WO2012130086A1 - 抑制rna干扰脱靶效应的特异性修饰 - Google Patents
抑制rna干扰脱靶效应的特异性修饰 Download PDFInfo
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- WO2012130086A1 WO2012130086A1 PCT/CN2012/072861 CN2012072861W WO2012130086A1 WO 2012130086 A1 WO2012130086 A1 WO 2012130086A1 CN 2012072861 W CN2012072861 W CN 2012072861W WO 2012130086 A1 WO2012130086 A1 WO 2012130086A1
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- WIPO (PCT)
- Prior art keywords
- sirna
- dna
- sense strand
- chemically modified
- artificial
- Prior art date
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- 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
- C12N2310/30—Chemical structure
- C12N2310/34—Spatial arrangement of the modifications
- C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end
-
- 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
- C12N2320/00—Applications; Uses
- C12N2320/50—Methods for regulating/modulating their activity
- C12N2320/53—Methods for regulating/modulating their activity reducing unwanted side-effects
Definitions
- the invention relates to the field of nucleic acid technology, in particular to a chemically modified double-stranded small interfering RNA (siRNA) molecule having the effect of reducing the sense of off-target of a sense strand, and a preparation method thereof, and the application of the modified siRNA molecule in preparing a pharmaceutical composition .
- siRNA small interfering RNA
- RNA interference is a phenomenon in which homologous gene expression is reduced at the mRNA level by double-stranded RNA (dsRNA).
- dsRNA double-stranded RNA
- RNA interference technology also known as knock-down or gene silencing, is a post-transcriptional gene expression regulation method, hence the name post-transcriptional gene silencing. , PTGS).
- RNA interference occurred around 1990, and two different research groups reported on RNA interference found in transgenic plants, followed by observation in almost all epigenetic organisms such as nematodes, fruit flies, zebrafish, and mice.
- RNA interference found in transgenic plants, followed by observation in almost all epigenetic organisms such as nematodes, fruit flies, zebrafish, and mice.
- Hamilton and Baulcombe detected double-stranded RNA fragments of 21 to 25 nucleotides in length in plants with RNA interference. These double-stranded RNA fragments have been shown to be required for RNA interference.
- Small interfering RNA siRNA
- Double-stranded siRNAs form cell-derived related enzymes and proteins.
- RISC RNA-induced silencing complex
- the sense strand (passenger strand) in the double-stranded siRNA is excreted by the RISC complex, and the antisense strand directs the RISC complex to bind to the homologous site on the target mRNA, and then from the complex Ribonuclease III degrades the target mRNA, thereby shutting down the expression of the target gene.
- both strands are involved in the binding of the RISC complex, that is, in addition to the antisense strand (guide strand) of the siRNA that mediates the expression silencing of homologous genes.
- the sense strand (passenger chain) of siRNA also mediates the silencing of expression of its homologous genes, triggering off-target effects mediated by the sense strand.
- this off-target effect of siRNA sense strands may lead to misinterpretation of siRNA inhibitory activity; in small nucleic acid pharmacies, it may trigger a series of consequences such as toxic side effects of siRNA drugs, which will seriously affect Application of RNA interference technology.
- the present invention relates to the following topics defined in the paragraphs numbered sequentially:
- siRNA small interfering RNA
- siRNA small interfering RNA
- siRNA small interfering RNA
- the chemically modified siRNA molecule according to paragraph 4 characterized in that the chemical modification is such that the phosphodiester bond between the nucleotides is replaced by a phosphorothioate bond.
- the chemically modified siRNA molecule according to paragraph 8 characterized in that the non-RNA base is selected from the group consisting of thymine, 5-methylcytosine, isocytosine, Pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 5-propynyl-6-fluorouracil (5-propyny-6 -fluoroluracil), 5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, 2,6-diaminopurine (2,6-( ⁇ 1 ⁇ ,1 ⁇ 116), 7-propyne-7-deazaadenine, 7-propynyl-7-deazaguanine (7-propyne-7-deazaguanine), 2-chloro-6-aminopurine.
- the base chemically modified to be a nucleotide is substituted with a DNA base.
- siRNA molecule of any of paragraphs 1-14, wherein the siRNA molecule is a blunt-ended siRNA molecule.
- siRNA molecule of any of paragraphs 1-14, wherein the siRNA molecule is a 3' overhanging siRNA molecule.
- siRNA molecule is an siRNA molecule having 1 to 6 3' overhangs.
- siRNA molecule is a siRNA molecule having 2-4 3' overhangs.
- a pharmaceutical composition comprising the chemically modified siRNA molecule of any of paragraphs 1-17 and a pharmaceutically acceptable carrier.
- a method for reducing the off-target effect of a sense strand of a siRNA molecule characterized in that the nucleotide of the 14th position of the sense strand of the siRNA molecule from the 5' end is chemically modified.
- a method for reducing the off-target effect of a sense strand of a siRNA molecule characterized in that the nucleotide of the 16th position of the sense strand of the siRNA molecule from the 5' end is chemically modified.
- a method for reducing the off-target effect of a sense strand of a siRNA molecule characterized in that the nucleotides of the sense strand of the siRNA molecule are chemically modified at positions 14 and 16 from the 5' end.
- non-RNA base is selected from the group consisting of thymine, 5-methylcytosine, and isopyrimidine ( Isocytosine), pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 5-propynyl-6-fluorouracil ( 5-propyny-6-fluoroluracil), 5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, 2, 6-Diaminopurine (2,6-( ⁇ 1 ⁇ ,1 ⁇ 116), 7-propynyl-7-deaza adenine (7 1" ( ⁇ 116-7-01632330161 ⁇ 116),
- 36. The method of reducing the off-target effect of a sense strand of a siRNA molecule according to paragraph 35, characterized in that the siRNA molecule is a siRNA molecule having 2-4 3' overhangs.
- the technical problem to be solved by the present invention is that the sense strand of the siRNA molecule mediates the silencing of the expression of its complementary gene transcript, thereby causing the problem of the off-target effect mediated by the sense strand.
- An object of the present invention is to provide a chemically modified siRNA molecule having an off-target effect caused by a sense strand and a method for reducing the off-target effect of a sense strand of a siRNA molecule.
- an aspect of the present invention provides an isolated chemically modified double-stranded small interfering RNA (siRNA) molecule comprising a sense strand (passenger strand) and an antisense strand (guide strand), the modified siRNA molecule
- siRNA small interfering RNA
- the nucleotide of the sense strand from the 5' end at the 14th or 16th position is a chemically modified nucleotide.
- the invention also provides an isolated chemically modified double-stranded small interfering RNA (siRNA) molecule comprising a sense strand (passenger strand) and an antisense strand (guide strand), the sense strand of the modified siRNA molecule from 5'
- the nucleotides at the 14th and 16th positions are all chemically modified nucleotides.
- the chemical modification described in the present invention is selected from one or more of the following modifications: (1) chemical modification of ribose in a nucleotide; (2) chemical modification of a base in a nucleotide; 3) Chemical modification of phosphodiester bonds between nucleotides.
- the chemical modification is a modification of the ribose 2'-OH in the nucleotide; more preferably, the chemical modification is such that the ribose 2'-OH in the nucleotide is replaced by a methoxy or fluoro . In another preferred aspect, the chemical modification is such that the phosphodiester bond between the nucleotides is replaced by a phosphorothioate linkage.
- the chemically modified base of the nucleotide is substituted with a non-RNA base; preferably, the non-RNA base is selected from the group consisting of thymine, 5-methylcytosine ( 5-methylcytosine), isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 5-propyne 5-propyny-6-fluoroluracil, 5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine , inosine, 2,6-diaminopurine, 7-propyne-7-deazaadenine, 7-propynyl- 7-propyne-7-deazaguanine, 2-chloro-6-aminopurine.
- the chemical modification is such that a base in a nucleotide is substituted with a DNA base.
- any of the above chemically modified siRNA molecules may have a sense strand and an antisense strand of 15-35 nucleotides in length, preferably 19-23 nucleotides in length, more preferably 21 nucleotides in length.
- Any of the above chemically modified siRNA molecules which may be blunt ends or 3' overhangs, and the 3' overhangs may be 1 to 6 nucleotides, preferably 2-4 nucleotides. More preferably, it is 2 nucleotides.
- Another aspect of the invention provides a pharmaceutical composition
- a pharmaceutical composition comprising any of the chemically modified siRNA molecules described above and a pharmaceutically acceptable carrier.
- the "pharmaceutically acceptable carrier” used in the present invention should be combined with the drug of the present invention.
- the small interfering RNA (siRNA) molecules in the composition are compatible with each other, i.e., can be blended therewith without substantially reducing the effect of the pharmaceutical composition on inhibiting gene expression.
- delivery vehicles include, but are not limited to, a variety of carriers useful for nucleic acid administration, such as liposomes, degradable polymeric compounds, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
- compositions of the present invention can be formulated into a variety of medically acceptable dosage forms, and can be administered by a physician to determine a dosage that is beneficial to the patient based on factors such as the type of patient, age, weight, and general condition, mode of administration, and the like.
- the preparation of the present invention refers to various liquid dosage forms such as an oral solution, an injection, a sublingual preparation, or the like, or a plurality of other dosage forms such as tablets and capsules are prepared by using appropriate excipients.
- the invention also provides the use of any of the above chemically modified siRNA molecules for the preparation of a pharmaceutical composition.
- a method of reducing off-target effects caused by a sense strand of a siRNA molecule is provided. Chemically modifying the nucleotide of the 14th or 16th position from the 5' end of the sense strand of the siRNA molecule, or the 14th and 16th positions of the sense strand of the siRNA molecule from the 5' end Simultaneous chemical modification of the nucleotides can significantly reduce the off-target effect caused by the sense strand of the modified siRNA molecule.
- the chemical modification described in the present invention is selected from one or more of the following modifications: (1) chemical modification of ribose in a nucleotide; (2) chemical modification of a base in a nucleotide; 3) Chemical modification of phosphodiester bonds between nucleotides.
- the chemical modification is a modification of the ribose 2'-OH in the nucleotide; more preferably, the chemical modification is such that the ribose 2'-OH in the nucleotide is replaced by a methoxy or fluoro . In another preferred aspect, the chemical modification is such that the phosphodiester bond between the nucleotides is replaced by a phosphorothioate linkage.
- the chemically modified base of the nucleotide is substituted with a non-RNA base; preferably, the non-RNA base is selected from the group consisting of thymine, 5-methylcytosine ( 5-methylcytosine), isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 5-propyne 5-propyny-6-fluoroluracil, 5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine , inosine, 2,6-diaminopurine, 7-propyne-7-deazaadenine, 7-propynyl- 7-propyne-7-deazaguanine, 2-chloro-6-aminopurine.
- the chemical modification is such that a base in a nucleotide is substituted with a DNA base.
- any of the above chemically modified siRNA molecules may have a sense strand and an antisense strand of 15 to 35 nucleotides in length, preferably 19 to 23 nucleotides in length, and optionally have a length of 21 nucleotides.
- Any of the above chemically modified siRNA molecules, which may be blunt ends or 3' overhangs, and the 3' overhangs may be 1 to 6 nucleotides, preferably 2 to 4 nucleotides, more It is preferably 2 nucleotides.
- the chemically modified siRNA molecule provided by the invention can significantly reduce the off-target effect caused by the sense strand of the siRNA molecule and enhance the specificity of the siRNA to silence the target gene expression.
- the researchers introduced an siRNA molecule into the cell, and the gene silencing mediated by the antisense strand (guide strand) is expected by the researchers; and the gene silencing mediated by the sense strand (passenger chain) is RNA.
- the side effects in the process of interference are called off-target effects caused by the sense chain.
- the inventors conducted a detailed study on the correlation between the chemical modification of the nucleotides at different sites of the siRNA sense strand and the off-target effect. It was found that the chemical modification of the nucleotide of the siRNA sense strand from the 5' end to the 14th or 16th position can significantly reduce the expression inhibitory activity of the sense strand on its complementary gene transcript, ie, inhibit the siRNA sense strand. The off-target effect; at the same time, the chemical modification does not affect the gene silencing activity of the unmodified antisense strand of the siRNA molecule. On this basis, the inventors completed the present invention.
- the term "antisense strand (guide strand)” refers to a siRNA-constituting strand of a double-stranded siRNA molecule which is complementary to a sequence of a target site in a transcript of a target gene.
- Sense strand (passenger strand) refers to another constituent strand of a siRNA molecule that is complementary to the antisense strand sequence of the siRNA molecule.
- the "sense strand target sequence” refers to a gene sequence complementary to a sense strand (passenger strand) in an siRNA molecule or a homologous sequence of the gene sequence.
- Antisense strand target sequence refers to a gene sequence that is complementary to an antisense strand (guide strand) in an siRNA molecule or a homologous sequence of the gene sequence.
- the modified small interfering RNA (siRNA) molecule may be composed of ribonucleotides, and may also include a hybrid molecule of a ribonucleotide and at least one deoxyribonucleotide.
- nucleic acid synthesis methods can be used to complete the synthesis of the nucleic acid molecules involved in the present invention, or a biotechnology company specialized in nucleic acid synthesis services can be entrusted to complete the synthesis of nucleic acid molecules, such as entrusting Guangzhou Ruibo Biotechnology Co., Ltd. Or Yingjun Biotechnology Co., Ltd. (invitrogen) for synthesis.
- oligonucleic acid molecules involves the following four steps: (1) synthesis of oligoribonucleotides; (2) deprotection; (3) purification separation; (4) desalting.
- siRNA having the nucleotide sequence shown in SEQ ID NO: 1 are as follows:
- a sense strand or antisense strand oligonucleotide sequence for synthesizing 1 millimolar amount of siRNA is set on an automatic DNA/RNA synthesizer (for example, Applied Biosystems EXPEDITE 8909), setting each The coupling time of the cycle is 10-15 minutes, the starting material is a solid phase-linked 5'-0-p-dimethoxy-thymidine support, and the first cycle connects a base on the solid support. And then at the nth time (2 ⁇ n ⁇ 35) In the loop, one base is ligated to the base to which the n-1th cycle is ligated, and this cycle is repeated until the synthesis of all nucleic acid sequences is completed.
- an automatic DNA/RNA synthesizer for example, Applied Biosystems EXPEDITE 8909
- the obtained crude product of the oligonucleotide was dissolved in 2 ml of an aqueous solution of ammonium acetate having a concentration of 1 mol/ml, and then separated by C18 high pressure liquid chromatography to obtain a purified oligonucleotide product.
- the manner of chemical modification is well known to those skilled in the art.
- the chemical modification of the nucleic acid molecule of the present invention is one or more of the following modifications:
- the modification of the phosphodiester bond includes, but is not limited to, modification of oxygen in the phosphodiester bond, such as Phosphorthioate and Boranophosphate, which are replaced by sulfur and boron quinone, respectively. Oxygen in the phosphodiester bond. Both modifications stabilize the structure of the siRNA molecule, maintaining the specificity and affinity of base pairing. Boron phosphate-modified siRNA is highly hydrophobic and easily forms hydrated proteins in plasma, and has less toxic side effects to human body than phosphorothioate-modified siRNA.
- Such ribose modifications include, but are not limited to, modifications to the hydroxyl group (2'-OH) in the nucleotide pentose.
- the introduction of certain substituents such as methoxy or fluorine at the hydroxyl position of the ribose gives the siRNA a stronger resistance to nuclease hydrolysis.
- Modifications to the hydroxyl group in the nucleotide pentose include 2'-fluro modification, 2'-oxymethyl modification (2'- ⁇ ), 2'-methoxyethyl modification (2'- ⁇ ), 2,4'-dinitrophenol modification (2'-DNP modification), locked nucleic acid (LNA), 2'-amino modification (Amina modification 2'-deoxy modification (2'-Deoxy modification).
- the base modifications include, but are not limited to, modifications to the bases of the nucleotides, such as 5'-bromo-uracil and 5'-, which introduce bromine or iodine at the 5 position of uracil.
- 5'-iodo-uracil modification is a commonly used base modification method, and other N3-methyl-uracil modification, 2,6-diaminopurine (2,6) -diaminopurine) modification, etc.
- N3-methyluracil 2,6-diaminopurine is preferably a modification of 2'-OH of ribose in a nucleotide of said nucleic acid molecule; further preferably, said modification is said The 2'-OH of the ribose in the nucleotide of the nucleic acid molecule is substituted with a methoxy group or a fluorine.
- the gene of interest of the siRNA molecule may be various genes, and a gene to be analyzed in a cell may be used as a gene of interest, and a gene required to inhibit expression may be used as a gene of interest, or may be associated with a disease or A dysfunction-related gene is used as a gene of interest, such as an oncogene, a viral gene, a cell membrane surface receptor gene, a nuclear receptor gene, or a gene on a cell signaling pathway.
- a person skilled in the art can design a target gene-specific siRNA molecule according to the sequence of the target gene, for example, input the sequence number of the target gene sequence or the target gene in the NCBI Genbank into various siRNA design programs, such as the Insert Design Tool for the shRNA Vectors (Ambion), shRNA Explorer (Gene Link), siDirect (Yuki Naito et al. University of Tokyo), SiRNA at Whitehead (Whitehead Institute for Biomedical Research), BLOCK-iT RNAi Designer (invitrogen), RNAi Design (IDT) , RNAi Explorer (Gene Link), siRNA Target Finder (Ambion), or siSearch (Stockholm Bioinformatics Center), etc.
- siRNA design programs such as the Insert Design Tool for the shRNA Vectors (Ambion), shRNA Explorer (Gene Link), siDirect (Yuki Naito et al. University of Tokyo), SiRNA at Whitehead (Whitehead Institute for Biomedical Research), BLOCK-iT RNAi Designer (invit
- the design program designs siRNA sequences for the provided gene sequences according to the designer's requirements and siRNA design principles. Some programs are also capable of performing genome-wide or transcript-based homology analysis of designed siRNA sequences to screen for siRNA molecules specific for the sequence of the gene of interest.
- the siRNA design procedures described above and the principles involved therein are well known to those skilled in the art, the entire disclosure of which is incorporated herein by reference.
- the invention will be described in further detail below with reference to examples. It is to be understood that the examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the reagents and culture media used in the present invention are all commercially available.
- the Invitrogen Beijing Branch was commissioned to synthesize the siRNA target sequence fragment in Table 1 for the construction of the fusion reporter expression vector.
- siRNA target site sequence was designed for the target site sequence in Table 1, and Guangzhou Ruibo Biotechnology Co., Ltd. was commissioned to synthesize the chemically modified and unmodified small interfering RNA (siRNA) listed in Table 2-11.
- siRNA target site sequence was designed for the target site sequence in Table 1, and Guangzhou Ruibo Biotechnology Co., Ltd. was commissioned to synthesize the chemically modified and unmodified small interfering RNA (siRNA) listed in Table 2-11.
- NPY-305 (As16, 69% 65% 5'-UGAGAGAAAGCACAGAAAAtt 5'-UUUUCUGUGCUUUC
- CM04-N27 (16, 76% 5'-GUGUCAGUGCGCAGCUGAAGGUUAUUGtt 5'-AGUAUUCUCAAUAAC(C) m UUCAGCU
- CM05-N27 (16, 82% 5'-GACUGGUGUUGUGAAGUUUACUCCGUAtt 5'-UCUUCGUCUACGGAG(U) m AAACUUC
- HEK293 human embryonic kidney cells (HEK293) cultured in DMEM medium (10% FBS, 2 mM L-glutamine, 100 units/ml penicillin and 100 g/ml streptomycin) were inoculated into a 24-well plate. Medium (1 0 5 cells / 0.5 ml medium / well). After the cells were grown for 24 hours, when the degree of cell fusion was about 50-70%, the medium was changed to Opti-MEM medium (Gibco).
- the recombinant firefly luciferase reporter plasmid carrying the siRNA isolated target site was transfected into the cells using Lipofectamine 2000 (Invitrogen, USA), and pRL-TK (encoded Renilla luciferase) was also transfected into the cells.
- Control plasmid Promega, Madison Wl, USA
- Each well contained 0.17 g of recombinant plasmid and 0.017 g of pRL-TK control plasmid with a final concentration of siRNA of 13 nM.
- DMEM medium (10% FBS, 2 mM L-glutamine, 100 units/ml penicillin and 100 g/ml streptomycin).
- siRNAs Two siRNAs (CM-01 and CM-11) were selected, and the recombinant firefly luciferase reporter plasmid containing the siRNA sense strand target site was constructed according to the protocol described in Example 2 (target sequence is shown in Table 1).
- the unmodified siRNA molecules shown in Table 2 were synthesized, as well as chemically modified siRNA molecules containing methoxy-modified nucleotides at positions 2-18 from the 5' end of the siRNA sense strand, respectively.
- the inhibitory activities of various siRNA molecules on the sense strand target site were examined, respectively.
- Each siRNA was made in parallel with 3 replicate wells per experiment, and each experiment was repeated at least 2 times.
- siRNA sequences were selected from each locus to synthesize their unmodified siRNA molecules, and in their sense strands from 5 'Chemically modified siRNA molecule containing a methoxy-modified nucleotide at positions 13-16; a recombinant firefly luciferase reporter gene containing the siRNA sense strand target site was constructed according to the experimental protocol described in Example 2 Plasmid (target site sequence is shown in Table 1); according to the protocol described in Example 2, the inhibitory activity of various modified or unmodified siRNA molecules in Table 3 on a target site complementary to its sense strand was tested; The siRNA was made in parallel for 3 replicates in each experiment, and each experiment was repeated at least 2 times.
- the experimental results shown in Table 3 indicate that when the nucleotide of the 14th position of the sense strand from the 5' end is U, C, or G, chemical modification can significantly reduce the modified siRNA molecule pair.
- the modified siRNA molecule reduces the number of genes complementary to its sense strand Inhibition activity; and the modification of the 13th or 15th point has little effect on the miss target effect of the sense strand.
- nucleic acid sequence-independent effect means that the modified nucleotides and modifications provided by the embodiments can be used with any siRNA sequence regardless of the specific sequence of the siRNA.
- Example 5 Effect of modification of the 14th/16th position of the siRNA positive/antisense strand on the off-target effect of siRNA sense strands As shown in Table 4, two siRNA sequences (cdc-2, NPY-305) were selected and synthesized.
- siRNA molecules An unmodified siRNA molecule, and an siRNA molecule that methoxy-modifies the nucleotide of the sense strand or the antisense strand of the siRNA from the 5' end to the 14th or 16th position, or both
- Modified siRNA molecules, modified and unmodified siRNA molecules are shown in Table 4.
- Recombinant firefly luciferase reporter plasmids containing the isolated target sites complementary to their sense or antisense strands were constructed according to the protocol described in Example 2 (target site sequences are shown in Table 1). The inhibitory activities of various siRNA molecules on isolated target sites complementary to their sense or antisense strands were tested, respectively, according to the protocol described in Example 2. Each siRNA was made in parallel with 3 replicate wells per experiment, and each experiment was repeated at least 2 times. The experimental results shown in Table 4 indicate that the chemical modification of one of the constituent strands of the siRNA only reduces the expression inhibitory activity of the modified strand against the complementary gene, without affecting the other unmodified siRNA consisting of a complementary strand of the gene. Expression of inhibitory activity.
- Example 6 Effect of simultaneous modification of the methoxy group at the 5' end of the siRNA sense strand from the 14th and 16th positions of the sense strand on the off-target effect of the sense strand
- siRNA sequences (CM-01, CM-02, CM-03, CM-04, CM-05) were selected to synthesize unmodified siRNA (Table 3) and simultaneously to the sense strand 5' The methoxy modified siRNA was picked up at positions 14 and 16 (Table 5).
- the recombinant firefly luciferase reporter plasmid containing the siRNA sense strand target sites was constructed according to the experimental protocol described in Example 2 (target sequence is shown in Table 1), and then various siRNAs were combined with recombinant firefly fluorescence.
- the prime enzyme reporter plasmid, pRL-TK (encoded Renilla luciferase) control plasmid was co-transfected into the cells, and 24 hours later, the cells were lysed to detect the activity of the two luciferases.
- siRNA sequences (CM-01, CM-02, CM-03, CM-04, CM-05) were selected to synthesize unmodified siRNA (Table 3) and the modified siRNA shown in Table 6;
- a recombinant firefly luciferase reporter plasmid containing the siRNA sense strand target site was constructed (the target site sequence is shown in Table 1).
- the inhibitory activities of various siRNA molecules on the target sites complementary to their sense strands were examined according to the protocol described in Example 2; each siRNA was made in parallel with 3 replicate wells per experiment, and each experiment was repeated at least 2 times.
- siRNA shown in Table 7 was selected, and unmodified siRNA (Table 3) and 5-methylcytosine modified modified siRNA shown in Table 7 were synthesized; according to the protocol described in Example 2, siRNA-containing siRNA was separately constructed. Recombinant firefly luciferase reporter plasmid at the target site of the chain (target sequence is shown in Table 1). The inhibitory activities of various siRNA molecules against target sites complementary to their sense strands were examined as described in Example 2. Each siRNA was made in parallel with 3 replicate wells per experiment, and each experiment was repeated at least 2 times.
- siRNAs shown in Table 8 were selected, and the unmodified siRNA (Table 3) and the modified siRNA shown in Table 8 were synthesized; according to the protocol described in Example 2, recombinant firefly containing the siRNA sense strand target site was separately constructed. Luciferase reporter plasmid (target sequence shown in Table 1). The inhibitory activities of various siRNA molecules on the target sites complementary to their sense strands were respectively examined according to the protocol described in Example 2; each siRNA was made in parallel with 3 replicate wells per experiment, and each experiment was repeated at least 2 times.
- siRNAs shown in Table 9 were selected, and the unmodified siRNA (Table 3) and the DNA base-modified siRNA shown in Table 9 were synthesized; the siRNA sense strand target sites were constructed according to the protocol described in Example 2, respectively.
- Recombinant firefly luciferase reporter plasmid target sequence shown in Table 1). According to the scheme described in Embodiment 2, separately check The inhibitory activity of various siRNA molecules on a target site complementary to its sense strand is measured. Each siRNA was made in parallel with 3 replicate wells per experiment, and each experiment was repeated at least 2 times.
- siRNAs having 3' overhangs of different lengths were synthesized (the number of nucleotides at the 3' overhang was 0, 1, 4, and 6, respectively, when "0" was blunt).
- the recombinant firefly luciferase reporter plasmid containing the siRNA sense strand target site was constructed according to the protocol described in Example 2 (the target site sequence is shown in Table 1), and then the siRNA in Table 10 and the recombinant firefly fluorescence were respectively prepared.
- the prime enzyme reporter plasmid, pRL-TK (encoded Renilla luciferase) control plasmid was co-transfected into the cells, and 24 hours later, the cells were lysed to detect the activity of the two luciferases.
- the experimental results shown in Table 10 indicate that in the siRNA molecules with 3' overhangs of different lengths, the 14th and/or 16th position at the 5' end of the sense strand is important for the off-target effect of the sense strand.
- Sites; compared to unmodified siRAN, separate or simultaneous modification of the two sites can reduce the inhibition of siRNA on the expression of the corresponding sense strand target site, ie, reduce the off-target effect of the sense strand.
- Example 12 Effect of siRNA sense strand modification on the off-target effect of sense strands of different lengths of siRNA
- siRNAs having different lengths were synthesized; according to the protocol described in Example 2, recombinant firefly luciferase reporter plasmids containing siRNA sense strand target sites were constructed (target sequence is shown in Table 1). .
- the siRNA in Table 11 was co-transfected into the cells with the recombinant firefly luciferase reporter plasmid and the pRL-TK (encoded Renilla luciferase) control plasmid. After 24 hours, the lysed cells were tested for two luciferases. active.
Description
抑制 RNA干扰脱靶效应的 性修饰
技术领域
本发明涉及核酸技术领域，具体涉及一种具有降低正义链脱靶效应的化学修饰的双链 小干扰 RNA (siRNA) 分子及其制备方法， 以及该种修饰的 siRNA分子在制备药物组合 物中的应用。 背景技术
RNA干扰 ( RNA interference, RNAi) 是由双链 RNA分子 (double-stranded RNA, dsRNA)在 mRNA水平降低同源基因表达的现象。 RNA干扰技术又被形象地称为基因敲 低 （knock-down ) 或基因沉默 （gene silencing ), 是一种转录后基因表达调控方法， 因 此又被称为转录后基因沉默 （post-transcriptional gene silencing, PTGS )。 最早有关
RNA干扰的报道出现在 1990年左右， 由两个不同的研究小组同时报道了在转基因植物 中发现的 RNA干扰现象， 随后又在线虫、 果蝇、 斑马鱼以及小鼠等几乎所有后生生物中 观察到 RNA干扰现象。 1999年， Hamilton和 Baulcombe在发生 RNA干扰现象的植物 中检测到长度为 21 -25个核苷酸的双链 RNA片段，这些双链 RNA片断被证明是产生 RNA 干扰现象所必需的， 被称作小干扰核酸 （small interfering RNA, siRNA)。 双链 siRNA与 细胞源性的相关酶和蛋白质形成 RNA 诱导的沉默复合体 （RNA-induced silencing complex, RISC)是 RNA干扰的效应分子。 通常认为， 在 RNA干扰过程中， 双链 siRNA 中的正义链（过客链）被 RISC复合体排出， 反义链指导 RISC复合体结合到目标 mRNA 上的同源位点， 然后由复合物中的核糖核酸酶 III降解目标 mRNA， 从而关闭靶基因的表 达。事实上， 对于一个标准的双链 siRNA, 它的两条链均能参与 RISC复合体的结合， 也 就是说， 除了 siRNA的反义链 （引导链） 能够介导同源基因的表达沉默外， siRNA的正 义链（过客链）也能介导其同源基因的表达沉默， 引发由正义链介导的脱靶效应。在基因 功能研究中， siRNA正义链的这种脱靶效应可能会导致错误解读 siRNA的抑制活性； 而 在小核酸制药中， 则有可能引发 siRNA药物的毒副作用等一系列后果， 这些都将严重影 响 RNA干扰技术的应用。
因此， 降低 siRNA分子正义链的脱靶效应， 提高 RNA干扰的特异性是目前迫切需要 解决的问题。
发明内容
本发明涉及以下按顺序编号的段落中定义的主题：
1、 分离的化学修饰的双链小干扰 RNA ( siRNA) 分子， 包含正义链 （过客链） 和 反义链（引导链）， 其特征在于所述修饰的 siRNA分子的正义链自 5' 端起第 14 位点的核苷酸为化学修饰的核苷酸。
2、 分离的化学修饰的双链小干扰 RNA ( siRNA) 分子， 包含正义链 （过客链） 和 反义链（引导链）， 其特征在于所述修饰的 siRNA分子的正义链自 5' 端起第 16 位点的核苷酸为化学修饰的核苷酸。
3、 分离的化学修饰的双链小干扰 RNA ( siRNA) 分子， 包含正义链 （过客链)和反 义链 （引导链）， 其特征在于所述修饰的 siRNA分子的正义链自 5' 端起第 14 位点和第 16位点的核苷酸为化学修饰的核苷酸。
4、 根据段落 1 -3任一项所述的化学修饰的 siRNA分子， 其特征在于所述化学修饰 选自以下修饰方式中的一种或几种：
(1 ) 对核苷酸中核糖的化学修饰；
(2) 对核苷酸中碱基的化学修饰；
(3) 对核苷酸之间磷酸二酯键的化学修饰。
5、 根据段落 4所述的化学修饰的 siRNA分子， 其特征在于所述化学修饰为对核苷 酸中核糖 2 ' -OH的修饰。
6、 根据段落 5所述的化学修饰的 siRNA分子， 其特征在于所述化学修饰为核苷酸 中核糖 2 ' -OH被甲氧基或氟取代。
根据段落 4所述的化学修饰的 siRNA分子， 其特征在于所述化学修饰为核苷酸 之间的磷酸二酯键被硫代磷酸酯键取代。
8、 根据段落 4所述的化学修饰的 siRNA分子， 其特征在于所述化学修饰为核苷酸 的碱基被非 RNA碱基取代。
9、 根据段落 8所述的化学修饰的 siRNA分子，其特征在于所述非 RNA碱基选自胸 腺嘧啶 (thymine)、 5-甲基胞嘧啶 (5-methylcytosine)、 异胞嘧啶（isocytosine)、 假 异胞嘧啶（pseudoisocytosine)、 5-溴尿嘧啶（5-bromouracil)、 5-丙炔基尿嘧啶 (5-propynyluracil)、 5-丙炔基 -6-氟代尿嘧啶（5-propyny-6-fluoroluracil)、 5-甲基 噻唑尿嘧啶 (5-methylthiazoleuracil)、 6-氨基嘌呤 (6-aminopurine)、 2-氨基嘌呤 (2-aminopurine)、 肌苷（inosine)、 2,6-二氨基嘌呤(2,6-(^1^,1^116)、 7-丙炔 基 -7-脱氮腺嘌呤（7-propyne-7-deazaadenine)、 7-丙炔基 -7-脱氮鸟嘌呤
(7-propyne-7-deazaguanine)、 2-氯 -6-氨基噪呤 (2-chloro-6-aminopurine)。 、 根据段落 8所述的化学修饰的 siRNA分子， 其特征在于所述化学修饰为核苷酸 的碱基被 DNA碱基取代。
、 根据段落 1 -10任一项所述的化学修饰的 siRNA分子， 其特征在于所述 siRNA 分子的正义链和反义链的长度为 15-35个核苷酸。
、 根据段落 11 所述的化学修饰的 siRNA分子， 其特征在于所述 siRNA分子的正 义链和反义链的长度为 19-23个核苷酸。
、 根据段落 12所述的化学修饰的 siRNA分子， 其特征在于所述 siRNA分子的正 义链和反义链的长度为 21个核苷酸。
、 根据段落 1 -14任一项所述的化学修饰的 siRNA分子， 其特征在于所述 siRNA 分子为平末端的 siRNA分子。
、 根据段落 1 -14任一项所述的化学修饰的 siRNA分子， 其特征在于所述 siRNA 分子为 3' 突出末端的 siRNA分子。
、 根据段落 15所述的化学修饰的 siRNA分子， 其特征在于所述 siRNA分子为具 有 1 -6个 3' 突出末端的 siRNA分子。
、 根据段落 16所述的化学修饰的 siRNA分子， 其特征在于所述 siRNA分子为具 有 2-4个 3' 突出末端的 siRNA分子。
、 一种药物组合物，其特征在于含有段落 1 -17任一项所述的化学修饰的 siRNA分 子及药学上可接受的载体。
、 段落 1 -17任一项所述的化学修饰的 siRNA分子在制备药物组合物中的应用。 、 一种降低 siRNA分子正义链脱靶效应的方法， 其特征在于对所述 siRNA分子的 正义链自 5' 端起第 14位点的核苷酸进行化学修饰。
、 一种降低 siRNA分子正义链脱靶效应的方法， 其特征在于对所述 siRNA分子的 正义链自 5' 端起第 16位点的核苷酸进行化学修饰。
、 一种降低 siRNA分子正义链脱靶效应的方法， 其特征在于对所述 siRNA分子的 正义链自 5' 端起第 14位点和第 16位点的核苷酸进行化学修饰。
、 根据段落 20-22任一项所述的降低 siRNA分子正义链脱靶效应的方法， 其特征 在于所述化学修饰选自以下修饰方式中的一种或几种：
(1 ) 对核苷酸中核糖的化学修饰；
(2) 对核苷酸中碱基的化学修饰；
(3) 对核苷酸之间磷酸二酯键的化学修饰。
、 根据段落 23所述的降低 siRNA分子正义链脱靶效应的方法，其特征在于所述化 学修饰为对核苷酸中核糖 2 ' -OH的修饰。
、 根据段落 24所述的降低 siRNA分子正义链脱靶效应的方法，其特征在于所述化 学修饰为核苷酸中核糖 2 ' -OH被甲氧基或氟取代。
、 根据段落 23所述的降低 siRNA分子正义链脱靶效应的方法，其特征在于所述化 学修饰为核苷酸之间磷酸二酯键被硫代磷酸酯键取代。
、 根据段落 23所述的降低 siRNA分子正义链脱靶效应的方法，其特征在于所述化 学修饰为核苷酸中碱基被非 RNA碱基取代。
、 根据段落 27所述的降低 siRNA分子正义链脱靶效应的方法，其特征在于所述非 RNA碱基选自胸腺嘧啶 (thymine)、 5-甲基胞嘧啶 (5-methylcytosine)、 异胞嘧啶 (isocytosine)、 假异胞喷锭 (pseudoisocytosine)、 5-溴尿喷锭 (5-bromouracil)、 5- 丙 炔 基 尿 嘧 啶 （5-propynyluracil) 、 5- 丙 炔 基 -6- 氟 代 尿 嘧 啶 (5-propyny-6-fluoroluracil)、 5-甲基噻唑尿嘧啶（5-methylthiazoleuracil)、 6-氨基 嘌呤 (6-aminopurine)、 2-氨基嘌呤 (2-aminopurine)、 肌苷（inosine)、 2, 6-二氨基 嘌呤 (2,6-(^1^,1^116)、7-丙炔基-7-脱氮腺嘌呤（7 1"(^116-7-01632330161^116)、
7-丙炔基 -7-脱氮鸟嘌呤（7-propyne-7-deazaguanine)、 2-氯 -6-氨基嘌呤 (2-chloro-6-aminopurine) o
、 根据段落 27所述的降低 siRNA分子正义链脱靶效应的方法，其特征在于所述化 学修饰为核苷酸中碱基被 DNA碱基取代。
、 根据段落 20-29任一项所述的降低 siRNA分子正义链脱靶效应的方法， 其特征 在于所述 siRNA分子的正义链和反义链的长度为 15-35个核苷酸。
、 根据段落 30所述的降低 siRNA分子正义链脱靶效应的方法， 其特征在于所述 siRNA分子的正义链和反义链的长度为 19-23个核苷酸。
、 根据段落 31 所述的降低 siRNA分子正义链脱靶效应的方法， 其特征在于所述 siRNA分子的正义链和反义链的长度为 21个核苷酸。
、 根据段落 20-32任一项所述的降低 siRNA分子正义链脱靶效应的方法， 其特征 在于所述 siRNA分子为平末端的 siRNA分子。
、 根据段落 20-32任一项所述的降低 siRNA分子正义链脱靶效应的方法， 其特征 在于所述 siRNA分子为 3' 突出末端的 siRNA分子。
、 根据段落 34所述的降低 siRNA分子正义链脱靶效应的方法， 其特征在于所述 siRNA分子为具有 1 -6个 3' 突出末端的 siRNA分子。
36、 根据段落 35所述的降低 siRNA分子正义链脱靶效应的方法， 其特征在于所述 siRNA分子为具有 2-4个 3' 突出末端的 siRNA分子。
本发明要解决的技术问题是 siRNA分子正义链介导其互补基因转录本的表达沉默， 从而引发由正义链介导的脱靶效应的问题。 本发明的目的是提供一种具有降低正义链 引起的脱靶效应的化学修饰的 siRNA分子及降低 siRNA分子正义链脱靶效应的方法。
为解决上述问题， 本发明一方面提供了一种分离的化学修饰的双链小干扰 RNA (siRNA) 分子， 包含正义链 （过客链） 和反义链 （引导链）， 所述修饰的 siRNA分子的 正义链自 5'端起第 14位点或第 16位点的核苷酸为化学修饰的核苷酸。 本发明还提供了 一种分离的化学修饰的双链小干扰 RNA (siRNA) 分子， 包含正义链 （过客链） 和反义 链 （引导链）， 所述修饰的 siRNA分子的正义链自 5'端起第 14位点和第 16位点的核苷 酸均为化学修饰的核苷酸。本发明中所述的化学修饰，选自以下修饰方式中的一种或几种： (1 ) 对核苷酸中核糖的化学修饰； （2) 对核苷酸中碱基的化学修饰； （3) 对核苷酸之 间磷酸二酯键的化学修饰。
在一种优选情况下， 所述化学修饰为对核苷酸中核糖 2'-OH 的修饰； 更优选地， 所 述化学修饰为核苷酸中核糖 2'-OH被甲氧基或氟取代。 在另一种优选情况下， 所述化学 修饰为核苷酸之间的磷酸二酯键被硫代磷酸酯键取代。 在另一种优选情况下， 所述化学 修饰为核苷酸的碱基被非 RNA 碱基取代； 优选地， 所述非 RNA 碱基选自胸腺嘧啶 (thymine)、 5-甲基胞嘧啶（5-methylcytosine)、 异胞嘧啶（isocytosine)、 假异胞嘧啶 (pseudoisocytosine)、 5-溴尿喷锭 (5-bromouracil)、 5-丙炔基尿喷锭 (5-propynyluracil)、 5-丙炔基 -6-氟代尿嘧 啶（5-propyny-6-fluoroluracil) 、 5- 甲 基噻唑尿嘧 啶 (5-methylthiazoleuracil)、 6-氨基嘌呤 (6-aminopurine)、 2-氨基嘌呤 (2-aminopurine)、 肌 苷（inosine)、 2,6-二氨基嘌呤（2,6-diaminopurine)、 7-丙炔基 -7-脱氮腺嘌呤 (7-propyne-7-deazaadenine)、 7-丙炔基 -7-脱氮鸟噪呤 (7-propyne-7-deazaguanine)、 2- 氯 -6-氨基嘌呤 (2-chloro-6-aminopurine)。 优选地， 所述化学修饰为核苷酸中碱基被 DNA 碱基取代。
上述任一种化学修饰的 siRNA分子， 其正义链和反义链的长度可以为 15-35个核苷 酸， 优选长度为 19-23个核苷酸， 更优选长度为 21个核苷酸。 上述任一种经过化学修饰 的 siRNA分子， 其可以为平末端， 也可以带有 3'突出末端， 3'突出末端可以为 1 -6个核 苷酸， 优选为 2-4个核苷酸， 更优选为 2个核苷酸。
本发明另一方面提供了一种药物组合物， 该组合物可包含上述任一种化学修饰的 siRNA分子及药学上可接受的载体。本发明所用的 "药学上可接受的载体"应当与本发明药
物组合物中的小干扰 RNA (siRNA) 分子相容， 即能与其共混而不会在通常情况下大幅 度降低药物组合物在抑制基因表达方面的效果。这类给药载体包括但不限于各种可用于核 酸给药的载体， 如脂质体、 可降解的高分子化合物、 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇及其组合。
本发明的药物组合物可以制成各种医学上可接受的剂型， 并可由医师根据患者种类、 年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。本发明所 述制剂是指口服液、注射剂、舌下含服剂等多种液体剂型， 或通过加以适当的赋形剂制备 成片剂、 胶囊剂等多种其他剂型。
本发明还提供了上述任一种化学修饰的 siRNA分子在制备药物组合物中的应用。 本发明的第三方面， 提供了一种降低 siRNA分子正义链引起的脱靶效应的方法。 通 过对 siRNA分子的正义链自 5'端起第 14位点或第 16位点的核苷酸进行化学修饰， 或者 对 siRNA分子的正义链自 5'端起第 14位点和第 16位点的核苷酸同时进行化学修饰， 能 够显著降低修饰后的 siRNA分子的正义链引起的脱靶效应。 本发明中所述的化学修饰， 选自以下修饰方式中的一种或几种： （1 ) 对核苷酸中核糖的化学修饰； （2) 对核苷酸中 碱基的化学修饰； （3) 对核苷酸之间磷酸二酯键的化学修饰。
在一种优选情况下， 所述化学修饰为对核苷酸中核糖 2'-OH 的修饰； 更优选地， 所 述化学修饰为核苷酸中核糖 2'-OH被甲氧基或氟取代。 在另一种优选情况下， 所述化学 修饰为核苷酸之间的磷酸二酯键被硫代磷酸酯键取代。 在再一种优选情况下， 所述化学 修饰为核苷酸的碱基被非 RNA 碱基取代； 优选地， 所述非 RNA 碱基选自胸腺嘧啶 (thymine)、 5-甲基胞嘧啶（5-methylcytosine)、 异胞嘧啶（isocytosine)、 假异胞嘧啶 (pseudoisocytosine)、 5-溴尿喷锭 (5-bromouracil)、 5-丙炔基尿喷锭 (5-propynyluracil)、 5-丙炔基 -6-氟代尿嘧 啶（5-propyny-6-fluoroluracil) 、 5- 甲 基噻唑尿嘧 啶 (5-methylthiazoleuracil)、 6-氨基嘌呤 (6-aminopurine)、 2-氨基嘌呤 (2-aminopurine)、 肌 苷（inosine)、 2,6-二氨基嘌呤（2,6-diaminopurine)、 7-丙炔基 -7-脱氮腺嘌呤 (7-propyne-7-deazaadenine)、 7-丙炔基 -7-脱氮鸟噪呤 (7-propyne-7-deazaguanine)、 2- 氯 -6-氨基嘌呤 (2-chloro-6-aminopurine)。 优选地， 所述化学修饰为核苷酸中碱基被 DNA 碱基取代。
上述任一种化学修饰的 siRNA分子， 其正义链和反义链的长度可以为 15-35个核苷 酸， 优选长度为 19-23个核苷酸， 更有选长度为 21 个核苷酸。 上述任一种化学修饰的 siRNA分子，其可以为平末端，也可以带有 3'突出末端， 3'突出末端可以为 1 -6个核苷酸， 优选为 2-4个核苷酸， 更优选为 2个核苷酸。
本发明的有益效果
本发明提供的化学修饰的 siRNA分子， 能够显著降低 siRNA分子正义链引起的脱靶 效应， 提高 siRNA对靶基因表达沉默的特异性。 具体实 ifc¾r式
在通常情况下， 研究人员将一个 siRNA分子导入细胞中， 由反义链 （引导链）介 导的基因沉默是研究人员所期望的； 而由正义链 （过客链） 介导的基因沉默是 RNA 干扰过程中的副作用， 被称为正义链引起的脱靶效应。
针对如何降低 siRNA分子正义链引起的脱靶效应的问题， 发明人对 siRNA正义链的 不同位点的核苷酸的化学修饰与脱靶效应之间的相关性进行了细致的研究。发现对 siRNA 正义链从 5'端起第 14位点或第 16位点的核苷酸进行化学修饰， 能显著降低正义链对与 其互补的基因转录本的表达抑制活性， 即抑制 siRNA正义链引起的脱靶效应； 同时该化 学修饰不影响 siRNA分子未修饰的反义链的基因沉默活性。 在此基础上， 发明人完成本 发明。
本发明中， 术语"反义链 （引导链） "是指双链 siRNA分子中与目标基因转录本中 的目标位点序列互补的 siRNA组成链。 "正义链 （过客链） "是指与 siRNA分子反义 链序列互补的 siRNA分子的另一条组成链。 "正义链靶序列"是指与 siRNA分子中正 义链 （过客链） 互补的基因序列或该基因序列的同源序列。 "反义链靶序列"是指与 siRNA分子中反义链 （引导链） 互补的基因序列或该基因序列的同源序列。
根据本发明， 所述修饰的小干扰 RNA ( siRNA) 分子可以由核糖核苷酸组成， 也 可以包括核糖核苷酸和至少一个脱氧核糖核苷酸的杂合分子。
本发明中， 可以应用各种常规的核酸合成方法完成本发明所涉及的核酸分子的合成， 或者委托专门从事核酸合成服务的生物技术公司完成核酸分子的合成，如委托广州锐博生 物科技有限公司或英骏生物技术有限公司 （invitrogen ) 进行合成。
一般来说，寡聚核酸分子的合成方法包括以下四个步骤：（1 )寡聚核糖核苷酸的合成； (2) 脱保护； （3) 纯化分离； （4) 脱盐。
例如， 具有 SEQ ID NO:1所示核苷酸序列的 siRNA的化学合成步骤如下：
( 1 ) siRNA组成链的合成： 在自动 DNA/RNA合成仪 （例如， Applied Biosystems EXPEDITE8909) 上设定合成 1 毫摩尔量的 siRNA的正义链或反义链寡聚核苷酸序 列， 设定每个循环的偶联时间为 10-15分钟， 起始物为固相连接的 5'-0-对二甲氧基- 胸苷支持物， 第一个循环在固相支持物上连接一个碱基， 然后在第 n次 (2 ≤n≤35)循
环中， 在第 n-1次循环所连接的碱基上连接一个碱基， 重复此循环直至完成全部核酸 序列的合成。
( 2 ) 脱保护
将连接有寡聚核苷酸的固相支持物加入到试管中， 并在此试管中加入 1毫升的乙 醇 /乙胺 (体积比为 1 :3)， 然后密封， 置于 55-70 °C温箱中， 孵育 2-30小时； 取出连接 有寡聚核苷酸的固相支持物， 用双蒸水淋洗 2次 (每次 1毫升)， 收集洗脱液； 室温下 干燥 30分钟后， 加入 1毫升四丁基氟化铵的四氢呋喃溶液（1 M )， 室温放置 4-12小 时； 再加入 2毫升乙醇， 收集沉淀即得到合成的寡聚核苷酸的粗产物。
( 3 ) 纯化分离
将得到的寡聚核苷酸的粗产物溶解于 2毫升浓度为 1 摩尔 /毫升的乙酸铵水溶液 中， 然后通过 C1 8高压液相色谱进行分离， 得到纯化的寡聚核苷酸产物。
( 4 ) 脱盐
用浓度为 75重量％的乙醇水溶液洗涤纯化的寡聚核苷酸产物 2-4次 （每次 2毫 升）， 除去盐份， 室温下干燥； 将正义链和反义链的寡聚核糖核酸混合溶解在 1 -2毫 升的缓冲液中（10mM Tris, pH = 7.5-8.0, 50mM NaCI)， 将此溶液加热至 95°C， 然后 缓缓将此溶液冷却至室温， 并维持在室温条件下 16-22小时， 即得到 siRNA溶液。
本发明中， 所述化学修饰的方式为本领域技术人员所公知。 例如， 本发明对所述核酸 分子进行的化学修饰为以下修饰方式中的一种或几种：
( 1 ) 对核苷酸中核糖的化学修饰；
( 2 ) 对核苷酸中碱基的化学修饰；
( 3 ) 对核苷酸之间磷酸二酯键的化学修饰。
所述对磷酸二酯键的修饰包括但不限于对磷酸二酯键中的氧进行修饰， 例如硫代 磷酸修饰 (Phosphorthioate)和硼垸化磷酸盐修饰 （Boranophosphate),分别用硫和硼 垸置换磷酸二酯键中的氧。 两种修饰都能稳定 siRNA分子的结构， 保持碱基配对的 特异性和亲和力。 硼垸化磷酸盐修饰的 siRNA疏水性强， 易于在血浆中形成水合蛋 白， 对人体的毒副作用低于硫代磷酸酯修饰的 siRNA。
所述核糖修饰包括但不限于对核苷酸戊糖中羟基 (2'-OH)的修饰。 在核糖的羟基位置 引入某些取代基如甲氧基或氟后， 使 siRNA具有更强的抵抗核酸酶水解的性能。 对核苷 酸戊糖中羟基的修饰包括 2'-氟修饰 （2'-fluro modification), 2'-氧甲基修饰 （2'-ΟΜΕ)、 2'- 甲氧乙基修饰 (2'-ΜΟΕ)、 2,4'-二硝基苯酚修饰 （2'-DNP modification),锁核酸 （LNA)、 2'- 氨基修饰 （Amina modification^ 2'-脱氧修饰 （2'-Deoxy modification)等。
2'-氟修饰 2'-氧甲基修饰
2'-甲氧乙基修饰 2， 4 '-二硝基苯酚修饰
2'-脱氧修饰
所述碱基修饰包括但不限于对核苷酸的碱基进行修饰， 如在尿嘧啶的 5 位点引入溴 或碘的 5'-溴尿嘧啶 (5'-bromo-uracil)和 5'-碘尿嘧啶 (5'-iodo-uracil)修饰是常使用的碱基修 饰方法， 其他还有 N3-甲基尿嘧啶（N3-methyl-uracil)修饰， 2,6-二氨基嘌呤 (2,6-diaminopurine)修饰等。
N3-甲基尿嘧啶 2, 6-二氨基嘌呤 优选情况下， 所述修饰为对所述核酸分子的核苷酸中核糖的 2'-OH的修饰； 进一步优 选为， 所述修饰为所述核酸分子的核苷酸中核糖的 2'-OH被甲氧基或氟取代。
根据本发明， 所述 siRNA分子的目的基因可以为各种基因， 可以将在细胞内的功能 有待分析的基因作为目的基因，可以将需要抑制其表达的基因作为目的基因，也可以将与 疾病或功能紊乱相关的基因作为目的基因， 如癌基因、 病毒基因、 细胞膜表面受体基因、 细胞核受体基因或细胞信号传导通路上的基因等。 本领域的技术人员根据目的基因的序 列，能够设计得到目的基因特异的 siRNA分子，例如，将目的基因序列或目的基因在 NCBI Genbank中的序列号输入各种 siRNA设计程序， 如 Insert Design Tool for the shRNA Vectors (Ambion)、 shRNA Explorer (Gene Link)、 siDirect (Yuki Naito et al. University of Tokyo)、 SiRNA at Whitehead (Whitehead Institute for Biomedical Research), BLOCK-iT RNAi Designer (invitrogen)、 RNAi Design (IDT), RNAi Explorer (Gene Link)、 siRNA Target Finder (Ambion)、 或 siSearch (Stockholm Bioinformatics Center)等， 该设计程序 根据设计者的要求和 siRNA的设计原则， 针对所提供的基因序列设计 siRNA序列。 有些 程序还能够对设计的 siRNA序列进行基于全基因组或转录组的同源性分析， 筛选出目的 基因序列特异的 siRNA分子。 以上所述的 siRNA设计程序及其涉及的原则为本领域技术 人员所公知， 其全部内容在此一并引入作为参考。 下面将结合实施例进一步详细描述本发明。应当理解， 列举这些实施例只是为了起说 明作用， 而并不是用来限制本发明的范围。 除非特别说明， 本发明所用到的试剂、培养基 均为市售商品。实施例中未注明具体条件的实验方法， 通常按照常规实验条件进行， 例如 Sambrook等人在 《分子克隆： 实验室手册》 （New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建议的条件。
实施例 1 . siRNA靶位点和 siRNA序列
委托 Invitrogen北京分公司合成表 1 中的 siRNA靶序列片段， 用于融合报告基因表 达载体的构建。
针对表 1 中靶位点序列设计 siRNA, 委托广州锐博生物科技有限公司合成表 2-11 中 所列的化学修饰的和未修饰的小干扰 RNA ( siRNA)。 siRNA靶位点序列
ιΐΘΘηηΘΘηηηοηηνοηηοηη-,9 8 ΚΛΙΟ :8 I> ON ai 03S
wonnowowovnovoono-.g
V3丄丄 i)W3W3VLi)V33丄 i)-<S Ζ ΚΛΙΟ
Hovoonovnonnonnowon-.g
■11 ON ai 03S
wnvnonvo909W9909V9-,g
VlV101V0909W9909V9-,g 9 ΚΛΙΟ
Hono90onno909nv9vnvn-,g
:9 I> ON ai 03S
wonoowoovononnnvno-.g
VO丄 33W3CW3丄 3丄丄 IV丄 3-<S 9 HAIO iiovnvwovonoonnoovon-.g
： S l> ON ai 03S
Hnno90von9nonon99vo9-,g
丄丄 丄 3丄 3丄 3丄 33V3i)-,S 1ΗΙΛΙ〇
}}09n00V9V9V0V9n909W-,9
： ON ai 03S
wovnoo9onn990onnn99-,g
V3X^L33i)3丄丄 3333丄丄丄 3i)-<S C HAIO
}}00VW9900W9099Vn9n-,9
： ε ι> ON αι 03S
Hnvnn99noov99onowo9-,g
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991V01V099V0901W99-,g UIAIO
}ioonnv909noo9nv9nvoo-,g
■11 ON ai 03S
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Honon9V990vnon9onnon-,g
■01 ON ai 03S
nnovnwovo n nvnnoovoo-.g
丄 OVIWOVi)丄 IV丄丄 33V33-<S 60ΙΛΙΟ
:6 ON ai 03S }}09noownwononnvn9v-,g
H9n99novonoo99nwvo9-,g
3丄 33丄 丄 3333丄 W 33-<S l98ZL0/Zl0Z l3/13d 9800£I Z OAV
owonoovoooonovonono-.g
丄 CWlWi)Vi)丄
novovonovoooonoo SS- OIAI〇 IV丄丄 丄 33V3i)3i)丄 3V3丄 3丄 0-<S
vonnoownwononnvn9v-,g
： ZON0I03S
nonoonovonoooonv
wono9ono99W9nown9-,g
3丄 33丄 丄 33 9ε-εοΐΛΐο novnnovonnooovoo
33丄 丄 333丄 33i)VVi)丄 3W丄 3-<S
9V9nnnvoo99V9n9voovo-,g
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nonnovwoovoovon
oonvovnnv90wn9n9V99-,g
。丄丄 3VW03V 9ε-30ΙΛΙΟ
Hoonovovnnoonwn
丄 丄 丄 3丄 3V33-<S
9nv99vono9no9nnnow9-,g
： SZON0I03S
nonoononnvonowo
nonvononvooovownnoo-.g
3丄 33丄 3丄 9ε-|-0ΙΛΙΟ
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onvov9nnovonw9V99vo-,g
： ^zoNaioas
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wonononnnoonononnnn-.g
： £ZONai03S
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:6I> ON ai 03S
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wowoovovnooono
vnnnowononnonoonovo-.g
V9W90V9V1 9ε-90ΙΛΙΟ 3VLLL3Wi)丄 3丄丄 3丄 33丄 一, S Honovoovowovonn
:8Z ON ai 03S owvn9V990vnon9onnon-,g
wnovnwovonnvnno l98ZL0/Zl0Z l3/13d 9800£I Z OAV
表 2. 对 siRNACMOI禾 P CM11正义链不同位点进行甲氧基修饰的序列表 修饰方式简写说明： 甲氧基修饰 （N m
T98Z.0/ZlOZN3/X3d 9800£1 Z OAV
l98ZLO/ZlOZ l3/13d 9800£I Z OAV
l98ZLO/ZlOZ l3/13d 9800£I Z OAV
表 4. 对 siRNA cdc-2和 NPY-305正义链和 /或反义链 5'-端起第 14和 /或 16位点进行甲氧基修饰的序列表
修饰方式简写说明： 甲氧基修饰 （N m 沉默效率 沉默效率
(siRNA正 (siRNA反
siRNA 编号 正义链序列 （5'-3') 编号 反义链序 义链靶序列 义链靶序列
作为靶点） 作为靶点）
Cdc-2 78% 87% SEQ ID NO 67 5'-UCGGGAAAUUUCUCUAUUAtt SEQ ID NO 68 5'-UAAUAGAGAAAUUU
Cdc-2(S14, 18% 86% 5'-UCGGGAAAUUUCU(C)mUAUUAtt S'-UAAUAGAGAAAUUUC
Cdc-2(As14m 78% 56% 5'-UCGGGAAAUUUCUCUAUUAtt 5'-UAAUAGAGAAAUU(U)
Cdc-2(S14m+As14m 21 % 64% 5'-UCGGGAAAUUUCU(C)mUAUUAtt 5'-UAAUAGAGAAAUU(U)
Cdc-2(S16, 56% 86% 5'-UCGGGAAAUUUCUCU(A)mUUAtt 5'-UAAUAGAGAAAUUUC
Cdc-2(As16, 77% 60% 5'-UCGGGAAAUUUCUCUAUUAtt 5'-UAAUAGAGAAAUUUC
Cdc-2(S16m+As16, 60% 62% 5'-UCGGGAAAUUUCUCU(A)mUUAtt 5'-UAAUAGAGAAAUUUC
NPY-305 70% 90% SEQ ID NO 69 5'-UGAGAGAAAGCACAGAAAAtt SEQ ID NO 70 5'-UUUUCUGUGCUUUC
NPY-305(S14, 8% 89% 5'-UGAGAGAAAGCAC(A)mGAAAAtt 5'-UUUUCUGUGCUUUC
NPY-305(As14, 72% 59% 5'-UGAGAGAAAGCACAGAAAAtt 5'-UUUUCUGUGCUUU(C
NPY-305(S14m+As14, 16% 62% 5'-UGAGAGAAAGCAC(A)mGAAAAtt 5'-UUUUCUGUGCUUU(C
NPY-305(S16, 36% 86% 5'-UGAGAGAAAGCACAG(A)m AAAtt 5'-UUUUCUGUGCUUUC
NPY-305(As16, 69% 65% 5'-UGAGAGAAAGCACAGAAAAtt 5'-UUUUCUGUGCUUUC
NPY-305(S16m+As16, 42% 67% 5'-UGAGAGAAAGCACAG(A)m AAAtt 5'-UUUUCUGUGCUUUC
2 表 5. 对 siRNA CM01 -05正义链 5'-端起第 14和 16位点同时进行甲氧基修饰的序列表
修饰方式简写说明： 甲氧基修饰 （N m
修饰方式简写说明： 氟代修饰 （N)F
表 9. 对 siRNA正义链 5'-端起第 14和 /或第 16位点进行 DNA取代修饰的序列表
修饰方式简写说明： DNA替换修饰 （N)D
//〇 ϊ98ί/-οίϊοίζ>1M
'(N) m '•mi m^
//〇 ϊ98ί/-οίϊοίζ>1><1M
//〇 ϊ98ί/-οίϊοίζ>1><1Μ
CM05-N15 (14, 44% 5'-GACUGGUGUUGUGAAtt 5'-UCUUCGUCUACGG(A)mGtt
CM01 -N23 95% SEQ ID NO 121 5'-GGUUAACAGCGAUCUGAUGUCAAtt SEQ ID NO 122 5'-CAGGAGAAUCACUUGACAUCAGAtt
CM02-N23 92% SEQ ID NO 123 5'-GGAGUGUAACGAUUACAUCCUGAtt SEQ ID NO 124 5'-GAACUUUGCUGCUCAGGAUGUAAtt
CM03-N23 92% SEQ ID NO 125 5'-GUAACUGAAGGCUCGCUCAAAUGtt SEQ ID NO 126 5'-CACCAGUGAGGCCAUUUGAGCGAtt
CM04-N23 94% SEQ ID NO 127 5'-GUGUCAGUGCGCAGCUGAAGGUUtt SEQ ID NO 128 5'-AGUAUUCUCAAUAACCUUCAGCUtt
CM05-N23 93% SEQ ID NO 129 5'-GACUGGUGUUGUGAAGUUUACUCtt SEQ ID NO 130 5'-UCUUCGUCUACGGAGUAAACUUCtt
CM01 -N23 (14, 64% 5'-GGUUAACAGCGAUCUGAUGUCAAtt 5'-CAGGAGAAUCACU(U)mGACAUCAGAtt
CM02-N23 (14, 30% 5'-GGAGUGUAACGAUUACAUCCUGAtt 5'-GAACUUUGCUGCU(C)mAGGAUGUAAt
CM03-N23 (14, 37% 5'-GUAACUGAAGGCUCGCUCAAAUGtt 5'-CACCAGUGAGGCC(A)mUUUGAGCGAt
CM04-N23 (14, 43% 5'-GUGUCAGUGCGCAGCUGAAGGUUtt 5'-AGUAUUCUCAAUA(A)mCCUUCAGCUtt
CM05-N23 (14, 57% 5'-GACUGGUGUUGUGAAGUUUACUCtt 5'-UCUUCGUCUACGG(A)mGUAAACUUCt
CM01 -N23 (16, 70% 5'-GGUUAACAGCGAUCUGAUGUCAAtt 5'-CAGGAGAAUCACUUG(A)m CAUCAGAt
CM02-N23 (16, 82% 5'-GGAGUGUAACGAUUACAUCCUGAtt 5'-GAACUUUGCUGCUCA(G)m GAUGUAA
CM03-N23 (16, 87% 5'-GUAACUGAAGGCUCGCUCAAAUGtt 5'-CACCAGUGAGGCCAU(U)m UGAGCGA
CM04-N23 (16, 79% 5'-GUGUCAGUGCGCAGCUGAAGGUUtt 5'-AGUAUUCUCAAUAAC(C)m UUCAGCUt
CM05-N23 (16, 82% 5'-GACUGGUGUUGUGAAGUUUACUCtt 5'-UCUUCGUCUACGGAG(U)m AAACUUCt
CM01 -N27 96% SEQ ID NO 131 5'-GGUUAACAGCGAUCUGAUGUCAAGUGAtt SEQ ID NO 132 5'-CAGGAGAAUCACUUGACAUCAGAUC
CM02-N27 91 % SEQ ID NO 133 5'-GGAGUGUAACGAUUACAUCCUGAGCAGtt SEQ ID NO 134 5'-GAACUUUGCUGCUCAGGAUGUAAUC
CM03-N27 93% SEQ ID NO 135 5'-GUAACUGAAGGCUCGCUCAAAUGGCCUtt SEQ ID NO 136 5'-CACCAGUGAGGCCAUUUGAGCGAGC
CM04-N27 91 % SEQ ID NO 137 5'-GUGUCAGUGCGCAGCUGAAGGUUAUUGtt SEQ ID NO 138 5'-AGUAUUCUCAAUAACCUUCAGCUGC
CM05-N27 93% SEQ ID NO 139 5'-GACUGGUGUUGUGAAGUUUACUCCGUAtt SEQ ID NO 140 5'-UCUUCGUCUACGGAGUAAACUUCAC
CM01 -N27 (14, 64% 5'-GGUUAACAGCGAUCUGAUGUCAAGUGAtt 5'-CAGGAGAAUCACU(U)mGACAUCAGAU
CM02-N27 (14, 30% 5'-GGAGUGUAACGAUUACAUCCUGAGCAGtt 5'-GAACUUUGCUGCU(C)mAGGAUGUAA
CM03-N27 (14, 38% 5'-GUAACUGAAGGCUCGCUCAAAUGGCCUtt 5'-CACCAGUGAGGCC(A)mUUUGAGCGA
CM04-N27 (14, 41 % 5'-GUGUCAGUGCGCAGCUGAAGGUUAUUGtt 5'-AGUAUUCUCAAUA(A)mCCUUCAGCU
CM05-N27 (14, 53% 5'-GACUGGUGUUGUGAAGUUUACUCCGUAtt 5'-UCUUCGUCUACGG(A)mGUAAACUUC
CM01 -N27 (16, 70% 5'-GGUUAACAGCGAUCUGAUGUCAAGUGAtt 5'-CAGGAGAAUCACUUG(A)m CAUCAGA
CM02-N27 (16, 86% 5'-GGAGUGUAACGAUUACAUCCUGAGCAGtt 5'-GAACUUUGCUGCUCA(G)m GAUGUAA
CM03-N27 (16, 90% 5'-GUAACUGAAGGCUCGCUCAAAUGGCCUtt 5'-CACCAGUGAGGCCAU(U)m UGAGCGA
CM04-N27 (16, 76% 5'-GUGUCAGUGCGCAGCUGAAGGUUAUUGtt 5'-AGUAUUCUCAAUAAC(C)m UUCAGCU
CM05-N27 (16, 82% 5'-GACUGGUGUUGUGAAGUUUACUCCGUAtt 5'-UCUUCGUCUACGGAG(U)m AAACUUC
5'-GGUUAACAGCGAUCUGAUGUCAAGUGAUU 5'-CAGGAGAAUCACUUGACAUCAGAUC
CM01 -N35 88% SEQ ID NO 141 SEQ ID NO 142
CUCCUGtt UAACCtt
5'-GGAGUGUAACGAUUACAUCCUGAGCAGCA 5'-GAACUUUGCUGCUCAGGAUGUAAUC
CM02-N35 89% SEQ ID NO 143 SEQ ID NO 144
AAGUUCtt ACUCCtt
5'-GUAACUGAAGGCUCGCUCAAAUGGCCUCA 5'-CACCAGUGAGGCCAUUUGAGCGAGC
CM03-N35 90% SEQ ID NO 145 SEQ ID NO 146
CUGGUGtt GUUACtt
5'-GUGUCAGUGCGCAGCUGAAGGUUAUUGA 5'-AGUAUUCUCAAUAACCUUCAGCUGC
CM04-N35 88% SEQ ID NO 147 SEQ ID NO 148
GAAUACUtt GACACtt
%9 Π3<V3W3 }} H
实施例 2. siRNA对靶位点抑制活性的检测
1 ) 构建带有 siRNA分离靶位点的重组萤火虫荧光素酶报告基因质粒 参见以下文献， 构建含有 siRNA分离靶位点的重组萤火虫荧光素酶报告基因质粒， 报告基因质粒载体可向论文作者索取。 （Du Q, Thonberg H, Zhang HY, Wahlestedt C & Liang Z. Validating siRNA Using a Reporter Made from Synthetic DNA Oligonucleotides. Biochem Biophys Res Commun. 325:243-9, 2004; Du Q, Thonberg H, Wang J, Wahlestedt C & Liang Z. A Systematic Analysis of the Silencing Effects of an Active siRNA at All Single-nucleotide Mismatched Target Sites. Nucleic Acids Res. 33:1671 -7, 2005; Li ZS, Qiao RP, Du Q, Yang ZJ, Zhang LR, Zhang PZ, Liang Z & Zhang LH. Studies on Aminoisonucleoside Modified siRNAs: Stability and Silencing Activity. Bioconjugate Chem. 18:1017-24, 2007; Dahlgren C, Zhang HY, Du Q, Grahn M, Norstedt G, Wahlestedt C & Liang Z. Analysis of siRNA specificity on targets with double-nucleotide mismatches. Nucleic Acids Res. 36:e53, 2008; Huang H, Qiao R, Zhao D, Zhang T, Li丫 Yi F, Lai F, Hong J, Ding X, Yang Z, Zhang L, Du Q*, Liang Z*. Profiling of mismatch discrimination in RNAi enabled rational design of allele-specific siRNAs. Nucleic Acids Res. 37:7560-9, 2009)。
2) 转染 参照上述文献中描述的方法， 进行细胞转染和 siRNA活性测定。 具体将在 DMEM培 养基 （10% FBS， 2mM L-谷氨酰胺， 100个单位 /毫升的青霉素和 100 g/ml的链霉素） 中培养的人胚胎肾细胞 (HEK293)接种到 24孔板中 （1 05细胞 /0.5 ml 培养基 /孔）。 待 细胞生长 24小时后， 细胞的融合度为 50-70%左右时， 将培养基换为 Opti-MEM培养基 ( Gibco公司）。 利用 Lipofectamine 2000 ( Invitrogen公司， 美国） 将带有 siRNA分离 靶位点的重组萤火虫荧光素酶报告基因质粒转染入细胞， 同时转染进入细胞的还有 pRL-TK (编码海肾萤光素酶） 的对照质粒 （Promega公司， Madison Wl, USA)， 以及化 学合成的修饰的或未修饰的 siRNA分子。 每孔含 0.17g重组质粒和 0.017g pRL-TK对照 质粒， siRNA的终浓度为 13 nM。 每种 siRNA平行转染三个复孔， 以只转染同样量的两 种报告基因质粒（不转染 siRNA) 的三个复孔作为对照。 4小时后再将转染介质换成 1 ml DMEM培养基 （10% FBS， 2mM L-谷氨酰胺， 100个单位 /毫升的青霉素和 100 g/ml 的链霉素）。
3) 荧光素酶活性测定
转染 24小时后收获细胞， 以 10 μΙ细胞裂解液裂解细胞， 利用双荧光素酶报告基因 分析系统 ( Dual-Luciferase Assay System, Promega公司） 禾口酶标仪 ( Novostar, BMG Labtechnologies GmbH, Germany) 测定两种荧光素酶的活性， 以未转染 siRNA的孔中 的报告基因的表达量作为对照， 通过以下公式计算得到 siRNA对靶位点的沉默效率。
沉默效率 = 1 - (实验组萤火虫荧光素酶报告基因的表达量 /实验组海肾荧光素酶报告基 因的表达量) /(对照组萤火虫荧光素酶报告基因的表达量 /对照组海肾荧光素酶报告基因的 表达量） 实施例 3. 甲氧基核苷酸修饰 siRNA正义链的不同位点对正义链脱靶效应的影响
选取 2条 siRNA (CM-01和 CM-11 )，按照实施例 2所述的方案分别构建含有 siRNA 正义链靶位点的重组萤火虫荧光素酶报告基因质粒 （靶位点序列如表 1所示）； 合成表 2 所示的未修饰的 siRNA分子， 以及分别在 siRNA正义链自 5'端起第 2-18位点含有甲氧 基修饰核苷酸的经化学修饰的 siRNA分子。 按照实施例 2所述的实验方案， 分别检测各 种 siRNA分子对其正义链靶位点的抑制活性。每种 siRNA每次实验平行做 3个复孔， 每 个实验重复至少 2次。 表 2所示的实验结果表明， 对 siRNA正义链自 5'端起第 14位点 的核苷酸的化学修饰能够显著降低修饰的 siRNA对与其正义链互补的基因的表达抑制活 性;对 siRNA正义链自 5'端起第 16位点的核苷酸的化学修饰也能够在一定程度上降低修 饰的 siRNA对与其正义链互补的基因的表达抑制活性； 而对其余位点的修饰基本上不影 响 siRNA对与其正义链互补的基因的表达抑制活性。 实施例 4. 甲氧基修饰 siRNA正义链的 13-16位点对正义链脱靶效应的影响
如表 3所示，在 siRNA正义链自 5'端起的第 13-16位点中，每个位点选取 12条 siRNA 序列， 合成其未修饰的 siRNA分子， 以及分别在其正义链自 5'端起第 13-16位点含有甲 氧基修饰核苷酸的化学修饰的 siRNA分子； 按照实施例 2 所述的实验方案， 构建含有 siRNA正义链靶位点的重组萤火虫荧光素酶报告基因质粒（靶位点序列如表 1所示）； 按 照实施例 2所述方案， 分别检测表 3中各种修饰或未修饰的 siRNA分子对与其正义链互 补的靶位点的抑制活性；每种 siRNA每次实验平行做 3个复孔，每个实验重复至少 2次。 表 3所示实验结果表明， 正义链从 5'端起第 14位点的核苷酸为 、 U、 C、 G中任一种 时， 对其进行化学修饰均能显著降低修饰的 siRNA分子对与其正义链互补的基因的表达 抑制活性； 此外， 正义链从 5'端起第 16位点的核苷酸为 、 U、 C、 G中任一种时， 对 其进行化学修饰也能在一定程度上降低修饰的 siRNA分子对与其正义链互补的基因的表
达抑制活性； 而第 13位点或第 15位点的修饰对正义链脱靶效应的影响较小。 这个实验 结果也表明， siRNA 的修饰对正义链脱靶效应的影响是一种与修饰位置相关， 而与具体 的核酸序列无关的现象， 即该效应是一种不依赖于核酸序列的效应。 本文中所使用的"不 依赖于核酸序列的效应"是指实施方案提供的修饰核苷酸和修饰方式可用于任何 siRNA序 列而不考虑该 siRNA的具体序列。 实施例 5. 修饰 siRNA正 /反义链的 14位点 /16位点对 siRNA正义链脱靶效应的影响 如表 4所示，选取 2条 siRNA序列（cdc-2, NPY-305),合成其未修饰的 siRNA分子， 以及对 siRNA的正义链或反义链从 5'端起第 14位点或第 16位点的核苷酸进行甲氧基修 饰的 siRNA分子，或同时对正义链和反义链从 5'端起第 14位点的核苷酸进行甲氧基修饰 的 siRNA分子，或同时对正义链和反义链从 5'端起第 16位点的核苷酸进行甲氧基修饰的 siRNA分子， 修饰和未修饰的 siRNA分子如表 4所示。 按照实施例 2所述的方案， 构建 分别含有与其正义链或反义链互补的分离靶位点的重组萤火虫荧光素酶报告基因质粒 (靶 位点序列如表 1所示）。按照实施例 2所述方案，分别检测各种 siRNA分子对与其正义链 或反义链互补的分离的靶位点的抑制活性。 每种 siRNA每次实验平行做 3个复孔， 每个 实验重复至少 2次。 表 4所示的实验结果表明， 对 siRNA中一条组成链的化学修饰仅降 低被修饰链对与其互补的基因的表达抑制活性， 而不影响另一条未修饰的 siRNA组成链 对与其互补的基因的表达抑制活性。 实施例 6. 甲氧基同时修饰 siRNA正义链 5'端起第 14和 16位点对正义链脱靶效应的影 响
如表 5 所示， 选择 5条 siRNA序列 (CM-01 , CM-02, CM-03, CM-04, CM-05), 合成未经修饰的 siRNA (表 3) 及同时对正义链 5'端起第 14和 16位点进行甲氧基修饰 的 siRNA (表 5)。 按照实施例 2所述实验方案， 分别构建含有这些 siRNA正义链靶位点 的重组萤火虫荧光素酶报告基因质粒（靶位点序列如表 1所示）， 然后分别将各种 siRNA 与重组萤火虫荧光素酶报告基因质粒、 pRL-TK (编码海肾萤光素酶） 对照质粒共转染入 细胞中， 24小时后裂解细胞检测两种荧光素酶的活性。 表 5所示实验结果表明， 在不同 的 siRNA分子中， 正义链 5'端起第 14位点和第 16位点是对正义链脱靶效应具有重要影 响作用的位点；与未经修饰的 siRAN相比，对该两个位点的同时修饰能够显著降低 siRNA 对相应的正义链靶位点基因表达的抑制作用；与单独修饰第 14位点或第 16位点的 siRNA 相比， 对两个位点的同时修饰具有一定的协同作用。
实施例 7. 氟代修饰对 siRNA正义链脱靶效应的影响
选择 5条 siRNA序列 （CM-01， CM-02, CM-03, CM-04, CM-05), 合成未经修饰 的 siRNA (表 3) 及表 6中所示的修饰的 siRNA; 按照实施例 2所述方案， 分别构建含 有 siRNA正义链靶位点的重组萤火虫荧光素酶报告基因质粒 （靶位点序列如表 1所示）。 按照实施例 2所述方案， 分别检测各种 siRNA分子对与其正义链互补的靶位点的抑制活 性； 每种 siRNA每次实验平行做 3个复孔， 每个实验重复至少 2次。 表 6所示的实验结 果表明， 对 siRNA分子正义链 5'端起第 14 和 /或第 16位点进行氟代修饰能显著降低修 饰的 siRNA分子的正义链对与其互补的基因的表达抑制活性。 实施例 8. 5-甲基胞嘧啶修饰对 siRNA正义链脱靶效应的影响
选择表 7中所示的 siRNA, 合成未经修饰的 siRNA (表 3) 及表 7中所示的 5-甲基 胞嘧啶修饰修饰的 siRNA; 按照实施例 2所述方案， 分别构建含有 siRNA正义链靶位点 的重组萤火虫荧光素酶报告基因质粒（靶位点序列如表 1所示）。按照实施例 2所述方案， 分别检测各种 siRNA分子对与其正义链互补的靶位点的抑制活性。每种 siRNA每次实验 平行做 3个复孔， 每个实验重复至少 2次。 表 7所示的实验结果表明， 对 siRNA分子正 义链 5'端起第 14 和 /或第 16位点进行 5-甲基胞嘧啶修饰能显著降低修饰的 siRNA分子 的正义链对与其互补的基因的表达抑制活性。 实施例 9. 5-溴尿嘧啶修饰对 siRNA正义链脱靶效应的影响
选择表 8中所示的 siRNA, 合成未经修饰的 siRNA (表 3) 及表 8中所示的修饰的 siRNA; 按照实施例 2所述方案， 分别构建含有 siRNA正义链靶位点的重组萤火虫荧光 素酶报告基因质粒 （靶位点序列如表 1 所示）。 按照实施例 2所述方案， 分别检测各种 siRNA分子对与其正义链互补的靶位点的抑制活性； 每种 siRNA每次实验平行做 3个复 孔， 每个实验重复至少 2次。 表 8所示的实验结果表明， 对 siRNA分子正义链 5'端起第 14 和 /或第 16位点进行 5-溴尿嘧啶修饰能显著降低修饰的 siRNA分子的正义链对与其互 补的基因的表达抑制活性。 实施例 10. DNA替换修饰对 siRNA正义链脱靶效应的影响
选择表 9中所示的 siRNA, 合成未经修饰的 siRNA (表 3)及表 9中所示的 DNA碱 基修饰的 siRNA; 按照实施例 2所述方案， 分别构建含有 siRNA正义链靶位点的重组萤 火虫荧光素酶报告基因质粒 （靶位点序列如表 1所示）。 按照实施例 2所述方案， 分别检
测各种 siRNA分子对与其正义链互补的靶位点的抑制活性。每种 siRNA每次实验平行做 3个复孔， 每个实验重复至少 2次。 表 9所示的实验结果表明， 对 siRNA分子正义链 5' 端起第 14 和 /或第 16位点的碱基进行 DNA替换修饰能显著降低修饰的 siRNA分子的正 义链对与其互补的基因的表达抑制活性。 以上实施例表明， 对 siRNA正义链 5'端起第 14 和 /或第 16位点进行不同方式的化 学修饰均能降低由正义链引起的脱靶效应； 这个结果进一步表明， siRNA 的修饰对正义 链脱靶效应的影响是一种与修饰位置相关的现象。 实施例 11 . siRNA正义链修饰对具有不同长度 3'突出末端的 siRNA的正义链脱靶效应的 影响
如表 10 所示， 合成具有不同长度 3'突出末端的 siRNA (3'突出末端的核苷酸数目分 别为 0 、 1、 4、 6，为" 0"时即为平末端）。按照实施例 2所述方案，分别构建含有 siRNA 正义链靶位点的重组萤火虫荧光素酶报告基因质粒 （靶位点序列如表 1所示）， 然后分别 将表 10中的 siRNA与重组萤火虫荧光素酶报告基因质粒、 pRL-TK (编码海肾萤光素酶) 对照质粒共转染入细胞中， 24小时后裂解细胞检测两种荧光素酶的活性。表 10所示的实 验结果表明，在具有不同长度的 3'突出末端的 siRNA分子中，正义链 5'端起第 14位点和 /或第 16位点是对正义链脱靶效应具有重要影响的位点； 与未经修饰的 siRAN相比， 对 该两个位点的单独修饰或同时修饰能够降低 siRNA对相应的正义链靶位点基因表达的抑 制作用， 即降低正义链的脱靶效应。 实施例 12. siRNA正义链修饰对不同长度 siRNA的正义链脱靶效应的影响
如表 11 所示， 合成具有不同长度的 siRNA; 按照实施例 2所述方案， 分别构建含有 siRNA正义链靶位点的重组萤火虫荧光素酶报告基因质粒（靶位点序列如表 1所示）。 分 别将表 11 中的 siRNA与重组萤火虫荧光素酶报告基因质粒、 pRL-TK (编码海肾萤光素 酶）对照质粒共转染入细胞中， 24小时后裂解细胞检测两种荧光素酶的活性。表 11所示 的实验结果表明， 在具有不同长度的 siRNA分子中， 正义链 5'端起第 14位点和 /或第 16 位点是对正义链脱靶效应具有重要影响的位点； 与未经修饰的 siRAN相比， 对该两个位 点的单独修饰或同时修饰能够降低 siRNA对相应的正义链靶位点基因表达的抑制作用， 即降低正义链的脱靶效应。
SEQUENCE LISTING
<110> 北京大学
<120> 抑制 RNA干扰脱靶效应的特异性修饰
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<210> 34
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uucagcugcgcacugacactt
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<400> 39
gacugguguugugaaguuutt
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aaacuucacaacaccaguctt
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<400> 41
ggcucaagugauucuccugtt
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<400> 42
caggagaaucacuugagcctt
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gaacuuugcugcucaguautt
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auacugagcagcaaaguuctt
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gcaaauggccucacuggugtt
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caccagugaggccauuugctt
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<400> 47
gcagguuauugagaauacutt
<210> 48
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<400> 48
aguauucucaauaaccugctt
<210> 49
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<400> 49
gagacuccguagacgaagatt 21
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<400> 50
ucuucgucuacggagucuctt 21
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gcaacucggaccugguuautt 21
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auaaccagguccgaguugctt 21
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gguuuccgguucgccuacatt 21
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uguaggcgaaccggaaacctt 21
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<400> 55
gcaggucucugucacgcuutt
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aagcgugacagagaccugctt
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guauuugucaccaagcucatt
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ugagcuuggugacaaauactt
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gagcggaagcgcaucuauatt
<210> 60
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<400> 60
uauagaugcgcuuccgcuctt
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<400> 61
guccaguacaacaaguucatt
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ugaacuuguuguacuggactt
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<400> 63
ccaaccaaagaaugaagaatt
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uucuucauucuuugguuggtt
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gg ag u cacag cu cu u ecu u tt
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aaggaagagcugugacucctt
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<400> 67
ucgggaaauuucucuauuatt
<210> 68
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<400> 68
uaauagagaaauuucccgatt
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<400> 69
ugagagaaagcacagaaaatt
<210> 70
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<400> 70
uuuucugugcuuucucucatt
<210> 71
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<400> 71
gguuaacagcgaucugaug 19
<210> 72
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<400> 72
caucagaucgcuguuaacc 19
<210> 73
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<400> 73
ggaguguaacgauuacauc 19
<210> 74
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<400> 74
gauguaaucguuacacucc 19
<210> 75
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<400> 75
guaacugaaggcucgcuca 19
<210> 76
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<400> 76
ugagcgagccuucaguuac 19
<210> 77
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<400> 77
gugucagugcgcagcugaa 19
<210> 78
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<400> 78
uucagcugcgcacugacac 19
<210> 79
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<400> 79
gacugguguugugaaguuu 19
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<400> 80
aaacuucacaacaccaguc 19
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<400> 81
gguuaacagcgaucugaugt
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<400> 82
caucagaucgcuguuaacct
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ggaguguaacgauuacauct
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gauguaaucguuacacucct
<210> 85
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guaacugaaggcucgcucat
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ugagcgagccuucaguuact
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gugucagugcgcagcugaat
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uucagcugcgcacugacact
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gacugguguugugaaguuut
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aaacuucacaacaccaguct
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gguuaacagc aucugaugt ttt
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caucagaucg cuguuaacct ttt
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ggaguguaacgauuacauct ttt
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gauguaaucg uacacucct ttt
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guaacugaaggcucgcucat ttt
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ugagcgagccuucaguuact ttt
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gugucagugcgcagcugaat ttt
<210> 98
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uucagcugcg cacugacact ttt
<210> 99
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gacugguguugugaaguuut ttt
<210> 100
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aaacuucaca acaccaguct ttt
<210> 101
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gguuaacagc gaucugaugt ttttt
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caucagaucg cuguuaacct ttttt
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ggaguguaac gauuacauct ttttt
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gauguaaucg uuacacucct ttttt
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guaacugaag gcucgcucat ttttt
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ugagcgagcc uucaguuact ttttt
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gugucagugc gcagcugaat ttttt
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uucagcugcg cacugacact ttttt
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gacugguguu gugaaguuut ttttt
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aaacuucaca acaccaguct ttttt
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gguuaacagcgaucutt
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caggagaaucacuugtt
<210> 113
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<400> 113
ggaguguaacgauuatt
<210> 114
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<400> 114
gaacuuugcugcucatt 17
<210> 115
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<400> 115
guaacugaaggcucgtt 17
<210> 116
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<400> 116
caccagugaggccautt 17
<210> 117
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<400> 117
gugucagugcgcagctt 17
<210> 118
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<400> 118
aguauucucaauaactt
<210> 119
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<400> 119
gacugguguugugaatt
<210> 120
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<400> 120
ucuucgucuacggagtt
<210> 121
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<400> 121
gguuaacagcgaucugaugucaatt
<210> 122
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<400> 122
caggagaaucacuugacaucagatt
<210> 123
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<400> 123
ggaguguaacgauuacauccugatt
<210> 124
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<400> 124
gaacuuugcugcucaggauguaatt
<210> 125
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guaacugaaggcucgcucaaaugtt
<210> 126
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<400> 126
caccagugaggccauuugagcgatt
<210> 127
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<400> 127
gugucagugcgcagcugaagguutt
<210> 128
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<400> 128
aguauucucaauaaccuucagcutt
<210> 129
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gacugguguugugaaguuuacuctt
<210> 130
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<400> 130
ucuucgucuacggaguaaacuuctt
<210> 131
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<400> 131
gguuaacagcgaucugaugucaagugatt
<210> 132
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<400> 132
caggagaaucacuugacaucagaucgctt
<210> 133
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<400> 133
ggaguguaacgauuacauccugagcagtt
<210> 134
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<400> 134
gaacuuugcugcucaggauguaaucgutt
<210> 135
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<400> 135
guaacugaaggcucgcucaaauggccutt
<210> 136
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<400> 136
caccagugaggccauuugagcgagccutt
<210> 137
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<400> 137
gugucagugcgcagcugaagguuauugtt
<210> 138
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<400> 138
aguauucucaauaaccuucagcugcgctt
<210> 139
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<400> 139
gacugguguugugaaguuuacuccguatt
<210> 140
<211 > 29
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<400> 140
ucuucgucuacggaguaaac ucacaatt
<210> 141
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<400> 141
gguuaacagcgaucugaugucaagugauucuccugtt
<210> 142
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<400> 142
caggagaaucacuugacaucagaucgcuguuaacctt
<210> 143
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<400> 143
ggaguguaacgauuacauccugagcagcaaaguuctt
<210> 144
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<400> 144
gaacuuugcugcucaggauguaaucguuacacucctt
<210> 145
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<400> 145
guaacugaaggcucgcucaaauggccucacuggugtt
<210> 146
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<213> artificial
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<400> 146
caccagugaggccauuugagcgagccuucaguuactt
<210> 147
<211 > 37
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<400> 147
gugucagugcgcagcugaagguuauugagaauacutt
<210> 148
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<400> 148
aguauucucaauaaccuucagcugcgcacugacactt
<210> 149
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<400> 149
gacugguguugugaaguuuacuccguagacgaagatt
<210> 150
<211 > 37
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<213> artificial
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<400> 150
ucuucgucuacggaguaaacuucacaacaccaguctt
Claims
1、 分离的化学修饰的双链小干扰 RNA (siRNA) 分子， 包含正义链 （过客链） 和反义 链（引导链）， 其特征在于所述修饰的 siRNA分子的正义链自 5'端起第 14位点的核 苷酸为化学修饰的核苷酸。
2、 分离的化学修饰的双链小干扰 RNA (siRNA) 分子， 包含正义链 （过客链） 和反义 链（引导链）， 其特征在于所述修饰的 siRNA分子的正义链自 5'端起第 16位点的核 苷酸为化学修饰的核苷酸。
3、 分离的化学修饰的双链小干扰 RNA (siRNA)分子， 包含正义链（过客链)和反义链
(引导链）， 其特征在于所述修饰的 siRNA分子的正义链自 5'端起第 14位点和第 16位点的核苷酸为化学修饰的核苷酸。
4、 根据权利要求 1 -3任一项所述的化学修饰的 siRNA分子， 其特征在于所述化学修饰 选自以下修饰方式中的一种或几种：
(1 ) 对核苷酸中核糖的化学修饰；
(2) 对核苷酸中碱基的化学修饰；
(3) 对核苷酸之间磷酸二酯键的化学修饰。
5、 根据权利要求 4所述的化学修饰的 siRNA分子， 其特征在于所述化学修饰为对核苷 酸中核糖 2'-OH的修饰。
6、 根据权利要求 5所述的化学修饰的 siRNA分子， 其特征在于所述化学修饰为核苷酸 中核糖 2'-OH被甲氧基或氟取代。
1、 根据权利要求 4所述的化学修饰的 siRNA分子，其特征在于所述化学修饰为核苷酸 之间的磷酸二酯键被硫代磷酸酯键取代。
8、 根据权利要求 4所述的化学修饰的 siRNA分子， 其特征在于所述化学修饰为核苷酸 的碱基被非 RNA碱基取代。
9、 根据权利要求 8所述的化学修饰的 siRNA分子， 其特征在于所述非 RNA碱基选自 胸腺嘧啶 (thymine)、 5-甲基胞嘧啶 (5-methylcytosine)、 异胞嘧啶 (isocytosine)、 假 异胞嘧啶（pseudoisocytosine)、 5-溴尿嘧啶（5-bromouracil)、 5-丙炔基尿嘧啶 (5-propynyluracil)、 5-丙炔基 -6-氟代尿嘧啶 (5-propyny-6-fluoroluracil)、 5-甲基噻唑 尿嘧啶（5-methylthiazoleuracil)、 6-氨基嘌呤（6-aminopurine)、 2-氨基嘌呤 (2-aminopurine)、 肌苷 (inosine)、 2,6-二氨基嘌呤(2,6-(^1^11(^1^116)、 7-丙炔基 -7- 脱 氮 腺 嘌 呤（7-propyne-7-deazaadenine) 、 7-丙 炔基 -7-脱 氮 鸟 嘌 呤 (7-propyne-7-deazaguanine)、 2-氯 -6-氨基噪呤 (2-chloro-6-aminopurine)。
、 根据权利要求 8所述的化学修饰的 siRNA分子，其特征在于所述化学修饰为核苷酸 的碱基被 DNA碱基取代。
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| US11633482B2 (en) | 2017-12-29 | 2023-04-25 | Suzhou Ribo Life Science Co., Ltd. | Conjugates and preparation and use thereof |
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