CN102719434A - Specific modification for inhibiting RNA interference off-target effect - Google Patents
Specific modification for inhibiting RNA interference off-target effect Download PDFInfo
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Abstract
The invention provides a chemically modified double-strand small interfering RNA (siRNA) molecule and its preparation and application methods. Chemically modified nucleotide is introduced into the 14th site or/and 16th site beginning from 5' terminal of the siRNA molecular sense strand, thereby reducing the off-target effect caused by the modified siRNA molecular sense strand and enhancing specificity of the modified siRNA molecule for silence of target gene expression.
Description
Technical field
The present invention relates to the nucleic acid technical field, be specifically related to a kind of have reduce positive-sense strand miss the target double-chain small disturbance RNA (siRNA) molecule and preparation method thereof of chemically modified of effect and the application of siRNA molecule in pharmaceutical compositions of this kind modification.
Background technology
(RNA interference is that (double-stranded RNA dsRNA) reduces the phenomenon that homologous gene is expressed in the mRNA level by double stranded rna molecule RNAi) in the RNA interference.The RNA perturbation technique is called clpp gene low (knock-down) or gene silencing (gene silencing) again visually; It is a kind of posttranscriptional gene expression regulation method; So the PTGS that is otherwise known as (post-transcriptional gene silencing, PTGS).Relevant the earliest RNA interferential report appeared at about nineteen ninety; Reported the RNA interference of in transgenic plant, finding simultaneously by two different research groups, given birth in the biology in nearly all back such as nematode, fruit bat, zebra fish and mouse again subsequently and observe the RNA interference.1999; Hamilton and Baulcombe detect the double-stranded RNA fragment that length is 21-25 Nucleotide in the plant that the RNA interference takes place; It is necessary that these double-stranded RNA segments are proved to be to produce the RNA interference; Be known as small RNA (small interfering RNA, siRNA).The relevant enzyme of double-stranded siRNA and cell source and protein form RNA inductive silencing complex, and (RNA-induced silencing complex RISC) is RNA interferential effector molecule.It has been generally acknowledged that; In the RNA interfering process; Positive-sense strand (passerby's chain) among the double-stranded siRNA is discharged by the RISC complex body; Antisense strand instructs the RISC complex body to be attached to the homologous site on the target mRNA, then by the degraded of the rnase iii in mixture target mRNA, thereby closes target gene expression.In fact; Double-stranded siRNA for a standard; Its two chains all can be participated in the combination of RISC complex body, that is to say, except the antisense strand (guiding chain) of siRNA can mediate the homogenic expression silencing; The positive-sense strand of siRNA (passerby's chain) also can mediate its homogenic expression silencing, causes the effect of missing the target by the positive-sense strand mediation.In gene functional research, the inhibition activity that this effect of missing the target of siRNA positive-sense strand may lead to errors and understand siRNA; And in the small nucleic acids pharmacy, then might cause a series of consequences such as toxic side effect of siRNA medicine, these all will have a strong impact on the application of RNA perturbation technique.
Therefore, reduce the effect of missing the target of siRNA molecule positive-sense strand, improving RNA interferential specificity is the problem that presses for solution at present.
Summary of the invention
The present invention relates to the theme that defines in the following paragraph that numbers in order:
1, the double-chain small disturbance RNA of isolating chemically modified (siRNA) molecule; Comprise positive-sense strand (passerby's chain) and antisense strand (guiding chain), it is characterized in that the Nucleotide of positive-sense strand from 5 ' end the 14th site of the siRNA molecule of said modification is the Nucleotide of chemically modified.
2, the double-chain small disturbance RNA of isolating chemically modified (siRNA) molecule; Comprise positive-sense strand (passerby's chain) and antisense strand (guiding chain), it is characterized in that the Nucleotide of positive-sense strand from 5 ' end the 16th site of the siRNA molecule of said modification is the Nucleotide of chemically modified.
3, the double-chain small disturbance RNA of isolating chemically modified (siRNA) molecule; Comprise positive-sense strand (passerby's chain) and antisense strand (guiding chain), it is characterized in that the Nucleotide of positive-sense strand from 5 ' end the 14th site and the 16th site of the siRNA molecule of said modification is the Nucleotide of chemically modified.
4,, it is characterized in that said chemically modified is selected from one or more in the following modification mode according to the siRNA molecule of each described chemically modified of paragraph 1-3:
(1) to the chemically modified of ribose in the Nucleotide;
(2) to the chemically modified of base in the Nucleotide;
(3) to the chemically modified of phosphodiester bond between the Nucleotide.
5,, it is characterized in that said chemically modified is the modification to ribose 2 ' in the Nucleotide-OH according to the siRNA molecule of paragraph 4 described chemically modifieds.
6,, it is characterized in that said chemically modified is that ribose 2 ' in the Nucleotide-OH is replaced by methoxyl group or fluorine according to the siRNA molecule of paragraph 5 described chemically modifieds.
7,, it is characterized in that said chemically modified is that phosphodiester bond between the Nucleotide is replaced by phosphorothioate bond according to the siRNA molecule of paragraph 4 described chemically modifieds.
8,, it is characterized in that said chemically modified is that the base of Nucleotide is replaced by non-RNA base according to the siRNA molecule of paragraph 4 described chemically modifieds.
9, according to the siRNA molecule of paragraph 8 described chemically modifieds; It is characterized in that said non-RNA base is selected from thymus pyrimidine (thymine), 5-methylcytosine (5-methylcytosine), iso-cytosine (isocytosine), false iso-cytosine (pseudoisocytosine), 5-bromouracil (5-bromouracil), 5-proyl uridylic (5-propynyluracil), 5-proyl-6-fluorouracil (5-propyny-6-fluoroluracil), 5-methylthiazol uridylic (5-methylthiazoleuracil), adenine (6-aminopurine), 2-aminopurine (2-aminopurine), inosine (inosine), 2; The 6-diaminopurine (2,6-diaminopurine), 7-proyl-7-denitrogenation VITAMIN B4 (7-propyne-7-deazaadenine), 7-proyl-7-deazaguanine (7-propyne-7-deazaguanine), 2-chloro-adenine (2-chloro-6-aminopurine).
10,, it is characterized in that said chemically modified is that the base of Nucleotide is replaced by the DNA base according to the siRNA molecule of paragraph 8 described chemically modifieds.
11,, it is characterized in that the positive-sense strand of said siRNA molecule and the length of antisense strand are 15-35 Nucleotide according to the siRNA molecule of each described chemically modified of paragraph 1-10.
12,, it is characterized in that the positive-sense strand of said siRNA molecule and the length of antisense strand are 19-23 Nucleotide according to the siRNA molecule of paragraph 11 described chemically modifieds.
13,, it is characterized in that the positive-sense strand of said siRNA molecule and the length of antisense strand are 21 Nucleotide according to the siRNA molecule of paragraph 12 described chemically modifieds.
14,, it is characterized in that said siRNA molecule is flat terminal siRNA molecule according to the siRNA molecule of each described chemically modified of paragraph 1-14.
15,, it is characterized in that the siRNA molecule that said siRNA molecule is 3 ' protruding terminus according to the siRNA molecule of each described chemically modified of paragraph 1-14.
16,, it is characterized in that said siRNA molecule is for having the siRNA molecule of 1-6 3 ' protruding terminus according to the siRNA molecule of paragraph 15 described chemically modifieds.
17,, it is characterized in that said siRNA molecule is for having the siRNA molecule of 2-4 3 ' protruding terminus according to the siRNA molecule of paragraph 16 described chemically modifieds.
18, a kind of pharmaceutical composition is characterized in that containing the siRNA molecule and the pharmaceutically acceptable carrier of each described chemically modified of paragraph 1-17.
19, the application of siRNA molecule in pharmaceutical compositions of each described chemically modified of paragraph 1-17.
20, a kind of siRNA of reduction molecule positive-sense strand method of effect of missing the target is characterized in that the Nucleotide of positive-sense strand from 5 ' end the 14th site of said siRNA molecule is carried out chemically modified.
21, a kind of siRNA of reduction molecule positive-sense strand method of effect of missing the target is characterized in that the Nucleotide of positive-sense strand from 5 ' end the 16th site of said siRNA molecule is carried out chemically modified.
22, a kind of siRNA of reduction molecule positive-sense strand method of effect of missing the target is characterized in that the Nucleotide of positive-sense strand from 5 ' end the 14th site and the 16th site of said siRNA molecule is carried out chemically modified.
23, according to the miss the target method of effect of each described reduction of paragraph 20-22 siRNA molecule positive-sense strand, it is characterized in that said chemically modified is selected from one or more in the following modification mode:
(1) to the chemically modified of ribose in the Nucleotide;
(2) to the chemically modified of base in the Nucleotide;
(3) to the chemically modified of phosphodiester bond between the Nucleotide.
24, according to the miss the target method of effect of the described reduction of paragraph 23 siRNA molecule positive-sense strands, it is characterized in that said chemically modified is the modification to ribose 2 ' in the Nucleotide-OH.
25, according to the miss the target method of effect of the described reduction of paragraph 24 siRNA molecule positive-sense strands, it is characterized in that said chemically modified is that ribose 2 ' in the Nucleotide-OH is replaced by methoxyl group or fluorine.
26, according to the miss the target method of effect of the described reduction of paragraph 23 siRNA molecule positive-sense strands, it is characterized in that said chemically modified is that phosphodiester bond is replaced by phosphorothioate bond between the Nucleotide.
27, according to the miss the target method of effect of the described reduction of paragraph 23 siRNA molecule positive-sense strands, it is characterized in that said chemically modified is that base is replaced by non-RNA base in the Nucleotide.
28, according to the miss the target method of effect of the described reduction of paragraph 27 siRNA molecule positive-sense strands; It is characterized in that said non-RNA base is selected from thymus pyrimidine (thymine), 5-methylcytosine (5-methylcytosine), iso-cytosine (isocytosine), false iso-cytosine (pseudoisocytosine), 5-bromouracil (5-bromouracil), 5-proyl uridylic (5-propynyluracil), 5-proyl-6-fluorouracil (5-propyny-6-fluoroluracil), 5-methylthiazol uridylic (5-methylthiazoleuracil), adenine (6-aminopurine), 2-aminopurine (2-aminopurine), inosine (inosine), 2; The 6-diaminopurine (2,6-diaminopurine), 7-proyl-7-denitrogenation VITAMIN B4 (7-propyne-7-deazaadenine), 7-proyl-7-deazaguanine (7-propyne-7-deazaguanine), 2-chloro-adenine (2-chloro-6-aminopurine).
29, according to the miss the target method of effect of the described reduction of paragraph 27 siRNA molecule positive-sense strands, it is characterized in that said chemically modified is that base is replaced by the DNA base in the Nucleotide.
30, according to the miss the target method of effect of each described reduction of paragraph 20-29 siRNA molecule positive-sense strand, it is characterized in that the positive-sense strand of said siRNA molecule and the length of antisense strand are 15-35 Nucleotide.
31, according to the miss the target method of effect of the described reduction of paragraph 30 siRNA molecule positive-sense strands, it is characterized in that the positive-sense strand of said siRNA molecule and the length of antisense strand are 19-23 Nucleotide.
32, according to the miss the target method of effect of the described reduction of paragraph 31 siRNA molecule positive-sense strands, it is characterized in that the positive-sense strand of said siRNA molecule and the length of antisense strand are 21 Nucleotide.
33, according to the miss the target method of effect of each described reduction of paragraph 20-32 siRNA molecule positive-sense strand, it is characterized in that said siRNA molecule is flat terminal siRNA molecule.
34, according to the miss the target method of effect of each described reduction of paragraph 20-32 siRNA molecule positive-sense strand, it is characterized in that the siRNA molecule that said siRNA molecule is 3 ' protruding terminus.
35, according to the miss the target method of effect of the described reduction of paragraph 34 siRNA molecule positive-sense strands, it is characterized in that said siRNA molecule is for having the siRNA molecule of 1-6 3 ' protruding terminus.
36, according to the miss the target method of effect of the described reduction of paragraph 35 siRNA molecule positive-sense strands, it is characterized in that said siRNA molecule is for having the siRNA molecule of 2-4 3 ' protruding terminus.
The technical problem that the present invention will solve is the expression silencing of its complementary gene transcript of siRNA molecule positive-sense strand mediation, thereby causes the problem by the effect of missing the target of positive-sense strand mediation.The purpose of this invention is to provide and a kind ofly have the siRNA molecule of the chemically modified that reduces the effect of missing the target that positive-sense strand causes and reduce the miss the target method of effect of siRNA molecule positive-sense strand.
For addressing the above problem; One aspect of the present invention provides a kind of double-chain small disturbance RNA (siRNA) molecule of isolating chemically modified; Comprise positive-sense strand (passerby's chain) and antisense strand (guiding chain), the positive-sense strand of the siRNA molecule of said modification is the Nucleotide of chemically modified from the Nucleotide in 5 ' end the 14th site or the 16th site.The present invention also provides a kind of double-chain small disturbance RNA (siRNA) molecule of isolating chemically modified; Comprise positive-sense strand (passerby's chain) and antisense strand (guiding chain), the Nucleotide of the positive-sense strand of the siRNA molecule of said modification from 5 ' end the 14th site and the 16th site is the Nucleotide of chemically modified.Chemically modified described in the present invention, be selected from the following modification mode one or more: (1) is to the chemically modified of ribose in the Nucleotide; (2) to the chemically modified of base in the Nucleotide; (3) to the chemically modified of phosphodiester bond between the Nucleotide.
Under a kind of preferable case, said chemically modified is the modification to ribose 2 ' in the Nucleotide-OH; More preferably, said chemically modified is that ribose 2 ' in the Nucleotide-OH is replaced by methoxyl group or fluorine.Under another kind of preferable case, said chemically modified is that the phosphodiester bond between the Nucleotide is replaced by phosphorothioate bond.Under another kind of preferable case, said chemically modified is that the base of Nucleotide is replaced by non-RNA base; Preferably; Said non-RNA base is selected from thymus pyrimidine (thymine), 5-methylcytosine (5-methylcytosine), iso-cytosine (isocytosine), false iso-cytosine (pseudoisocytosine), 5-bromouracil (5-bromouracil), 5-proyl uridylic (5-propynyluracil), 5-proyl-6-fluorouracil (5-propyny-6-fluoroluracil), 5-methylthiazol uridylic (5-methylthiazoleuracil), adenine (6-aminopurine), 2-aminopurine (2-aminopurine), inosine (inosine), 2; The 6-diaminopurine (2,6-diaminopurine), 7-proyl-7-denitrogenation VITAMIN B4 (7-propyne-7-deazaadenine), 7-proyl-7-deazaguanine (7-propyne-7-deazaguanine), 2-chloro-adenine (2-chloro-6-aminopurine).Preferably, said chemically modified is that base is replaced by the DNA base in the Nucleotide.
The siRNA molecule of above-mentioned any chemically modified, the length of its positive-sense strand and antisense strand can be 15-35 Nucleotide, and preferred length is a 19-23 Nucleotide, and more preferably length is 21 Nucleotide.Above-mentioned any siRNA molecule through chemically modified, it can also can have 3 ' protruding terminus for flat terminal, and 3 ' protruding terminus can be 1-6 Nucleotide, is preferably 2-4 Nucleotide, more preferably 2 Nucleotide.
The present invention provides a kind of pharmaceutical composition on the other hand, and said composition can comprise the siRNA molecule and the pharmaceutically acceptable carrier of above-mentioned any chemically modified.Used " the pharmaceutically acceptable carrier " of the present invention should be compatible with siRNA (siRNA) molecule in the pharmaceutical composition of the present invention, can not reduce pharmaceutical composition under normal conditions significantly in the effect aspect the inhibition of gene expression with its blend.This type drug administration carrier includes but not limited to the various carriers that can be used for the nucleic acid administration, like liposome, degradable macromolecular compound, salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.
Pharmaceutical composition of the present invention can be processed various medically acceptable formulations, and can by the doctor according to patient's kind, age, body weight and roughly factor such as disease condition, administering mode confirm the useful dosage of patient is used.Preparation according to the invention is meant various liquid formulations such as oral liquid, injection, sublingual administration agent, or is prepared into multiple other formulations such as tablet, capsule through appropriate excipients in addition.
The present invention also provides the application of siRNA molecule in pharmaceutical compositions of above-mentioned any chemically modified.
The third aspect of the invention provides the method for the effect of missing the target that a kind of siRNA of reduction molecule positive-sense strand causes.The Nucleotide of positive-sense strand from 5 ' end the 14th site or the 16th site through to the siRNA molecule carries out chemically modified; Perhaps the positive-sense strand of the siRNA molecule Nucleotide from 5 ' end the 14th site and the 16th site is carried out chemically modified simultaneously, can significantly reduce the effect of missing the target that the positive-sense strand of the siRNA molecule after the modification causes.Chemically modified described in the present invention, be selected from the following modification mode one or more: (1) is to the chemically modified of ribose in the Nucleotide; (2) to the chemically modified of base in the Nucleotide; (3) to the chemically modified of phosphodiester bond between the Nucleotide.
Under a kind of preferable case, said chemically modified is the modification to ribose 2 ' in the Nucleotide-OH; More preferably, said chemically modified is that ribose 2 ' in the Nucleotide-OH is replaced by methoxyl group or fluorine.Under another kind of preferable case, said chemically modified is that the phosphodiester bond between the Nucleotide is replaced by phosphorothioate bond.Under another preferable case, said chemically modified is that the base of Nucleotide is replaced by non-RNA base; Preferably; Said non-RNA base is selected from thymus pyrimidine (thymine), 5-methylcytosine (5-methylcytosine), iso-cytosine (isocytosine), false iso-cytosine (pseudoisocytosine), 5-bromouracil (5-bromouracil), 5-proyl uridylic (5-propynyluracil), 5-proyl-6-fluorouracil (5-propyny-6-fluoroluracil), 5-methylthiazol uridylic (5-methylthiazoleuracil), adenine (6-aminopurine), 2-aminopurine (2-aminopurine), inosine (inosine), 2; The 6-diaminopurine (2,6-diaminopurine), 7-proyl-7-denitrogenation VITAMIN B4 (7-propyne-7-deazaadenine), 7-proyl-7-deazaguanine (7-propyne-7-deazaguanine), 2-chloro-adenine (2-chloro-6-aminopurine).Preferably, said chemically modified is that base is replaced by the DNA base in the Nucleotide.
The siRNA molecule of above-mentioned any chemically modified, the length of its positive-sense strand and antisense strand can be 15-35 Nucleotide, and preferred length is a 1g-23 Nucleotide, and more selecting length is 21 Nucleotide.The siRNA molecule of above-mentioned any chemically modified, it can also can have 3 ' protruding terminus for flat terminal, and 3 ' protruding terminus can be 1-6 Nucleotide, is preferably 2-4 Nucleotide, more preferably 2 Nucleotide.
Beneficial effect of the present invention
The siRNA molecule of chemically modified provided by the invention can significantly reduce the effect of missing the target that siRNA molecule positive-sense strand causes, improves the siRNA specificity reticent to expression of target gene.
Embodiment
Under normal conditions, the researchist is with in the siRNA molecule transfered cell, and the gene silencing that is mediated by antisense strand (guiding chain) is that the researchist is desired; And the gene silencing that is mediated by positive-sense strand (passerby's chain) is the spinoff in the RNA interfering process, is called as the effect of missing the target that positive-sense strand causes.
To the problem that how to reduce the effect of missing the target that siRNA molecule positive-sense strand causes, the contriver has carried out careful research to the chemically modified of the Nucleotide of the different loci of siRNA positive-sense strand and the dependency of missing the target between the effect.Discovery is carried out chemically modified to the siRNA positive-sense strand from the Nucleotide in 5 ' end the 14th site or the 16th site, can significantly reduce the expression inhibiting activity of positive-sense strand pair and its complementary gene transcripts, promptly suppresses the effect of missing the target that the siRNA positive-sense strand causes; This chemically modified does not simultaneously influence the active for gene silencing of the antisense strand of siRNA molecule unmodified.On this basis, the contriver accomplishes the present invention.
Among the present invention, term " antisense strand (guiding chain) " be meant in the double-stranded siRNA molecule with the target gene transcript in target site sequence complementary siRNA form chain." positive-sense strand (passerby's chain) " is meant another composition chain with siRNA molecule antisense strand sequence complementary siRNA molecule." positive-sense strand target sequence " be meant with the siRNA molecule in the homologous sequence of positive-sense strand (passerby's chain) complementary gene order or this gene order." antisense strand target sequence " be meant with the siRNA molecule in the homologous sequence of antisense strand (guiding chain) complementary gene order or this gene order.
According to the present invention, the siRNA of said modification (siRNA) molecule can be made up of ribonucleotide, also can comprise the hybrid molecule of ribonucleotide and at least one deoxyribonucleotide.
Among the present invention; Can use the nucleic acid compound method of various routines and accomplish the synthetic of nucleic acid molecule involved in the present invention; Perhaps entrust the biotech company that specializes in the synthetic service of nucleic acid to accomplish the synthetic of nucleic acid molecule, as entrusting sharp rich bio tech ltd in Guangzhou or Ying Jun Bioisystech Co., Ltd (invitrogen) to synthesize.
In general, the compound method of nucleic acid oligomer molecule comprises following four steps: (1) oligomerization ribonucleotide synthetic; (2) deprotection; (3) purifies and separates; (4) desalination.
For example, it is following to have the chemosynthesis step of the siRNA of nucleotide sequence shown in the SEQ ID NO:1:
(1) siRNA forms the synthetic of chain: in that automated DNA/the RNA synthesizer (for example; Applied Biosystems EXPEDITE8909) goes up positive-sense strand or the antisense strand oligonucleotide sequence that the siRNA of 1 mmole amount is synthesized in setting; Setting each round-robin coupling time is 10-15 minute; Initiator be 5 '-O-of connecting of solid phase to dimethoxy-thymidine upholder, first circulates in and connects a base on the solid support, then in circulate the n time (2≤n≤35); On the base that the n-1 time circulation connected, connect a base, repeat this circulation until accomplishing the synthetic of whole nucleotide sequences.
(2) deprotection
The solid support that is connected with oligonucleotide is joined in the test tube, and in this test tube, add ethanol/ethamine (volume ratio is 1: 3) of 1 milliliter, sealing places 55-70 ℃ of incubator then, hatches 2-30 hour; Taking-up is connected with the solid support of oligonucleotide, with distilled water drip washing 2 times (each 1 milliliter), collects elutriant; After dry 30 minutes, add the tetrahydrofuran solution (1M) of 1 milliliter of tetrabutyl ammonium fluoride under the room temperature, room temperature was placed 4-12 hour; Add 2 milliliters of ethanol again, collecting precipitation promptly obtains the crude product of synthetic oligonucleotide.
(3) purifies and separates
It is in 1 mole/milliliter the ammonium acetate solution that the crude product of the oligonucleotide that obtains is dissolved in 2 ml concns, separates through the C18 HPLC then, obtains the oligonucleotide product of purifying.
(4) desalination
Using concentration is oligonucleotide product 2-4 time (each 2 milliliters) of the aqueous ethanolic solution washing purifying of 75 weight %, removes salt, dry under the room temperature; Oligomerization Yeast Nucleic Acid mixed dissolution (10mM Tris in the damping fluid of 1-2 milliliter with positive-sense strand and antisense strand; PH=7.5-8.0; 50mM NaCl), this solution is heated to 95 ℃, slowly this solution is cooled to room temperature then; And kept 16-22 hour at ambient temperature, promptly obtain siRNA solution.
Among the present invention, the mode of said chemically modified is conventionally known to one of skill in the art.For example, the present invention's chemically modified that said nucleic acid molecule is carried out is one or more in the following modification mode:
(1) to the chemically modified of ribose in the Nucleotide;
(2) to the chemically modified of base in the Nucleotide;
(3) to the chemically modified of phosphodiester bond between the Nucleotide.
Said modification to phosphodiester bond includes but not limited to the oxygen in the phosphodiester bond is modified; For example thiophosphoric acid is modified (Phosphorthioate) and borine phosphoric acid salt is modified (Boranophosphate), respectively with the oxygen in sulphur and the borine displacement phosphodiester bond.Two kinds of modifications can both be stablized the structure of siRNA molecule, keep the specificity and the avidity of base pairing.The siRNA hydrophobicity that borine phosphoric acid salt is modified is strong, is easy in blood plasma, form hydrated protein, the toxic side effect of human body is lower than the siRNA of thiophosphatephosphorothioate modification.
Thiophosphoric acid is modified borine phosphoric acid salt and is modified
Said ribose modify include but not limited to hydroxyl in the Nucleotide pentose (2 '-OH) modification.After the hydroxy position of ribose is introduced some substituting group such as methoxyl group or fluorine, make siRNA have the performance of stronger opposing nucleicacidase hydrolysis.To the modification of hydroxyl in the Nucleotide pentose comprise 2 '-fluorine modify (2 '-fluro modification), 2 '-the oxygen methyl modify (2 '-OME), 2 '-methoxyethyl modify (2 '-MOE), 2,4 '-dinitrophenol(DNP) modify (2 '-DNP modification), lock nucleic acid (LNA), 2 '-amido modified (Amina modification), 2 '-deoxidation modifies (2 '-Deoxy modification) etc.
2 '-fluorine modification 2 '-modification of oxygen methyl
2 '-methoxyethyl modification 2,4 '-the dinitrophenol(DNP) modification
Lock nucleic acid 2 '-amido modified
2 '-the deoxidation modification
Said base modification includes but not limited to the base of Nucleotide is modified; As introduce in 5 sites of uridylic 5 of bromine or iodine '-bromouracil (5 '-bromo-uracil) with 5 '-iodouracil (5 '-iodo-uracil) to modify be the normal base modification method of using; Other also have N3-6-Methyl Uracil (N3-methyl-uracil) to modify; 2,6-diaminopurine (2,6-diaminopurine) modify etc.
5 '-bromouracil 5 '-iodouracil
N3-6-Methyl Uracil 2,6-diaminopurine
Under the preferable case, the said modification that is modified to 2 '-OH of ribose in the Nucleotide of said nucleic acid molecule; Further be preferably, 2 '-OH of ribose is replaced by methoxyl group or fluorine in the said Nucleotide that is modified to said nucleic acid molecule.
According to the present invention; The goal gene of said siRNA molecule can be range gene; Can be with gene to be analyzed being arranged as goal gene in intracellular function; Can be with the gene that need to suppress its expression as goal gene, also can be with the gene relevant as goal gene with disease or dysfunction, like the gene on oncogene, virogene, surface of cell membrane acceptor gene, nuclear receptor gene or the cell signaling path etc.Those skilled in the art is according to the sequence of goal gene; Can design and obtain the special siRNA molecule of goal gene; For example; Target gene sequences or the sequence number of goal gene in NCBI Genbank are imported various siRNA designs program; Like Insert Design Tool for the shRNA Vectors (Ambion), shRNA Explorer (Gene Link), siDirect (Yuki Naito et al.University of Tokyo), SiRNA at Whitehead (Whitehead lnstitute 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., this is designed program according to planner's requirement and the principle of design of siRNA, to the gene order design siRNA sequence that is provided.Some program can also be carried out based on full genome or transcribes the homology analysis of group the siRNA sequence of design, filters out the special siRNA molecule of target gene sequences.Above-described siRNA designs program and the principle that relates to is conventionally known to one of skill in the art, and its full content is incorporated herein by reference at this in the lump.
To combine embodiment that the present invention is described in further detail below.Should be appreciated that and enumerate these embodiment, and be not to be used for limiting scope of the present invention just for an illustration.Unless stated otherwise, the used reagent of the present invention, substratum are the commercial goods.The experimental technique of unreceipted actual conditions among the embodiment; Usually carry out according to the normal experiment condition; For example people such as Sambrook is at " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1.siRNA target site and siRNA sequence
Entrust the siRNA target sequence fragment in the synthetic table 1 of Invitrogen Beijing Company, be used to merge the structure of reporter gene expression carrier.
To the table 1 site sequence design siRNA that hits, entrust listed chemically modified and siRNA unmodified (siRNA) among the synthetic table of the sharp rich bio tech ltd in the Guangzhou 2-11.
Table 1.siRNA target site sequence
Embodiment 2.siRNA suppresses active detection to target site
1) makes up the reorganization Photinus pyralis LUC reporter gene plasmid that has siRNA separation target site
Referring to following document, make up and contain the reorganization Photinus pyralis LUC reporter gene plasmid that siRNA separates target site, the reporter gene plasmid vector can be asked for to the paper author.(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?Y，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) transfection
With reference to the method for describing in the above-mentioned document, carry out cell transfecting and siRNA determination of activity.Specifically will be in the DMEM substratum (10%FBS, 2mM L-glutaminate, the Streptomycin sulphate of the penicillium mould of 100 units per ml and 100 μ g/ml) cultured human embryo tire kidney cell (HEK293) be inoculated in 24 orifice plates (1 * 10
5Cell/0.5ml substratum/hole).Treat that the cell growth after 24 hours, when the degrees of fusion of cell is the 50-70% left and right sides, is changed to Opti-MEM substratum (Gibco company) with substratum.Utilize Lipofectamine 2000 (Invitrogen company; The reorganization Photinus pyralis LUC reporter gene plasmid transfection that will have siRNA separation target site the U.S.) is gone into cell; Transfection simultaneously gets into control plasmid (the Promega company that also has pRL-TK (coding sea pansy luciferase) of cell; Madison WI, USA), and the siRNA molecule modification of chemosynthesis or unmodified.Every hole contains 0.17g recombinant plasmid and 0.017g pRL-TK control plasmid, and the final concentration of siRNA is 13nM.Three multiple holes of every kind of parallel transfection of siRNA, with three multiple holes of two kinds of reporter gene plasmids (not transfection siRNA) of the same amount of a transfection as contrast.Change transfection medium into 1mlDMEM substratum (10%FBS, 2mM L-glutaminate, penicillium mould that 100 units/person of outstanding talent rises and the Streptomycin sulphate of 100 μ g/ml) again after 4 hours.
3) uciferase activity is measured
Transfection is harvested cell after 24 hours; With 10 μ l cell pyrolysis liquid lysing cell, utilize two luciferase reporter gene analytical systems (Dual-Luciferase Assay System, Promega company) and ELIASA (Novosta r; BMG Labtechnologies GmbH; Germany) measure the activity of two kinds of luciferases, as contrast, calculate the reticent efficient of siRNA target site through following formula with the expression amount of the reporter gene in the hole of untransfected siRNA.
Reticent efficient=1-(expression amount of the expression amount of experimental group Photinus pyralis LUC reporter gene/experimental group renilla luciferase reporter gene)/(expression amount of the expression amount of control group Photinus pyralis LUC reporter gene/control group renilla luciferase reporter gene)
The different loci of embodiment 3. methoxyl group nucleotide modification siRNA positive-sense strands is to the miss the target influence of effect of positive-sense strand
Choose 2 siRNA (CM-01 and CM-11), make up the reorganization Photinus pyralis LUC reporter gene plasmid (the target site sequence is as shown in table 1) that contains siRNA positive-sense strand target site according to embodiment 2 described schemes respectively; The siRNA molecule of the unmodified shown in the synthetic table 2, and respectively the siRNA positive-sense strand contain from 5 ' end 2-18 site the methoxyl group modified nucleotide through the siRNA of chemically modified molecule.According to embodiment 2 described experimental programs, it is active to the inhibition of its positive-sense strand target site to detect various siRNA molecules respectively.Every kind of siRNA tests parallel 3 the multiple holes of doing at every turn, and each experiment repeats 2 times at least.Experimental result shown in the table 2 shows, can significantly reduce siRNA pair and its positive-sense strand complementary expression of gene inhibition activity of modification from the chemically modified of the Nucleotide in 5 ' end the 14th site to the siRNA positive-sense strand; Also can reduce siRNA pair and its positive-sense strand complementary expression of gene inhibition activity of modification to a certain extent from the chemically modified of the Nucleotide in 5 ' end the 16th site to the siRNA positive-sense strand; And the modification in all the other sites is not influenced siRNA pair and its positive-sense strand complementary expression of gene inhibition activity basically.
The 13-16 site that embodiment 4. methoxyl groups are modified the siRNA positive-sense strand is to the miss the target influence of effect of positive-sense strand
As shown in table 3; The siRNA positive-sense strand from 5 ' end the 13-16 site in; 12 siRNA sequences are chosen in each site, the siRNA molecule of synthetic its unmodified, and the siRNA molecule that contains the chemically modified of methoxyl group modified nucleotide respectively at its positive-sense strand from 5 ' end 13-16 site; According to embodiment 2 described experimental programs, make up the reorganization Photinus pyralis LUC reporter gene plasmid (the target site sequence is as shown in table 1) that contains siRNA positive-sense strand target site; According to embodiment 2 said schemes, the siRNA molecule that detects various modifications in the table 3 or unmodified respectively is pair active with the inhibition of its positive-sense strand complementary target site; Every kind of siRNA tests parallel 3 the multiple holes of doing at every turn, and each experiment repeats 2 times at least.Experimental result shows shown in the table 3, positive-sense strand from the Nucleotide in 5 ' end the 14th site be A, U, C, the G any the time, it is carried out the siRNA molecule pair that chemically modified all can significantly reduce modification suppresses activity with its positive-sense strand complementary expression of gene; In addition, positive-sense strand from the Nucleotide in 5 ' end the 16th site be A, U, C, the G any the time, it is carried out the siRNA molecule pair that chemically modified also can reduce modification to a certain extent suppresses activity with its positive-sense strand complementary expression of gene; And the modification in the 13rd site or the 15th site is less to the miss the target influence of effect of positive-sense strand.This experimental result also shows, the miss the target influence of effect is a kind of with to modify the position relevant to positive-sense strand in the modification of siRNA, and the phenomenon that has nothing to do with concrete nucleotide sequence, promptly this effect is a kind of effect that does not rely on nucleotide sequence." not relying on the effect of nucleotide sequence " used herein is meant that modified nucleotide that embodiment provides and modification mode can be used for any siRNA sequence and do not consider the concrete sequence of this siRNA.
Embodiment 5. modify siRNA just/14 sites/16 sites of antisense strand are to the miss the target influence of effect of siRNA positive-sense strand
As shown in table 4; Choose 2 siRNA sequence (cdc-2; NPY-305); The siRNA molecule of synthetic its unmodified, and the positive-sense strand of siRNA or antisense strand carried out the siRNA molecule that methoxyl group is modified from the Nucleotide in 5 ' end the 14th site or the 16th site, or simultaneously to positive-sense strand and antisense strand from 5 ' hold the Nucleotide in the 14th site to carry out the methoxyl group modification the siRNA molecule; Or simultaneously positive-sense strand and antisense strand being carried out the siRNA molecule that methoxyl group is modified from the Nucleotide in 5 ' end the 16th site, the siRNA molecule of modification and unmodified is as shown in table 4.According to embodiment 2 described schemes, make up and contain the reorganization Photinus pyralis LUC reporter gene plasmid (the target site sequence is as shown in table 1) that separates target site with its positive-sense strand or antisense strand complementary respectively.According to embodiment 2 said schemes, it is active with the inhibition of its positive-sense strand or the isolating target site of antisense strand complementary to detect various siRNA molecules pair respectively.Every kind of siRNA tests parallel 3 the multiple holes of doing at every turn, and each experiment repeats 2 times at least.Experimental result shown in the table 4 shows; The chemically modified of a composition chain among the siRNA only reduced modified chain and pair suppress active, and the siRNA that does not influence another unmodified forms chain and pair suppresses active with its complementary expression of gene with its complementary expression of gene.
Embodiment 6. methoxyl groups are modified siRNA positive-sense strand 5 ' simultaneously and have been held the 14th and 16 sites to the miss the target influence of effect of positive-sense strand
As shown in table 5, select 5 siRNA sequences (CM-01, CM-02, CM-03, CM-04, CM-05), synthetic without the siRNA (table 3) that modifies and hold the 14th and 16 sites to carry out the siRNA (table 5) of methoxyl group modification to positive-sense strand 5 ' simultaneously.According to embodiment 2 said experimental programs; Make up the reorganization Photinus pyralis LUC reporter gene plasmid (the target site sequence is as shown in table 1) that contains these siRNA positive-sense strand target sites respectively; Respectively various siRNA are gone in the cell with reorganization Photinus pyralis LUC reporter gene plasmid, pRL-TK (coding sea pansy luciferase) control plasmid cotransfection then, lysing cell detects the activity of two kinds of luciferases after 24 hours.Experimental result shows shown in the table 5, and in different siRNA molecules, it is that the positive-sense strand effect of missing the target is had the site of material impact effect that positive-sense strand 5 ' has been held the 14th site and the 16th site; Compare with siRAN, modify in the time of to these two sites and can significantly reduce the restraining effect that siRNA expresses corresponding positive-sense strand target point gene without modification; Compare with the siRNA in independent modification the 14th site or the 16th site, modification has certain synergy in the time of to two sites.
Embodiment 7. fluoro are modified the miss the target influence of effect of siRNA positive-sense strand
Select 5 siRNA sequences (CM-01, CM-02, CM-03, CM-04, CM-05), synthetic without the siRNA (table 3) that modifies and the siRNA of the modification shown in the table 6; According to embodiment 2 said schemes, make up the reorganization Photinus pyralis LUC reporter gene plasmid (the target site sequence is as shown in table 1) that contains siRNA positive-sense strand target site respectively.According to embodiment 2 said schemes, it is active with the inhibition of its positive-sense strand complementary target site to detect various siRNA molecules pair respectively; Every kind of siRNA tests parallel 3 the multiple holes of doing at every turn, and each experiment repeats 2 times at least.Experimental result shown in the table 6 shows, held the 14th and/or the 16th site to carry out fluoro to siRNA molecule positive-sense strand 5 ' and modified the positive-sense strand of the siRNA molecule that can significantly reduce modification and pair suppress active with its complementary expression of gene.
Embodiment 8.5-methylcystein is modified the miss the target influence of effect of siRNA positive-sense strand
SiRNA shown in the option table 7, synthetic siRNA without siRNA (table 3) that modifies and the modification of the 5-methylcytosine shown in the table 7; According to embodiment 2 said schemes, make up the reorganization Photinus pyralis LUC reporter gene plasmid (the target site sequence is as shown in table 1) that contains siRNA positive-sense strand target site respectively.According to embodiment 2 said schemes, it is active with the inhibition of its positive-sense strand complementary target site to detect various siRNA molecules pair respectively.Every kind of siRNA tests parallel 3 the multiple holes of doing at every turn, and each experiment repeats 2 times at least.Experimental result shown in the table 7 shows, held the 14th and/or the 16th site to carry out 5-methylcytosine to siRNA molecule positive-sense strand 5 ' and modified the positive-sense strand of the siRNA molecule that can significantly reduce modification and pair suppress active with its complementary expression of gene.
Embodiment 9.5-bromouracil is modified the miss the target influence of effect of siRNA positive-sense strand
SiRNA shown in the option table 8, synthetic without the siRNA (table 3) that modifies and the siRNA of the modification shown in the table 8; According to embodiment 2 said schemes, make up the reorganization Photinus pyralis LUC reporter gene plasmid (the target site sequence is as shown in table 1) that contains siRNA positive-sense strand target site respectively.According to embodiment 2 said schemes, it is active with the inhibition of its positive-sense strand complementary target site to detect various siRNA molecules pair respectively; Every kind of siRNA tests parallel 3 the multiple holes of doing at every turn, and each experiment repeats 2 times at least.Experimental result shown in the table 8 shows, held the 14th and/or the 16th site to carry out 5-bromouracil to siRNA molecule positive-sense strand 5 ' and modified the positive-sense strand of the siRNA molecule that can significantly reduce modification and pair suppress active with its complementary expression of gene.
Embodiment 10.DNA replacement is modified the miss the target influence of effect of siRNA positive-sense strand
SiRNA shown in the option table 9, synthetic without the siRNA (table 3) that modifies and the siRNA of the DNA base modification shown in the table 9; According to embodiment 2 said schemes, make up the reorganization Photinus pyralis LUC reporter gene plasmid (the target site sequence is as shown in table 1) that contains siRNA positive-sense strand target site respectively.According to embodiment 2 said schemes, it is active with the inhibition of its positive-sense strand complementary target site to detect various siRNA molecules pair respectively.Every kind of siRNA tests parallel 3 the multiple holes of doing at every turn, and each experiment repeats 2 times at least.Experimental result shown in the table 9 shows, held the base in the 14th and/or the 16th site to carry out the DNA replacement to siRNA molecule positive-sense strand 5 ' and modified the positive-sense strand of the siRNA molecule that can significantly reduce modification and pair suppress active with its complementary expression of gene.
Above embodiment shows that the chemically modified of having held the 14th and/or the 16th site to carry out different modes to siRNA positive-sense strand 5 ' all can reduce the effect of missing the target that is caused by positive-sense strand; This result further shows, the modification of siRNA to positive-sense strand miss the target the influence of effect be a kind of with modify the relevant phenomenon in position.
Embodiment 11.siRNA positive-sense strand is modified the miss the target influence of effect of positive-sense strand to the siRNA with different lengths 3 ' protruding terminus
As shown in table 10, synthetic siRNA (the Nucleotide number of 3 ' protruding terminus is respectively 0,1,4,6, is flat terminal during for " 0 ") with different lengths 3 ' protruding terminus.According to embodiment 2 said schemes; Make up the reorganization Photinus pyralis LUC reporter gene plasmid (the target site sequence is as shown in table 1) that contains siRNA positive-sense strand target site respectively; Respectively the siRNA in the table 10 is gone in the cell with reorganization Photinus pyralis LUC reporter gene plasmid, pRL-TK (coding sea pansy luciferase) control plasmid cotransfection then, lysing cell detects the activity of two kinds of luciferases after 24 hours.Experimental result shown in the table 10 shows, in the siRNA molecule of 3 ' protruding terminus with different lengths, it is that the positive-sense strand effect of missing the target is had the site of material impact that positive-sense strand 5 ' has been held the 14th site and/or the 16th site; Compare with siRAN, can reduce the restraining effect that siRNA expresses corresponding positive-sense strand target point gene, promptly reduce the effect of missing the target of positive-sense strand the independent modification in these two sites or modification simultaneously without modification.
Embodiment 12.siRNA positive-sense strand is modified the miss the target influence of effect of positive-sense strand to different lengths siRNA
As shown in table 11, synthetic siRNA with different lengths; According to embodiment 2 said schemes, make up the reorganization Photinus pyralis LUC reporter gene plasmid (the target site sequence is as shown in table 1) that contains siRNA positive-sense strand target site respectively.Respectively the siRNA in the table 11 is gone in the cell with reorganization Photinus pyralis LUC reporter gene plasmid, pRL-TK (coding sea pansy luciferase) control plasmid cotransfection, lysing cell detects the activity of two kinds of luciferases after 24 hours.Experimental result shown in the table 11 shows, in having the siRNA molecule of different lengths, it is that the positive-sense strand effect of missing the target is had the site of material impact that positive-sense strand 5 ' has been held the 14th site and/or the 16th site; Compare with siRAN, can reduce the restraining effect that siRNA expresses corresponding positive-sense strand target point gene, promptly reduce the effect of missing the target of positive-sense strand the independent modification in these two sites or modification simultaneously without modification.
Claims (10)
1. the double-chain small disturbance RNA of isolating chemically modified (siRNA) molecule; Comprise positive-sense strand (passerby's chain) and antisense strand (guiding chain), it is characterized in that the Nucleotide of positive-sense strand from 5 ' end the 14th site of the siRNA molecule of said modification is the Nucleotide of chemically modified.
2. the double-chain small disturbance RNA of isolating chemically modified (siRNA) molecule; Comprise positive-sense strand (passerby's chain) and antisense strand (guiding chain), it is characterized in that the Nucleotide of positive-sense strand from 5 ' end the 16th site of the siRNA molecule of said modification is the Nucleotide of chemically modified.
3. the double-chain small disturbance RNA of isolating chemically modified (siRNA) molecule; Comprise positive-sense strand (passerby's chain) and antisense strand (guiding chain), it is characterized in that the Nucleotide of positive-sense strand from 5 ' end the 14th site and the 16th site of the siRNA molecule of said modification is the Nucleotide of chemically modified.
4. according to the siRNA molecule of each described chemically modified of claim 1-3, it is characterized in that said chemically modified is selected from one or more in the following modification mode:
(1) to the chemically modified of ribose in the Nucleotide;
(2) to the chemically modified of base in the Nucleotide;
(3) to the chemically modified of phosphodiester bond between the Nucleotide.
5. the siRNA molecule of chemically modified according to claim 4 is characterized in that said chemically modified is the modification to ribose 2 ' in the Nucleotide-OH.
6. the siRNA molecule of chemically modified according to claim 5 is characterized in that said chemically modified is that ribose 2 ' in the Nucleotide-OH is replaced by methoxyl group or fluorine.
7. the siRNA molecule of chemically modified according to claim 4 is characterized in that said chemically modified is that phosphodiester bond between the Nucleotide is replaced by phosphorothioate bond.
8. the siRNA molecule of chemically modified according to claim 4 is characterized in that said chemically modified is that the base of Nucleotide is replaced by non-RNA base.
9. the siRNA molecule of chemically modified according to claim 8; It is characterized in that said non-RNA base is selected from thymus pyrimidine (thymine), 5-methylcytosine (5-methylcytosine), iso-cytosine (isocytosine), false iso-cytosine (pseudoisocytosine), 5-bromouracil (5-bromouracil), 5-proyl uridylic (5-propynyluracil), 5-proyl-6-fluorouracil (5-propyny-6-fluoroluracil), 5-methylthiazol uridylic (5-methylthiazoleuracil), adenine (6-aminopurine), 2-aminopurine (2-aminopurine), inosine (inosine), 2; The 6-diaminopurine (2,6-diaminopurine), 7-proyl-7-denitrogenation VITAMIN B4 (7-propyne-7-deazaadenine), 7-proyl-7-deazaguanine (7-propyne-7-deazaguanine), 2-chloro-adenine (2-chloro-6-aminopurine).
10. the siRNA molecule of chemically modified according to claim 8 is characterized in that said chemically modified is that the base of Nucleotide is replaced by the DNA base.
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| US11660347B2 (en) | 2017-12-01 | 2023-05-30 | Suzhou Ribo Life Science Co., Ltd. | Nucleic acid, composition and conjugate containing same, preparation method, and use thereof |
| US11633482B2 (en) | 2017-12-29 | 2023-04-25 | Suzhou Ribo Life Science Co., Ltd. | Conjugates and preparation and use thereof |
| US11918600B2 (en) | 2018-08-21 | 2024-03-05 | Suzhou Ribo Life Science Co., Ltd. | Nucleic acid, pharmaceutical composition and conjugate containing nucleic acid, and use thereof |
| US11896674B2 (en) | 2018-09-30 | 2024-02-13 | Suzhou Ribo Life Science Co., Ltd. | SiRNA conjugate, preparation method therefor and use thereof |
| WO2023138659A1 (en) * | 2022-01-20 | 2023-07-27 | 上海拓界生物医药科技有限公司 | Dsrna, use thereof and preparation method therefor |
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