as follows 1) -11):
  • the sequence of X unit from X1-X4 is the sequence 54, 57, 58, 59 of Table 1.
  • the polymerized nucleic acid whose target gene is TP53 is as follows 1) -11):
  • the sequence of X units from X1-X4 is the sequence 86-89 of Table 2.
  • VEGFA UGUACUCGAUCUCAUCA GUGGacaucuu 62 VEGFA AAGAUGUCCACCAGGGU UGCGgaucaaa 63 VEGFA UUUGAUCCGCAUAAUCU GCCAgcacaua 64 VEGFA UAUGUGCUGGCCUUGGU AACUuucugcu 65 VEGFA AGCAGAAAGUUCAUGG UUGggugcau 66 VEGFA AUGCACCCAAGACAGC AUCgaguaca 67 VEGFA UGUACUCGAUCUCAUC UGGacaucuu 68 VEGFA AAGAUGUCCACCAGGG GCGgaucaaa 69 VEGFA UUUGAUCCGCAUAAUC CCAgcacaua 70 VEGFA UAUGUGCUGGCCUUGG ACUuucugcu 71
  • Target gene Sequence (5'-3 ') Serial number TP53 UGUGGAAUCAACCCACAG UUUGCgugugga 72 TP53 UCCACACGCAAAUUUCCU ACAGAaacacuu 73 TP53 AAGUGUUUCUGUCAUCCA ACUACaugugua 74 TP53 UACACAUGUAGUUGUAGU UGGUAaucuacu 75 TP53 AGUAGAUUACCACUGGAG UCUCCgcaagaa 76 TP53 UUCUUGCGGAGAUUCUCU GUUGAuuccaca 77 TP53 UGUGGAAUCAACCCACA UUUGCgugugga 78 TP53 UCCACACGCAAAUUUCC ACAGAaacacuu 79 TP53 AAGUGUUUCUGUCAUCC ACUACaugugua 80 TP53 UACACAUGUAGUUGUAG UGGUAaucuacu 81 TP53 AGUAGAUUACCACUGGA UCUCCgcaagaa 82
  • HeLa cells 5 ⁇ 10 5 HeLa cells (ATCC No. CRL-1958) were seeded in a 12-well culture plate containing 10% fetal bovine serum in DMEM medium, and the polymerized nucleic acids prepared in step 1 were transfected into HeLa cells: Nucleic acid is mixed with transfection reagents and buffers (Guangzhou Ruibo Biological Technology Co., Ltd., name riboFECT TM CP Buffer, article number C10511-1), and then added to the cell culture medium, the volume of each well is 1mL, so that the final concentration of polymerized nucleic acid transfection 50 nM, the culture plate was placed in a 5% CO 2 , 37 ° C. incubator for 48 h.
  • transfection reagents and specific steps refer to the method in riboFect TM (Guangzhou Ruibo Biotechnology Co., Ltd.).
  • NC negative control group
  • NT untreated control group
  • the control sequence of the NC group is siRNA, which is a double-stranded RNA molecule obtained by complementary binding of the following two single-stranded RNA molecules: 5'-UUCUCCGAACGUGUCACGUdTdT-3 'and 5'-ACGUGACACGUUCGGAGAAdTdT-3'.
  • RNA of the transfected cells was extracted by rizol method for real-time quantitative PCR to detect the target gene mRNA expression level.
  • the q-PCR was repeated 3 times. SD means.
  • the sequences of real-time quantitative PCR primers for detecting target genes are shown in the attached table.
  • the test results are shown in Table 3.
  • the experimental results show that the polymer molecules with different structures can effectively inhibit the expression of target genes.
  • the X unit sequence for preparing VEGFA-R6-A polymerized nucleic acid and the X unit sequence for polymerized nucleic acid TP53-R6-A are the same as in step (1); the X unit sequence for preparing P65-R6-A polymerized nucleic acid is in order from Table X1-X6 in Table 5 Sequences 15, 16, 17, 23, 24, 25.
  • test results are shown in Table 4.
  • the experimental results show that the polymer nucleic acid of the present invention at a low concentration can also achieve the purpose of reducing the expression level of the target gene.
  • a polymerized nucleic acid of the following structure is prepared, and the targeted nucleic acid unit of each structure targets four or six different regions of the PPIB or P65 gene, respectively.
  • the polymerized nucleic acid whose target gene is PPIB is as follows 1) -5):
  • the polymerized nucleic acid whose target gene is P65 is as follows 1) -5):
  • Unit X T1 from the 1st to 12th position; T2 from the 13th to 18th position; A3 from the 19th to 30th position.
  • Unit H1 Bits 1-4 are Hy; Bits 5-16 are Hx.
  • Unit H4 bits 1-12 are Hx; bits 13-16 are Hy.
  • Unit C C4 at positions 1-6, C3 at positions 7-12, C2 at positions 13-18, and C1 at positions 19-24.
  • step (a) The inhibition experiment steps are the same as step (a).
  • the suppression results are shown in Figure 7.
  • the results showed that the polymerized nucleic acids of the five structures all effectively inhibited the target gene expression.
  • the level of inhibition of the target gene PPIB was above 80%, and the level of inhibition of the target gene P65 was above 75%.
  • a polymerized nucleic acid of the R structure was prepared that targeted 4 or 6 different target genes.
  • BCCC-R4 The target genes are BIRC5, CTNNB, COPS5, and CLU.
  • the sequence of the X unit from X1-X4 is the sequence 28-31 in Table 3.
  • BCCCEH-R6 The target genes are BIRC5, CTNNB, COPS5, CLU, EIF4E, and HIF1A.
  • the sequence of the X unit from X1-X6 is the sequence 32, 33, 28, 29, 30, and 34 in Table 6.
  • step (a) The inhibition experiment steps are the same as step (a).
  • Target gene Sequence (5'-3 ') Serial number BIRC5 AGAAGAAACACUGGGCCA ACUGCugaucuu 28 CTNNB1 AAGAUCAGCAGUCUCAUU UGGUCaggucuu 29 COPS5 AAGACCUGACCAGUGGUA UUUGAcucugau 30 CLU AUCAGAGUCAAAGAGCUU AGUGUuucuucu 31 HIF1A UCAAGUUGCUGGUCAUCAGGAGGuugcuaa 32 EIF4E UUAGCAACCUCCUGAUUA AGUGUuucuucu 33 CLU AUCAGAGUCAAAGAGCUU CCAGCaacuuga 34
  • Unit X T1 from the 1st to 12th position; T2 from the 13th to 18th position; A3 from the 19th to 30th position.
  • Polymeric nucleic acids of the following structures were prepared, which targeted four different target genes.
  • SPPV-R4 The target genes are SOD1, PPIB, P65, and VEGFA, and the sequence of the X unit from X1-X4 is the sequence 35-38 in Table 7.
  • SPPV-L4 The target genes are SOD1, PPIB, P65, and VEGFA.
  • the sequence of X unit from X1-X4 is sequence 35-38 in Table 7, the sequence of H1 unit is sequence 39, and the sequence of H4 unit is sequence 40.
  • SPPV-Cr4 The target genes are SOD1, PPIB, P65, and VEGFA.
  • the sequence of X unit from X1-X4 is sequence 35-38 in Table 7, and the sequence of C unit is sequence 41.
  • Fig. 9 The results of the inhibition experiment are shown in Fig. 9. The results showed that the polymerized nucleic acids with three structures all effectively inhibited the expression of four target genes. And found that among the three kinds of polymer structures, the polymer nucleic acid of Cr4 structure had the best inhibitory effect, and the expression level of each target gene was the lowest.
  • Target gene Sequence (5'-3 ') Serial number SOD1 UACUUUCUUCAUUUCCAC CUGUUccaaaa 35 PPIB UUUUUGGAACAGUCUUUC UGAGAccuucaa 36 P65 UUGAAGGUCUCAUAUGUC CAUGCagauuau 37 VEGFA AUAAUCUGCAUGGUGAUG AUGAAgaaagua 38 H GGAAAUGAAgaagua 39 H4 uacuuucuucaucauc 40 C caucACgacaUAgaaaGAguggAA 41
  • Unit X T1 from the 1st to 12th position; T2 from the 13th to 18th position; A3 from the 19th to 30th position.
  • Unit H1 Bits 1-4 are Hy; Bits 5-16 are Hx.
  • Unit H4 bits 1-12 are Hx; bits 13-16 are Hy.
  • Unit C C4 at positions 1-6, C3 at positions 7-12, C2 at positions 13-18, and C1 at positions 19-24.
  • the invention provides a novel nucleic acid unit that can be used to construct a polymerized nucleic acid and a polymerized nucleic acid that can be used to interfere with the expression of a target gene.
  • the present invention realizes multi-target interference by designing and constructing a novel nucleic acid unit and its self-assembling polymerized nucleic acid, which can be used to inhibit the expression of multiple genes in a signal pathway for the occurrence or development of a disease, or simultaneously inhibit the expression of multiple target genes of a disease, It has broad application prospects in many disciplines such as biology and chemistry.
  • the polymerized nucleic acid can simultaneously target multiple sequences, and the sequences may be in one gene or located in multiple genes.
  • the advantages of the present invention are: 1) high RNAi efficiency; 2) good stability; 3) reduced off-target rate; 4) enhanced ability to introduce cells; 5) modular design.

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Abstract

本发明公开了新型的可用于构建聚合核酸的核酸单元及可用于干扰目的基因表达的聚合核酸。本发明通过设计和构建新型的核酸单元及其自组装聚合核酸，实现了多靶标干扰，可用于抑制疾病发生或发展的信号通路中的多个基因表达，或同时抑制多个疾病靶基因表达，在生物学、化学等多个学科领域具有广阔的应用前景。该聚合核酸可实现同时靶向多个序列，所述序列可在一个基因中，或位于多个基因中。本发明优势在于：1)RNAi效能高；2)稳定性好；3)降低脱靶率；4)导入细胞能力增强；5)可模块化设计。

Description

核酸单元及其聚合核酸与应用 技术领域
本发明属于核酸技术领域，具体涉及新型结构的核酸单元与其自组装聚合核酸，所述聚合核酸可干扰一个或多个目的基因的表达。
背景技术
CON(互补寡核苷酸)技术使得人工合成的寡核苷酸分子能够通过序列互补作用与靶向分子结合，并改变靶向分子的生物特性。CON技术包括RNAi(RNA干扰)技术和ASO(反义寡核苷酸)技术两大类，目前已广泛应用于功能基因组学研究，并有望成为除小分子化合物和生物制剂以外的第三大治疗手段。如用于治疗纯合子型家族性高胆固醇血症(homozygous familial hypercholesterolemia，FoFH)的米泊美生(mipomersen)，其是Genzyme研发的一种合成的硫代磷酸寡核苷酸，通过与Apo B-100蛋白mRNA的编码区互补配对，抑制Apo B-100蛋白(LDL和VLDL的主要载脂蛋白)的翻译合成，从而有效降低FoFH患者的LDL-C、TC、Non-HDL-C水平。尽管CON技术已经取得了一定成功，但这项技术仍有待完善之处。如传统的RNAi试剂有下述不足：1)繁琐的合成步骤及相对高的生产成本；2)对内切酶和外切酶高度敏感，稳定性不高；3)抑制有效率不够高，不能保证单个分子一定能抑制靶基因表达；4)非特异活性导致的副作用，该非特异性的作用主要源自正义链；5)难以导入细胞尤其是动物体内。
纳米技术研究的是直径在1～100nm纳米范围内物质的性质和应用。纳米结构物质所具有的小尺寸效应、表面效应、高扩散率等特性为科学研究和技术应用开拓了新领域。寡核苷酸结构灵活，如RNA结构，能够自我组装形成纳米结构。以DNA和RNA为代表的核酸纳米技术，是纳米技术领域发展的一个新方向。
发明公开
本发明要解决的技术问题是如何抑制或降低或干扰靶基因的表达。
为了解决上述技术问题，本发明首先提供了一种用于抑制或降低或干扰靶基因表达的聚合核酸分子。
本发明提供的用于抑制或干扰靶基因表达的聚合核酸分子由n条X型核酸分子组成；
每条所述X型核酸分子自5’端起依次由靶向片段一、靶向片段二和接头片段三组成。每条所述X型核酸分子的靶向片段一与其相邻的X型核酸分子的接头片段三互补配对。具体的，将n条X型核酸分子分别命名为X1单元、X2单元、X3单元，依次类推，Xn-1单元、Xn单元；所述X1单元的接头片段三与所述X2单元 的靶向片段一互补配对(完全互补)，所述X2单元的接头片段三与所述X3单元的靶向片段一互补配对(完全互补)，依次类推，所述Xn-1单元的接头片段三与所述Xn单元的靶向片段一互补配对(完全互补)，所述Xn单元的接头片段三与所述X1单元的靶向片段一互补配对(完全互补)。每条所述X型核酸分子的靶向片段一与其他X型核酸分子序列均不互补。
每条所述聚合核酸分子的靶向片段一和靶向片段二均与靶基因互补配对；每条所述聚合核酸分子通过靶向片段一和靶向片段二与靶基因的特定序列结合，实现抑制或降低或干扰靶基因的表达。在某些特定的情况下，与靶基因序列互补的区域可以延长至接头片段三的部分序列。同一条X型核酸分子中的靶向片段一和靶向片段二可以相同，也可不同。两条不同的X型核酸分子中的靶向片段一或靶向片段二可以相同，也可不同。
每条所述X型核酸分子的长度相同(结构也相同)。
所述n为大于或等于3的整数。
在本发明的具体实施例中，所述n条X型核酸分子依次首尾相接(通过前一条X型核酸分子的接头片段三与下一条相邻的X型核酸分子的靶向片段一互补实现)，最终形成具有环状二级结构的聚合核酸分子。
上述聚合核酸分子中，所述n条X型核酸分子还可形成具有线性结构的聚合核酸分子，所述具有线性结构的聚合核酸分子还包括H型核酸分子；所述H型核酸分子由H1型核酸分子和Hn型核酸分子组成。
将n条X型核酸分子分别命名为X1单元、X2单元、X3单元，依次类推，Xn-1单元、Xn单元；所述X1单元的接头片段三与所述X2单元的靶向片段一互补配对，所述X2单元的接头片段三与所述X3单元的靶向片段一互补配对，依次类推，所述Xn-1单元的接头片段三与所述Xn单元的靶向片段一互补配对；所述H1型核酸分子与所述X1单元的靶向片段一互补配对；所述Hn型核酸分子与所述Xn单元的接头片段三互补配对。
进一步的，所述H型核酸分子的5’端或3’端还包括Hy片段；所述H型核酸分子由Hx片段和Hy片段组成。
所述H1型核酸分子与所述X1单元的互补区可延伸至所述X1单元的靶向片段二的部分或全部序列；所述Hn型核酸分子与所述Xn单元的互补区可延伸至所述Xn单元的靶向片段二的部分或全部序列。
所述H1型核酸分子的Hx片段与所述X1单元的靶向片段一互补配对；所述Hn型核酸分子的Hx片段与所述Xn单元的接头片段三互补配对；所述Hy片段与所述X1单元或所述Xn单元的靶向片段二自5’端或3’端起1个、2个或多个连续的核酸分子互补配对。
上述聚合核酸分子中，具有环状结构的聚合核酸分子还包括C型核酸分子；所述C型核酸分子由n个片段依次连接而成，n个片段分别为n条所述X型核酸分子中的靶向片段二的反向互补序列。具体的，所述C型核酸分子自3’端起依次由帽 端片段1、帽端片段2、帽端片段3，依次类推，帽端片段Cn-1、帽端片段n组成；将n条X型核酸分子分别命名为X1单元、X2单元、X3单元，依次类推，Xn-1单元、Xn单元；
所述X1单元的接头片段三与所述X2单元的靶向片段一互补配对，所述X2单元的接头片段三与所述X3单元的靶向片段一互补配对，依次类推，所述Xn-1单元的接头片段三与所述Xn单元的靶向片段一互补配对，所述Xn单元的接头片段三与所述X1单元的靶向片段一互补配对；所述帽端片段1与所述X1单元的靶向片段二互补配对，所述帽端片段2与所述X2单元的靶向片段二互补配对，依次类推，所述帽端片段Cn-1与所述Xn-1单元的靶向片段二互补配对，所述帽端片段n与所述Xn单元的靶向片段二互补配对。
上述聚合核酸分子中，所述核酸分子(X型核酸分子、H型核酸分子或C型核酸分子)可为DNA或RNA或由DNA和RNA组成的寡核苷酸。
进一步的，所述核酸分子(X型核酸分子、H型核酸分子或C型核酸分子)为单链RNA分子。
上述聚合核酸分子中，所述X型核酸分子的长度为15-50nt，优选为24-36nt；
所述靶向片段一的长度为5-24nt；
所述靶向片段二的长度为1-20nt；
所述接头片段三的长度为5-24nt；
所述靶向片段一和所述靶向片段二的长度总和至少为14-16nt；
所述Hy片段的长度为2-6nt或更长。
进一步的，所述X型核酸分子的长度为24-36nt。
上述聚合核酸分子中，所述聚合核酸分子至少包括一个修饰的核苷酸。
进一步的，所述修饰为磷酸骨架修饰、碱基修饰和/或核糖修饰。所述核糖修饰为核糖2位羟基基团被卤素基团或O-烷基基团取代。所述烷基为甲基、乙基、丙基或甲乙基。
更进一步的，所述X型核酸分子的接头片段三自3’端第一位核苷酸起连续的5-9个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代；
所述H型核酸分子的Hx片段自3’端第一位核苷酸起连续的5-9个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代；
所述H型核酸分子自3’端第一位核苷酸起连续的8-30个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代；优选的，所述H型核酸分子自3’端第一位核苷酸起连续的14-18个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代；
所述C型核酸分子中每个帽端片段自5’端第一位核苷酸起连续的2-6个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代。
上述聚合核酸分子中，所述n为3或4或5或6或7或8。
进一步的，所述n为4或5或6。
上述聚合核酸分子中，所述靶基因的个数为一个或两个或多个，且所述靶 基因的个数不超过n个；每条X型核酸分子对应一个靶基因，或者多条X型核酸分子对应同一个靶基因的不同区域。
进一步的，所述靶基因的个数为1个或4个或6个。
所述靶基因为如下基因中至少一种：PPIB基因、p65基因、BIRC5基因、CTNNB基因、COPS5基因、CLU基因、EIF4E基因、HIF1A基因、TP53基因、VEGFA基因和SOD1基因。
更进一步的，用于干扰靶基因PPIB表达的聚合核酸分子为如下a1)-a5)：
a1)由序列1、序列2、序列3和序列4所示的单链RNA分子组成；
a2)由序列1、序列2、序列3、序列4、序列20和序列21所示的单链RNA分子组成；
a3)由序列1、序列2、序列3、序列4和序列8所示的单链RNA分子组成；
a4)由序列2、序列3、序列9、序列10、序列11和序列12所示的单链RNA分子组成；
a5)由序列2、序列3、序列6、序列9、序列10、序列11、序列13和序列14所示的所示的单链RNA分子组成；
用于干扰靶基因P65表达的聚合核酸分子为如下b1)-b5)：
b1)由序列15、序列16、序列17和序列18所示的单链RNA分子组成；
b2)由序列6、序列7、序列15、序列16、序列17和序列19所示的单链RNA分子组成；
b3)由序列15、序列16、序列17、序列18和序列22所示的单链RNA分子组成；
b4)由序列15、序列16、序列17、序列23、序列24和序列25所示的单链RNA分子组成；
b5)由序列15、序列16、序列17、序列20、序列23、序列24、序列26和序列27所示的所示的单链RNA分子组成；
用于同时干扰靶基因BIRC5、CTNNB、COPS5和CLU表达的聚合核酸分子为由序列28、序列29、序列30和序列31所示的单链RNA分子组成；
用于同时干扰靶基因BIRC5、CTNNB、COPS5、CLU、EIF4E和HIF1A表达的聚合核酸分子为由序列28、序列29、序列30、序列32、序列33和序列34所示的单链RNA分子组成；
用于同时干扰靶基因SOD1、PPIB、P65和VEGFA表达的聚合核酸分子为如下c1)-c3)：
c1)由序列35、序列36、序列37和序列38所示的单链RNA分子组成；
c2)由序列35、序列36、序列37、序列38、序列39和序列40所示的单链RNA分子组成；
c3)由序列35、序列36、序列37、序列38和序列41所示的单链RNA分子组成；
用于干扰靶基因VEGFA表达的聚合核酸分子为如下d1)-d11)：
d1)由序列42、序列43、序列44、序列45、序列46和序列47所示的单链RNA分子组成；
d2)由序列48、序列49、序列50、序列51、序列52和序列53所示的单链RNA分子组成；
d3)由序列54、序列55、序列56、序列57、序列58和序列59所示的单链RNA分子组成；
d4)由序列42、序列43、序列45、序列46和序列47所示的单链RNA分子组成；
d5)由序列48、序列49、序列51、序列52和序列53所示的单链RNA分子组成；
d6)由序列54、序列55、序列57、序列58和序列59所示的单链RNA分子组成；
d7)由序列42、序列45、序列46和序列47所示的单链RNA分子组成；
d8)由序列48、序列51、序列52和序列53所示的单链RNA分子组成；
d9)由序列54、序列57、序列58和序列59所示的单链RNA分子组成；
d10)由序列60、序列61、序列62、序列63、序列64和序列65所示的单链RNA分子组成；
d11)由序列66、序列67、序列68、序列69、序列70和序列71所示的单链RNA分子组成；
用于干扰靶基因TP53表达的聚合核酸分子为如下e1)-e11)：
e1)由序列72、序列73、序列74、序列75、序列76和序列77所示的单链RNA分子组成；
e2)由序列78、序列79、序列80、序列81、序列82和序列83所示的单链RNA分子组成；
e3)由序列84、序列85、序列86、序列87、序列88和序列89所示的单链RNA分子组成；
e4)由序列73、序列74、序列75、序列76和序列77所示的单链RNA分子组成；
e5)由序列79、序列80、序列81、序列82和序列83所示的单链RNA分子组成；
e6)由序列54、序列55、序列57、序列58和序列59所示的单链RNA分子组成；
e7)由序列74、序列75、序列76和序列77所示的单链RNA分子组成；
e8)由序列80、序列81、序列82和序列83所示的单链RNA分子组成；
e9)由序列序列86、序列87、序列88和序列89所示的单链RNA分子组成；
e10)由序列90、序列91、序列92、序列93、序列94和序列95所示的单链RNA 分子组成；
e11)由序列96、序列97、序列98、序列99、序列100和序列101所示的单链RNA分子组成。
为了解决上述技术问题，本发明又提供了上述聚合核酸分子的衍生物。
本发明提供的上述聚合核酸分子的衍生物为如下(m1)-(m5)中任一种：
(m1)将上述聚合核酸分子删除或增加一个或几个核苷酸，得到与所述聚合核酸分子具有相同功能的聚合核酸分子的衍生物；
(m2)将上述聚合核酸分子进行核苷酸取代或修饰，得到与所述聚合核酸分子具有相同功能的聚合核酸分子的衍生物；
(m3)将上述聚合核酸分子的骨架改造为硫代磷酸脂骨架，得到与所述聚合核酸分子具有相同功能的聚合核酸分子的衍生物；
(m4)由上述聚合核酸分子编码的肽核酸、锁核酸或解锁核酸，得到与所述聚合核酸分子具有相同功能的聚合核酸分子的衍生物；
(m5)将上述聚合核酸分子的一端或中间接上信号分子和/或活性分子和/或功能基团，得到与所述聚合核酸分子具有相同功能的聚合核酸分子的衍生物。
为了解决上述技术问题，本发明还提供了上述聚合核酸分子的制备方法。
本发明提供的上述聚合核酸分子的制备方法包括如下步骤：
M1)合成上述X型核酸分子和/或H型核酸分子和/或C型核酸分子；
M2)将所述X型核酸分子和/或所述H型核酸分子和/或所述C型核酸分子退火，得到所述聚合核酸。
上述方法中，所述退火的反应体系为将等摩尔量的各单链RNA分子、RNA退火buffer和水混匀得到的体系。所述退火的反应条件为PCR仪中90℃，2min使其充分变性，随后PCR仪中降温至25℃使其退火。
n为4时，退火体系如下(总体积为100μL)：各单链RNA分子溶液(浓度为100μM)20μL、RNA退火buffer(5X)15μL、DEPC水5μL。
n为5时，退火体系如下(总体积为150μL)：各单链RNA分子溶液(浓度为150μM)20μL、RNA退火buffer(5X)30μL、DEPC水20μL。
n为6时，退火体系如下(总体积为200μL)：各单链RNA分子溶液(浓度为200μM)20μL、RNA退火buffer(5X)40μL、DEPC水40μL。
为了解决上述技术问题，本发明还提供了上述聚合核酸分子或衍生物的新用途。
本发明提供了上述聚合核酸分子或衍生物在如下A1)或A2)中的应用；
A1)调控细胞中靶基因表达水平；
A2)制备预防和/或缓解和/或治疗由靶基因表达引起的疾病的产品。
上述应用中，所述调控为抑制或降低或干扰。
上述应用中，所述细胞为肿瘤细胞。
上述应用中，所述靶基因为疾病相关基因；所述疾病相关基因具体为肿瘤相关基因；所述肿瘤相关基因具体为如下基因中至少一种：PPIB基因、p65基因、BIRC5基因、CTNNB基因、COPS5基因、CLU基因、EIF4E基因、HIF1A基因、TP53基因、VEGFA基因和SOD1基因。
为了解决上述技术问题，本发明还提供了一种抑制或降低或干扰细胞中靶基因表达水平的试剂或试剂盒或药物。
本发明提供的抑制或降低或干扰细胞中靶基因表达水平的试剂或试剂盒或药物包括上述聚合核酸分子或上述衍生物。
为了解决上述技术问题，本发明最后提供了一种抑制或降低或干扰细胞中靶基因表达水平的方法。
本发明提供的抑制或降低或干扰细胞中靶基因表达水平的方法包括如下步骤：将上述聚合核酸分子或衍生物导入所述细胞，实现抑制或降低所述细胞中靶基因的表达水平。
上述方法中，所述导入的方法为将所述聚合核酸分子、转染试剂和缓冲液混匀后加入细胞培养基中，得到反应体系；所述聚合核酸分子在所述反应体系中的终浓度为1-300nM。
上述方法中，所述细胞为肿瘤细胞。
上述方法中，所述靶基因为疾病相关基因；所述疾病相关基因具体为肿瘤相关基因；所述肿瘤相关基因具体为如下基因中至少一种：PPIB基因、p65基因、BIRC5基因、CTNNB基因、COPS5基因、CLU基因、EIF4E基因、HIF1A基因、TP53基因、VEGFA基因和SOD1基因。
本发明优势在于：
1)RNAi效能提高；
2)结构更稳定，化学稳定性好，抗核酸酶降解能力增强，尤其是血液中的稳定性增强，半衰期延长；
3)降低脱靶率；
4)能形成纳米颗粒结构，导入细胞能力增强；
5)可模块化设计核酸分子。
6)一些结构的聚合核酸发挥RNAi作用不需要Dicer酶的参与，因此对化学修饰的耐受程度更高，可以部分修饰或完全修饰。
本发明将CON技术与纳米技术结合，通过构建具有可准确设计的，且具有自我组装能力的核酸结构，从而实现多靶标干扰作用，可用于抑制疾病发生或发展的信号通路中的多个基因表达，或同时抑制多个疾病靶基因表达，在生物学、化学等多个学科领域具有广阔的应用前景。
附图说明
图1为靶向核酸单元-X型示意图。
图2为侧翼核酸单元-H型示意图。
图3为帽端核酸单元-C型示意图。
图4为R结构聚合核酸平面和成环示意图。上图为R结构聚合核酸平面示意图。下图为R结构聚合核酸成环示意图(左：R＝(Xi)4；右：R＝(Xi)6)。
图5为L结构聚合核酸平面示意图。
图6为Cr结构聚合核酸平面示意图。
图7为单基因相对表达水平。
图8为多基因相对表达水平。
图9为多基因相对表达水平(n＝4)。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、核酸单元、聚合核酸的设计与合成
一、核酸单元的设计
本发明设计了3种型态的核酸单元，分别命名为X型靶向核酸单元、H型侧翼核酸单元、C型帽端核酸单元。这些核酸单元可以自由组合为多种结构的聚合核酸。
1、靶向核酸单元：X型
X型靶向核酸单元的结构如下：5’-T1-T2-A3-3’。其中T1为靶向片段一，T2为靶向片段二，A3为接头片段三，T1和T2组成一段与靶基因序列互补的序列。在某些特定的情况下，与靶基因序列互补的区域可以延长至A3的部分序列。X型靶向核酸单元的长度为15-50nt，优选为24-36nt。其中T1长度为5-24nt；T2长度为1-20nt，A3长度为5-24nt。X型靶向核酸单元示意图见图1。
2、侧翼核酸单元：H型
H型侧翼核酸单元的结构如下：3’-Hx-Hy-5’或3’-Hy-Hx-5’。其中Hx为侧翼主体片段，Hy为侧翼延伸片段(Hy可以不存在)。Hx与Hy通过磷酸二酯键连接。Hy位于Hx的5’端或3’端。Hx与X单元的T1片段或A3片段互补配对；Hy与X单元的T2片段自5’端或3’端起1个、2个或多个连续的核苷酸互补配对。Hy的长度可为2-6nt或更长。H型侧翼核酸单元示意图见图2。
3、帽端核酸单元：C型
C型帽端核酸单元的结构如下：3’-C1-C2-……Cn-5’。其中C1为帽端片段1，与X1单元的T2片段反向互补；C2为帽端片段2，与X2单元的T2片段反向互补，依次类推，Cn为帽端片段n，与Xn单元的T2片段反向互补。C型帽端核酸单元示意图见图3。
二、聚合核酸分子的设计
本发明根据步骤一的三种核酸单元设计了3种结构的聚合核酸，分别命名为 R结构聚合核酸、L结构聚合核酸、Cr结构聚合核酸。这些结构能够同时靶向同一个基因的不同位点，也能够同时靶向不同基因的不同位点，实现干扰靶基因的表达。
1、聚合核酸结构一：R结构
R结构聚合核酸的结构如下：(Xi)n。其中Xi为靶向核酸单元。n为大于或等于3的整数，优选为3-8的整数，更优选为4、5、6。
将n个X型靶向核酸单元分别命名为X1单元、X2单元、X3单元，依次类推，Xn-1单元、Xn单元；
本发明的R结构聚合核酸中，每个靶向核酸单元均通过序列互补作用与聚合核酸中除自身外的另外两个靶向核酸单元形成双链区域，即相邻X单元通过T1片段和A3片段互补配对连接，具体的，X1单元的A3片段与X2单元的T1片段互补配对，X2单元的A3片段与X3单元的T1片段互补配对，依次类推，Xn单元的A3片段与X1单元的T1片段互补配对(每个靶向核酸单元的T1片段与其相邻的靶向核酸单元的A3片段互补配对，且每个靶向核酸单元的T1片段与其他靶向核酸单元序列均不互补配对)，从而形成环状二级结构。在这个环状结构中，每个靶向核酸单元的结构和长度相同。
每个靶向核酸单元能通过其T1和T2与靶基因的特定序列结合，从而调控靶基因的表达。
R结构聚合核酸的平面和成环示意图见图4。
2、聚合核酸结构二：L结构
L结构聚合核酸的结构如下：H1-(Xi)n-Hn。其中H1、Hn均为侧翼核酸单元，Xi为靶向核酸单元。n为大于或等于3的整数，优选为3-8的整数，更优选为4、5、6。
将n个X型靶向核酸单元分别命名为X1单元、X2单元、X3单元，依次类推，Xn-1单元、Xn单元。
本发明的L结构聚合核酸中，X1单元的A3片段与X2单元的T1片段互补配对，X2单元的A3片段与X3单元的T1片段互补配对，依次类推，Xn-1单元的A3片段与Xn单元的T1片段互补配对；H1单元与X1单元的T1片段互补配对。H1单元与X1单元的互补区还可延伸至X1单元的T2片段的部分或全部；Hn单元与Xn单元的A3片段互补配对，Hn单元与Xn单元的互补区还可延伸至Xn单元的T2片段的部分或全部。H1单元的5’端或Hn单元的3’端还包括Hy片段，Hy片段与所述X1单元或所述Xn单元的靶向片段二自5’端或3’端起1个、2个或多个连续的核酸分子互补配对。
L结构聚合核酸的平面示意图见图5。
3、聚合核酸结构三：Cr结构
Cr结构聚合核酸的结构如下：(Xi)n-C。其中Xi为靶向核酸单元，C为帽端核酸单元。n为大于或等于3的整数，优选为3-8的整数，更优选为4、5、6。
将n个X型靶向核酸单元分别命名为X1单元、X2单元、X3单元，依次类推，Xn-1单元、Xn单元。
本发明的Cr结构聚合核酸中，X1单元的A3片段与X2单元的T1片段互补配对，X2单元的A3片段与X3单元的T1片段互补配对，依次类推，Xn-1单元的A3片段与Xn单元的T1片段互补配对；帽端核酸单元C由帽端片段1、帽端片段2，依次类推，帽端片段n组成，帽端片段1与X1单元的T2片段互补配对，帽端片段2与X2单元的T2片段互补配对，依次类推，帽端片段n与Xn单元的T2片段互补配对，帽端片段1、帽端片段2、……和帽端片段n均通过磷酸二酯键连接为一条单链核酸。
Cr结构聚合核酸的平面示意图见图6。
三、聚合核酸的合成方法及核酸单元的修饰方法
1、聚合核酸的合成方法
本发明的核酸单元以序列特异性方式彼此退火，其互补性促进此聚合核酸的自装配，形成具有二级结构的聚合核酸分子。具体合成方法如下：
1)合成聚合核酸结构所需的核酸单元；
2)将核酸单元置于退火条件中，使其相互退火，自组装形成二级结构体。
退火反应体系为将等摩尔量的各核酸单元、RNA退火buffer(5X)(碧云天Annealing Buffer for RNA oligos(5X)，R0051)和水(DEPC水)混匀得到的体系。
退火反应条件为PCR仪中90℃，2min使其充分变性，随后PCR仪中降温至25℃使其退火。
2、核酸单元的修饰方法
为了增加核酸酶稳定性和生物学活性可在核酸中引入适当的核苷酸糖、碱基和磷酸部分的修饰，包括对核糖进行化学修饰，如2’核糖羟基基团被卤素基团或O-烷基基团取代，烷基包括甲基、乙基、丙基或甲乙基等，该类核酸修饰不会降低聚合核酸的干扰活性。本发明设计的核酸单元包括如下修饰：
1)靶向核酸单元中的A3接头片段自3’端第一位核苷酸起连续的5-9个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代。
2)侧翼核酸单元的侧翼主体片段自3’端第一位核苷酸起连续的5-9个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代；或侧翼核酸单元自3’端第一位核苷酸起连续的8-30个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代。优选的，侧翼核酸单元自3’端第一位核苷酸起连续的14-18个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代。
3)帽端核酸单元的每个帽端核酸片段从5’端到3’端的2-6个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代。
实施例2、聚合核酸的制备及其在干扰靶基因表达中的应用
(一)R结构聚合核酸：靶向同一基因的不同区域的抑制实验
1、聚合核酸
制备如下结构的聚合核酸，每个结构的靶向核酸单元分别靶向VEGFA、TP53基因的4个、5个或6个不同区域。
A表示靶向核酸单元的T1和A3片段为12nt，T2片段为6nt。
B表示靶向核酸单元的T1和A3片段为12nt，T2片段为5nt。
C表示靶向核酸单元的T1和A3片段为12nt，T2片段为3nt。
D表示靶向核酸单元的T1和A3片段为11nt，T2片段为6nt。
E表示靶向核酸单元的T1和A3片段为10nt，T2片段为6nt。
n为4时，退火体系如下(总体积为100μL)：各核酸单元溶液(浓度为100μM)20μL、RNA退火buffer(5X)15μL、DEPC水5μL。
n为5时，退火体系如下(总体积为150μL)：各核酸单元溶液(浓度为150μM)20μL、RNA退火buffer(5X)30μL、DEPC水20μL。
n为6时，退火体系如下(总体积为200μL)：各核酸单元溶液(浓度为200μM)20μL、RNA退火buffer(5X)40μL、DEPC水40μL。
靶基因为VEGFA的聚合核酸为如下1)-11)：
1)VEGFA-R6-A(R结构，n＝6)：其X单元序列从X1-X6依次为表1的序列42-47。
2)VEGFA-R6-B(R结构，n＝6)：其X单元序列从X1-X6依次为表1的序列48-53。
3)VEGFA-R6-C(R结构，n＝6)：其X单元序列从X1-X6依次为表1的序列54-59。
4)VEGFA-R5-A(R结构，n＝5)：其X单元序列从X1-X5依次为表1的序列42、43、45、46、47。
5)VEGFA-R5-B(R结构，n＝5)：其X单元序列从X1-X5依次为表1的序列48、49、51、52、53。
6)VEGFA-R5-C(R结构，n＝5)：其X单元序列从X1-X5依次为表1的序列54、55、57、58、59。
7)VEGFA-R4-A(R结构，n＝4)：其X单元序列从X1-X4依次为表1的序列42、45、46、47。
8)VEGFA-R4-B(R结构，n＝4)：其X单元序列从X1-X4依次为表1的序列48、51、52、53。
9)VEGFA-R4-C(R结构，n＝5)：其X单元序列从X1-X4依次为表1的序列54、57、58、59。
10)VEGFA-R6-D(R结构，n＝6)：其X单元序列从X1-X6依次为表1的60-65。
11)VEGFA-R6-E(R结构，n＝6)：其X单元序列从X1-X6依次为表1的序列66-71。
靶基因为TP53的聚合核酸为如下1)-11)：
1)TP53-R6-A(R结构，n＝4)：其X单元序列从X1-X6依次为表2的序列72-77。
2)TP53-R6-B(R结构，n＝6)：其X单元序列从X1-X6依次为表2的序列78-83。
3)TP53-R6-C(R结构，n＝6)：其X单元序列从X1-X6依次为表2的序列84-89。
4)TP53-R5-A(R结构，n＝5)：其X单元序列从X1-X5依次为表2的序列73-77。
5)TP53-R5-B(R结构，n＝5)：其X单元序列从X1-X5依次为表2的序列79-83。
6)TP53-R5-C(R结构，n＝5)：其X单元序列从X1-X5依次为表2的序列85-89。
7)TP53-R4-A(R结构，n＝4)：其X单元序列从X1-X4依次为表2的序列74-77。
8)TP53-R4-B(R结构，n＝4)：其X单元序列从X1-X4依次为表2的序列80-83。
9)TP53-R4-C(R结构，n＝5)：其X单元序列从X1-X4依次为表2的序列86-89。
10)TP53-R6-D(R结构，n＝6)：其X单元序列从X1-X6依次为表2的序列90-95。
11)TP53-R6-E(R结构，n＝6)：其X单元序列从X1-X6依次为表2的序列96-101。
表1、核酸单元的序列
靶基因 序列(5'-3') 序列编号
VEGFA AGCAGAAAGUUCAUGGUUUCUUGggugcau 42
VEGFA AUGCACCCAAGACAGCAGAGAUCgaguaca 43
VEGFA UGUACUCGAUCUCAUCAGGGUGGacaucuu 44
VEGFA AAGAUGUCCACCAGGGUCAUGCGgaucaaa 45
VEGFA UUUGAUCCGCAUAAUCUGGGCCAgcacaua 46
VEGFA UAUGUGCUGGCCUUGGUGGAACUuucugcu 47
VEGFA AGCAGAAAGUUCAUGGUUCUUGggugcau 48
VEGFA AUGCACCCAAGACAGCAAGAUCgaguaca 49
VEGFA UGUACUCGAUCUCAUCAGGUGGacaucuu 50
VEGFA AAGAUGUCCACCAGGGUAUGCGgaucaaa 51
VEGFA UUUGAUCCGCAUAAUCUGGCCAgcacaua 52
VEGFA UAUGUGCUGGCCUUGGUGAACUuucugcu 53
VEGFA AGCAGAAAGUUCAUGUCUUGggugcau 54
VEGFA AUGCACCCAAGACAGAGAUCgaguaca 55
VEGFA UGUACUCGAUCUCAUGGUGGacaucuu 56
VEGFA AAGAUGUCCACCAGGAUGCGgaucaaa 57
VEGFA UUUGAUCCGCAUAAUGGCCAgcacaua 58
VEGFA UAUGUGCUGGCCUUGGAACUuucugcu 59
VEGFA AGCAGAAAGUUCAUGGUCUUGggugcau 60
VEGFA AUGCACCCAAGACAGCAGAUCgaguaca 61
VEGFA UGUACUCGAUCUCAUCAGUGGacaucuu 62
VEGFA AAGAUGUCCACCAGGGUUGCGgaucaaa 63
VEGFA UUUGAUCCGCAUAAUCUGCCAgcacaua 64
VEGFA UAUGUGCUGGCCUUGGUAACUuucugcu 65
VEGFA AGCAGAAAGUUCAUGGUUGggugcau 66
VEGFA AUGCACCCAAGACAGCAUCgaguaca 67
VEGFA UGUACUCGAUCUCAUCUGGacaucuu 68
VEGFA AAGAUGUCCACCAGGGGCGgaucaaa 69
VEGFA UUUGAUCCGCAUAAUCCCAgcacaua 70
VEGFA UAUGUGCUGGCCUUGGACUuucugcu 71
表2、核酸单元序列
靶基因 序列(5'-3') 序列编号
TP53 UGUGGAAUCAACCCACAGUUUGCgugugga 72
TP53 UCCACACGCAAAUUUCCUACAGAaacacuu 73
TP53 AAGUGUUUCUGUCAUCCAACUACaugugua 74
TP53 UACACAUGUAGUUGUAGUUGGUAaucuacu 75
TP53 AGUAGAUUACCACUGGAGUCUCCgcaagaa 76
TP53 UUCUUGCGGAGAUUCUCUGUUGAuuccaca 77
TP53 UGUGGAAUCAACCCACAUUUGCgugugga 78
TP53 UCCACACGCAAAUUUCCACAGAaacacuu 79
TP53 AAGUGUUUCUGUCAUCCACUACaugugua 80
TP53 UACACAUGUAGUUGUAGUGGUAaucuacu 81
TP53 AGUAGAUUACCACUGGAUCUCCgcaagaa 82
TP53 UUCUUGCGGAGAUUCUCGUUGAuuccaca 83
TP53 UGUGGAAUCAACCCAUUUGCgugugga 84
TP53 UCCACACGCAAAUUUACAGAaacacuu 85
TP53 AAGUGUUUCUGUCAUACUACaugugua 86
TP53 UACACAUGUAGUUGUUGGUAaucuacu 87
TP53 AGUAGAUUACCACUGUCUCCgcaagaa 88
TP53 UUCUUGCGGAGAUUCGUUGAuuccaca 89
TP53 UGUGGAAUCAACCCACAUUGCgugugga 90
TP53 UCCACACGCAAAUUUCCCAGAaacacuu 91
TP53 AAGUGUUUCUGUCAUCCCUACaugugua 92
TP53 UACACAUGUAGUUGUAGGGUAaucuacu 93
TP53 AGUAGAUUACCACUGGACUCCgcaagaa 94
TP53 UUCUUGCGGAGAUUCUCUUGAuuccaca 95
TP53 UGUGGAAUCAACCCACUGCgugugga 96
TP53 UCCACACGCAAAUUUCAGAaacacuu 97
TP53 AAGUGUUUCUGUCAUCUACaugugua 98
TP53 UACACAUGUAGUUGUAGUAaucuacu 99
TP53 AGUAGAUUACCACUGGUCCgcaagaa 100
TP53 UCUUGCGGAGAUUCUUGAuuccaca 101
注：小写字母表示该核苷酸核糖经过2’‐O-甲基核糖修饰。下划线标记的序列为靶序列。
2、抑制实验
将5×10 5个HeLa细胞(ATCC编号为CRL-1958)接种于含10％胎牛血清的DMEM培养基的12孔培养板中，分别将步骤1制备的聚合核酸转染HeLa细胞：将聚合核酸与转染试剂、缓冲液(广州市锐博生物科技有限公司，名称riboFECT TM CP Buffer，货号C10511-1)混合后加入细胞培养基中，每孔体积1mL，使聚合核酸的转染终浓度为50nM，将培养板置于5％CO 2、37℃恒温培养箱中培养48h。转染试剂及转染具体步骤参照riboFect TM(广州市锐博生物科技有限公司)中的方法。
每次细胞铺板除了试验组，还设置阴性对照组(NC)和未处理对照组(NT)。试验组和对照组均有3个重复。NC组的对照序列为siRNA，为如下两条单链RNA分子互补结合得到的双链RNA分子：5'-UUCUCCGAACGUGUCACGUdTdT-3'和5'-ACGUGACACGUUCGGAGAAdTdT-3'。
37℃、5％CO 2孵育48h后收集转染后细胞，rizol法提取转染后细胞的RNA进行实时定量PCR，检测靶基因mRNA表达水平，q-PCR重复3次，结果均用平均值±SD表示。检测靶基因的实时定量PCR引物序列如附表所示。
检测结果见表3。实验结果表明，以上不同结构的聚合分子均可高效抑制靶基因的表达。
表3、mRNA相对表达水平
  VEGFA TP53
R6-A 0.05 0.l0
R6-B 0.02 0.06
R6-C 0.13 0.21
R5-A 0.06 0.06
R5-B 0.03 0.02
R5-C 0.12 0.12
R4-A 0.04 0.04
R4-B 0.06 0.01
R4-C 0.23 0.13
R6-D 0.06 0.22
R6-E 0.06 0.07
注：阴性对照表达水平为1。
(二)R结构聚合核酸：低浓度抑制实验
1、聚合核酸
制备VEGFA-R6-A聚合核酸的X单元序列和聚合核酸TP53-R6-A的X单元序列同步骤(一)；制备P65-R6-A聚合核酸的X单元序列从X1-X6依次为表5中的序列15、16、17、23、24、25。
2、抑制实验
仅将步骤(一)中的抑制实验的聚合核酸的转染终浓度变为1nM，其他步骤保持不变。
检测结果见表4。实验结果表明，低浓度的本发明的聚合核酸也可实现降低靶基因表达水平的目的。
表4、mRNA相对表达水平
  VEGFA TP53 P65
R6-A(1nM) 0.53 0.64 0.51
注：阴性对照表达水平为1。
(三)三种结构聚合核酸：靶向同一基因的不同区域的抑制实验
1、聚合核酸
制备如下结构的聚合核酸，每个结构的靶向核酸单元分别靶向PPIB或P65基因的4个或6个不同区域。
靶基因为PPIB的聚合核酸为如下1)-5)：
1)PPIB-R4(R结构，n＝4)：其X单元序列从X1-X4依次为表5中的序列1-4。
2)PPIB-L4(L结构，n＝4)：其X单元序列从X1-X4依次为表5中的序列1-3和序列5，其H1单元序列为序列20，其H4单元序列为序列21。
3)PPIB-Cr4(Cr结构，n＝4)：其X单元序列从X1-X4依次为表5中的序列1-4，其C单元序列为序列8。
4)PPIB-R6(R结构，n＝6)：其X单元序列从X1-X6依次为表5中的序列2、3、9、10、11、12。
5)PPIB-L6(L结构，n＝6)：其X单元序列从X1-X6依次为表5中的序列2、3、9、10、11、13，其H1单元序列为序列6，其H6单元序列为序列14。
靶基因为P65的聚合核酸为如下1)-5)：
1)P65-R4(R结构，n＝4)：其X单元序列从X1-X4依次为表5中的序列15-18。
2)P65-L4(L结构，n＝4)：其X单元序列从X1-X4依次为表5中的序列15-17 和序列19，其H1单元序列为序列6，其H4单元序列为序列7。
3)P65-Cr4(Cr结构，n＝4)：其X单元序列从X1-X4依次为表5中的序列15-18，其C单元序列为序列22。
4)P65-R6(R结构，n＝6)：其X单元序列从X1-X6依次为表5中的序列15、16、17、23、24、25。
5)P65-L6(L结构，n＝6)：其X单元序列从X1-X6依次为表5中的序列15、16、17、23、24、26，其H1单元序列为序列20，其H6单元序列为序列27。
表5、核酸单元的序列
靶基因 序列(5'-3') 序列编号
PPIB AGAUGCUCUUUCCUCCUGCAAGGuguauuu 1
PPIB AAAUACACCUUGACGGUGGAUGAagaugua 2
PPIB UACAUCUUCAUCUCCAAUUCUCUucggaaa 3
PPIB UUUCCGAAGAGACCAAAGGAAAGagcaucu 4
PPIB UUUCCGAAGAGACCAAAGUCUACgagaaag 5
PPIB GGAGGAAAGagcaucu 6
PPIB cuuucucguagacuuu 7
PPIB cuuuGGauugGAcaccGUcaggAG 8
PPIB AGAUGCUCUUUCCUCCUGCAUGAaggugcu 9
PPIB AGCACCUUCAUGUUGCGUCAAGGuguauuu 10
PPIB UUUCCGAAGAGACCAAAGCGUGUaaucaag 11
PPIB CUUGAUUACACGAUGGAAGAAAGagcaucu 12
PPIB CUUGAUUACACGAUGGAAUCUACgagaaag 13
PPIB cuuucucguagauucc 14
P65 UGUGUAGCCAUUGAUCUUGCAUCaugaaga 15
P65 UCUUCAUGAUGCUCUUGAAUACCaccaaga 16
P65 UCUUGGUGGUAUCUGUGCUCGUCaccggau 17
P65 AUCCGGUGACGAUCGUCUAAUGGcuacaca 18
P65 AUCCGGUGACGAUCGUCUACACAucgguaa 19
P65 GAUCAAUGGcuacaca 20
P65 uuaccgauguguagac 21
P65 agacGAgcacAGucaaGAaagaUC 22
P65 AUCCGGUGACGAUCGUCUUCAGGagaugaa 23
P65 UUCAUCUCCUGAAAGGAGAUCAGcuccuaa 24
P65 UUAGGAGCUGAUCUGACUAAUGGcuacaca 25
P65 UUAGGAGCUGAUCUGACUACACAucgguaa 26
P65 uuaccgauguguaguc 27
注：小写字母表示该核苷酸核糖经过2-O-甲基核糖修饰。下划线标记的序列为靶序列。X单元：第1-12位为T1；第13-18位为T2；第19-30为A3。H1单元：第1-4位为Hy；第5-16位为Hx。H4单元：第1-12位为Hx；第13-16位为Hy。C单元：第1-6位为C4，第7-12位为C3，第13-18位为C2，第19-24位为C1。
2、抑制实验
抑制实验步骤同步骤(一)。
抑制结果见图7。结果表明，5种结构的聚合核酸均有效抑制了靶基因的表达。针对靶基因PPIB的抑制水平均在80％以上，针对靶基因P65的抑制水平均在75％以上。
(四)R结构聚合核酸靶向不同基因的抑制实验(n＝4和6)
1、聚合核酸
制备如下R结构的聚合核酸，该聚合核酸靶向4个或6个不同的靶基因。
BCCC-R4：靶基因为BIRC5、CTNNB、COPS5、CLU，其X单元序列从X1-X4依次为表3中的序列28-31。
BCCCEH-R6：靶基因为BIRC5、CTNNB、COPS5、CLU、EIF4E、HIF1A，其X单元序列从X1-X6依次为表6中的序列32、33、28、29、30、34。
2、抑制实验
抑制实验步骤同步骤(一)。
表6、核酸单元的序列
靶基因 序列(5'-3') 序列编号
BIRC5 AGAAGAAACACUGGGCCAACUGCugaucuu 28
CTNNB1 AAGAUCAGCAGUCUCAUUUGGUCaggucuu 29
COPS5 AAGACCUGACCAGUGGUAUUUGAcucugau 30
CLU AUCAGAGUCAAAGAGCUUAGUGUuucuucu 31
HIF1A UCAAGUUGCUGGUCAUCAGGAGGuugcuaa 32
EIF4E UUAGCAACCUCCUGAUUAAGUGUuucuucu 33
CLU AUCAGAGUCAAAGAGCUUCCAGCaacuuga 34
注：小写字母表示该核苷酸核糖经过2-O-甲基核糖修饰。下划线标记的序列为靶序列。X单元：第1-12位为T1；第13-18位为T2；第19-30为A3。
抑制实验结果见图8。结果表明，2种结构的聚合核酸均有效抑制了4个或6靶基因的表达。R4结构的聚合核酸靶向的4种基因的表达水平均降低至0.2以下。
(五)R结构、L结构和Cr结构聚合核酸靶向不同基因的抑制实验(n＝4)
1、聚合核酸
制备如下各结构的聚合核酸，该聚合核酸靶向4个不同的靶基因。
SPPV-R4：靶基因为SOD1、PPIB、P65、VEGFA，其X单元序列从X1-X4依次为表7中的序列35-38。
SPPV-L4：靶基因为SOD1、PPIB、P65、VEGFA，其X单元序列从X1-X4依次为表7中的序列35-38，其H1单元序列为序列39，其H4单元序列为序列40。
SPPV-Cr4：靶基因为SOD1、PPIB、P65、VEGFA，其X单元序列从X1-X4依次为表7中的序列35-38，其C单单元序列为序列41。
2、抑制实验
同步骤(一)中的抑制实验。
抑制实验结果见图9。结果表明，3种结构的聚合核酸均有效抑制了4种靶基因的表达。并发现，在三种聚合结构中，Cr4结构的聚合核酸的抑制效果最好，其每个靶基因的表达水平均为最低。
表7、核酸单元的序列
靶基因 序列(5'-3') 序列编号
SOD1 UACUUUCUUCAUUUCCACCUGUUccaaaaa 35
PPIB UUUUUGGAACAGUCUUUCUGAGAccuucaa 36
P65 UUGAAGGUCUCAUAUGUCCAUGCagauuau 37
VEGFA AUAAUCUGCAUGGUGAUGAUGAAgaaagua 38
H GGAAAUGAAgaaagua 39
H4 uacuuucuucaucauc 40
C caucACgacaUAgaaaGAguggAA 41
注：小写字母表示该核苷酸核糖经过2-O-甲基核糖修饰。下划线标记的序列为靶序列。X单元：第1-12位为T1；第13-18位为T2；第19-30为A3。H1单元：第1-4位为Hy；第5-16位为Hx。H4单元：第1-12位为Hx；第13-16位为Hy。C单元：第1-6位为C4，第7-12位为C3，第13-18位为C2，第19-24位为C1。
上述各实验中检测靶基因的实时定量PCR引物序列如表8所示。
表8、检测靶基因的实时定量PCR引物序列
引物名称 引物序列(5'-3')
H-TP53-qPCR-F TTGTGCCTGTCCTGGGAGAG
H-TP53-qPCR-R GGAGAGGAGCTGGTGTTGTTG
H-HIF1A-qPCR-F GCCCTAACGTGTTATCTGTC
H-HIF1A-qPCR-R CGCTTTCTCTGAGCATTCTG
h-EIF4E-qPCR-F GGAGGTTGCTAACCCAGAACAC
h-EIF4E-qPCR-R GGAGATCAGCCGCAGGTTTG
h-VEGFA-qPCR-F GAGGGCAGAATCATCACGAAG
h-VEGFA-qPCR-R ACTCGATCTCATCAGGGTACTC
h-PPIB-qPCR-F GGCAAGCATGTGGTGTTTGG
h-PPIB-qPCR-R GGTTTATCCCGGCTGTCTGTC
h-p65-qPCR-F GGGAAGGAACGCTGTCAGAG
h-p65-qPCR-R TAGCCTCAGGGTACTCCATCA
h-SOD1-qPCR-F GCAGGGCATCATCAATTTCG
h-SOD1-qPCR-R GAATCCATGCAGGCCTTCAG
H-BIRC5-qPCR-F AGAACTGGCCCTTCTTGGAG
H-BIRC5-qPCR-R GAAACACTGGGCCAAGTCTG
H-CTNNB1-qPCR-F GCTCGGGATGTTCACAACC
H-CTNNB1-qPCR-R CCCTGCAGCTACTCTTTGG
H-COPS5-qPCR-F TGGAATAAATACTGGGTGAATACG
H-COPS5-qPCR-R GGCTTCTGACTGCTCTAAC
H-CLU-qPCR-F CAAGGCGAAGACCAGTACTATC
H-CLU-qPCR-R CAGTGACACCGGAAGGAAC
工业应用
本发明提供了新型的可用于构建聚合核酸的核酸单元及可用于干扰目的基因表达的聚合核酸。本发明通过设计和构建新型的核酸单元及其自组装聚合核酸，实现了多靶标干扰，可用于抑制疾病发生或发展的信号通路中的多个基因表达，或同时抑制多个疾病靶基因表达，在生物学、化学等多个学科领域具有广阔的应用前景。该聚合核酸可实现同时靶向多个序列，所述序列可在一个基因中，或位于多个基因中。本发明优势在于：1)RNAi效能高；2)稳定性好；3)降低脱靶率；4)导入细胞能力增强；5)可模块化设计。

Claims (26)

  1. 一种用于干扰靶基因表达的聚合核酸分子，所述聚合核酸分子由n条X型核酸分子组成；
    每条所述X型核酸分子自5’端起依次由靶向片段一、靶向片段二和接头片段三组成；
    每条所述X型核酸分子的靶向片段一与其相邻的X型核酸分子的接头片段三互补配对；
    每条所述聚合核酸分子的靶向片段一和靶向片段二均与靶基因互补配对；
    每条所述X型核酸分子的长度相同；
    所述n为大于或等于3的整数。
  2. 根据权利要求1所述的聚合核酸分子，其特征在于：所述聚合核酸分子还包括H型核酸分子；所述H型核酸分子由H1型核酸分子和Hn型核酸分子组成；
    将n条X型核酸分子分别命名为X1单元、X2单元、X3单元，依次类推，Xn-1单元、Xn单元；
    所述X1单元的接头片段三与所述X2单元的靶向片段一互补配对，所述X2单元的接头片段三与所述X3单元的靶向片段一互补配对，依次类推，所述Xn-1单元的接头片段三与所述Xn单元的靶向片段一互补配对；
    所述H1型核酸分子与所述X1单元的靶向片段一互补配对，所述Hn型核酸分子与所述Xn单元的接头片段三互补配对。
  3. 根据权利要求2所述的聚合核酸分子，其特征在于：
    所述H型核酸分子的5’端或3’端还包括Hy片段；所述H型核酸分子由Hx片段和Hy片段组成；
    所述H1型核酸分子的Hx片段与所述X1单元的靶向片段一互补配对；所述Hn型核酸分子的Hx片段与所述Xn单元的接头片段三互补配对；
    所述Hy片段与所述X1单元或所述Xn单元的靶向片段二自5’端或3’端起1个、2个或多个连续的核酸分子互补配对。
  4. 根据权利要求1所述的聚合核酸分子，其特征在于：所述聚合核酸分子还包括C型核酸分子；
    所述C型核酸分子由n个片段依次连接而成，n个片段分别为n条所述X型核酸分子中的靶向片段二的反向互补序列。
  5. 根据权利要求1-4任一所述的聚合核酸分子，其特征在于：所述X型核酸分子、所述H型核酸分子和所述C型核酸分子均为单链RNA分子。
  6. 根据权利要求1或3所述的聚合核酸分子，其特征在于：
    所述X型核酸分子的长度为15-50nt；
    所述靶向片段一的长度为5-24nt；
    所述靶向片段二的长度为1-20nt；
    所述接头片段三的长度为5-24nt；
    所述靶向片段一和所述靶向片段二的长度总和至少为14-16nt。
  7. 根据权利要求6所述的聚合核酸分子，其特征在于：所述X型核酸分子的长度为24-36nt。
  8. 根据权利要求1所述的聚合核酸分子，其特征在于：所述聚合核酸分子至少包括一个修饰的核苷酸。
  9. 根据权利要求8所述的聚合核酸分子，其特征在于：所述修饰为磷酸骨架修饰、碱基修饰和/或核糖修饰。
  10. 根据权利要求9所述的聚合核酸分子，其特征在于：所述核糖修饰为核糖2位羟基基团被卤素基团或O-烷基基团取代。
  11. 根据权利要求10所述的聚合核酸分子，其特征在于：所述烷基为甲基、乙基、丙基或甲乙基。
  12. 根据权利要求10所述的聚合核酸分子，其特征在于：所述X型核酸分子的接头片段三自3’端第一位核苷酸起连续的5-9个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代；
    或，所述H型核酸分子的Hx片段自3’端第一位核苷酸起连续的5-9个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代；
    或，所述H型核酸分子自3’端第一位核苷酸起连续的8-30个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代；
    或，所述C型核酸分子中与X型核酸分子的靶向片段二互补配对的每个片段自5’端第一位核苷酸起连续的2-6个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代。
  13. 根据权利要求12所述的聚合核酸分子，其特征在于：所述H型核酸分子自3’端第一位核苷酸起连续的14-18个核苷酸的核糖2位羟基基团被卤素基团或O-烷基基团取代。
  14. 根据权利要求1所述的聚合核酸分子，其特征在于：所述n为3或4或5或6或7或8。
  15. 根据权利要求14所述的聚合核酸分子，其特征在于：所述n为4或5或6。
  16. 根据权利要求1所述的聚合核酸分子，其特征在于：所述靶基因的个数为一个或两个或多个，且所述靶基因的个数不超过n个；
    或，所述靶基因的个数为1个或4个或6个。
  17. 根据权利要求1所述的聚合核酸分子，其特征在于：所述靶基因为如下基因中至少一种：PPIB基因、p65基因、BIRC5基因、CTNNB基因、COPS5基因、CLU基因、EIF4E基因、HIF1A基因、TP53基因、VEGFA基因和SOD1基因。
  18. 根据权利要求17所述的聚合核酸分子，其特征在于：
    用于干扰靶基因PPIB表达的聚合核酸分子为如下a1)-a5)：
    a1)由序列1、序列2、序列3和序列4所示的单链RNA分子组成；
    a2)由序列1、序列2、序列3、序列4、序列20和序列21所示的单链RNA分子组成；
    a3)由序列1、序列2、序列3、序列4和序列8所示的单链RNA分子组成；
    a4)由序列2、序列3、序列9、序列10、序列11和序列12所示的单链RNA分子组成；
    a5)由序列2、序列3、序列6、序列9、序列10、序列11、序列13和序列14所示的所示的单链RNA分子组成；
    或，用于干扰靶基因P65表达的聚合核酸分子为如下b1)-b5)：
    b1)由序列15、序列16、序列17和序列18所示的单链RNA分子组成；
    b2)由序列6、序列7、序列15、序列16、序列17和序列19所示的单链RNA分子组成；
    b3)由序列15、序列16、序列17、序列18和序列22所示的单链RNA分子组成；
    b4)由序列15、序列16、序列17、序列23、序列24和序列25所示的单链RNA分子组成；
    b5)由序列15、序列16、序列17、序列20、序列23、序列24、序列26和序列27所示的所示的单链RNA分子组成；
    或，用于同时干扰靶基因BIRC5、CTNNB、COPS5和CLU表达的聚合核酸分子为由序列28、序列29、序列30和序列31所示的单链RNA分子组成；
    或，用于同时干扰靶基因BIRC5、CTNNB、COPS5、CLU、EIF4E和HIF1A表达的聚合核酸分子为由序列28、序列29、序列30、序列32、序列33和序列34所示的单链RNA分子组成；
    或，用于同时干扰靶基因SOD1、PPIB、P65和VEGFA表达的聚合核酸分子为如下c1)-c3)：
    c1)由序列35、序列36、序列37和序列38所示的单链RNA分子组成；
    c2)由序列35、序列36、序列37、序列38、序列39和序列40所示的单链RNA分子组成；
    c3)由序列35、序列36、序列37、序列38和序列41所示的单链RNA分子组成；
    或，用于干扰靶基因VEGFA表达的聚合核酸分子为如下d1)-d11)：
    d1)由序列42、序列43、序列44、序列45、序列46和序列47所示的单链RNA分子组成；
    d2)由序列48、序列49、序列50、序列51、序列52和序列53所示的单链RNA分子组成；
    d3)由序列54、序列55、序列56、序列57、序列58和序列59所示的单链RNA分子组成；
    d4)由序列42、序列43、序列45、序列46和序列47所示的单链RNA分子组成；
    d5)由序列48、序列49、序列51、序列52和序列53所示的单链RNA分子组成；
    d6)由序列54、序列55、序列57、序列58和序列59所示的单链RNA分子组成；
    d7)由序列42、序列45、序列46和序列47所示的单链RNA分子组成；
    d8)由序列48、序列51、序列52和序列53所示的单链RNA分子组成；
    d9)由序列54、序列57、序列58和序列59所示的单链RNA分子组成；
    d10)由序列60、序列61、序列62、序列63、序列64和序列65所示的单链RNA分子组成；
    d11)由序列66、序列67、序列68、序列69、序列70和序列71所示的单链RNA分子组成；
    或，用于干扰靶基因TP53表达的聚合核酸分子为如下e1)-e11)：
    e1)由序列72、序列73、序列74、序列75、序列76和序列77所示的单链RNA分子组成；
    e2)由序列78、序列79、序列80、序列81、序列82和序列83所示的单链RNA分子组成；
    e3)由序列84、序列85、序列86、序列87、序列88和序列89所示的单链RNA分子组成；
    e4)由序列73、序列74、序列75、序列76和序列77所示的单链RNA分子组成；
    e5)由序列79、序列80、序列81、序列82和序列83所示的单链RNA分子组成；
    e6)由序列54、序列55、序列57、序列58和序列59所示的单链RNA分子组成；
    e7)由序列74、序列75、序列76和序列77所示的单链RNA分子组成；
    e8)由序列80、序列81、序列82和序列83所示的单链RNA分子组成；
    e9)由序列序列86、序列87、序列88和序列89所示的单链RNA分子组成；
    e10)由序列90、序列91、序列92、序列93、序列94和序列95所示的单链RNA分子组成；
    e11)由序列96、序列97、序列98、序列99、序列100和序列101所示的单链RNA分子组成。
  19. 权利要求1-18任一所述的聚合核酸分子的衍生物，为如下(m1)-(m5)中任一种：
    (m1)将权利要求1-18任一所述的聚合核酸分子删除或增加一个或几个核苷酸，得到与所述聚合核酸分子具有相同功能的聚合核酸分子的衍生物；
    (m2)将权利要求1-18任一所述的聚合核酸分子进行核苷酸取代或修饰，得到与所述聚合核酸分子具有相同功能的聚合核酸分子的衍生物；
    (m3)将权利要求1-18任一所述的聚合核酸分子的骨架改造为硫代磷酸脂骨架，得到与所述聚合核酸分子具有相同功能的聚合核酸分子的衍生物；
    (m4)由权利要求1-18任一所述的聚合核酸分子编码的肽核酸、锁核酸或解锁核酸，得到与所述聚合核酸分子具有相同功能的聚合核酸分子的衍生物；
    (m5)将权利要求1-18任一所述的聚合核酸分子的一端或中间接上信号分子和/或活性分子和/或功能基团，得到与所述聚合核酸分子具有相同功能的聚合核酸分子的衍生物。
  20. 权利要求1-18任一所述的聚合核酸分子的制备方法，包括如下步骤：
    M1)合成权利要求1-18中任一所述的X型核酸分子和/或H型核酸分子和/或C型核酸分子；
    M2)将所述X型核酸分子和/或所述H型核酸分子和/或所述C型核酸分子退火，得到所述聚合核酸分子。
  21. 权利要求1-18任一所述的聚合核酸分子或权利要求19所述的衍生物在如下A1)或A2)中的应用；
    A1)调控细胞中靶基因表达水平；
    A2)制备预防或缓解或治疗由靶基因表达引起的疾病的产品。
  22. 根据权利要求21所述的应用，其特征在于：所述调控为抑制或降低或干扰。
  23. 根据权利要求21所述的应用，其特征在于：所述细胞为肿瘤细胞。
  24. 根据权利要求21所述的应用，其特征在于：所述靶基因为疾病相关基因；
    或，所述疾病相关基因为肿瘤相关基因；
    或，所述肿瘤相关基因为如下基因中至少一种：PPIB基因、p65基因、BIRC5基因、CTNNB基因、COPS5基因、CLU基因、EIF4E基因、HIF1A基因、TP53基因、VEGFA基因和SOD1基因。
  25. 一种抑制或降低或干扰细胞中靶基因表达水平的试剂或试剂盒或药物，包括权利要求1-18任一所述的聚合核酸分子或权利要求19所述的衍生物。
  26. 一种抑制或降低或干扰细胞中靶基因表达水平的方法，包括如下步骤：将权利要求1-18任一所述的聚合核酸分子或权利要求19所述的衍生物导入所述细胞，实现抑制或降低所述细胞中靶基因的表达水平。
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