Small interfering RNA (Ribonucleic Acid) as well as preparation method and application thereof

Abstract

According to the oligonucleotide with the pseudouracil nucleotide residue at the 5'terminal of the siRNA positive-sense strand, the situation that the positive-sense strand is combined by AGO2 protein can be obviously reduced through the design scheme, and the siRNA gene silencing capacity mediated by the antisense strand is also obviously improved. The 5'end modified pseudouracil and N1-methyl pseudouracil can enable the siRNA positive-sense strand design to be more effective. Meanwhile, in the double-stranded RNA, the pseudouracil and the N1-methyl pseudouracil are paired with adenine to form U: A-like pairing. On the basis, different from common G: C pairing, during siRNA design, U: A pairing can also be selected at the 5'end of a positive-sense strand, during chemical synthesis, uracil is replaced with pseudouracil or N1-methyl pseudouracil, pU: A or NpU: A pairing is realized, so that the necessity of selecting G or C at the 5 'end of the positive-sense strand is obviously reduced, and the siRNA design space is directly increased.

Description

Technical Field

[0001] This specification relates to the field of biomedicine, and in particular to a small interfering RNA and its preparation method and application. Background Art

[0002] Small interfering RNA (siRNA) is an emerging therapeutic modality, a class of double-stranded, approximately 21-nucleotide (nt) RNAs. After forming an RNA-induced silencing complex (RISC) with proteins such as Argonaute-2 (AGO2), siRNA separates into two single strands: a guide strand (antisense) and a follower strand (sense). The guide strand (antisense) perfectly matches the target gene mRNA. AGO2 specifically recognizes and cleaves disease-associated mRNA, directly blocking the production of disease-related proteins at the translational level and exerting therapeutic effects. The follower strand (sense) is released from the RISC complex and degraded by intracellular RNases.

[0003] As a key protein for siRNA to function, the AGO2 protein has three functional domains, namely the PAZ domain, the MID domain, and the PIWI domain. After AGO2 binds to double-stranded siRNA, a strand selection process occurs (Strand selection). Ideally, the guide strand (antisense strand) is preferentially loaded into RISC to perform the targeting function, but the follower strand (sense strand) can also be loaded into RISC, resulting in nonspecific targeting and thus causing off-target effects. Both the siRNA antisense and sense strands bind to the MID domain through their 5′-terminal nucleotides. The binding of the siRNA sense strand to the MID domain of the AGO2 protein weakens the binding of the antisense strand to the AGO2 protein, resulting in a decrease in the effectiveness of the siRNA; at the same time, it leads to off-target effects of the siRNA sense strand.

[0004] Theoretically, AMP, UMP, CMP, and GMP can all bind to the AGO2 MID domain. However, UMP and AMP have higher affinity for the AGO2 MID domain (Kd is generally 0.3-0.6 mM), while GMP and CMP have much lower affinity for the AGO2 MID domain (Kd is generally 3-6 mM). Therefore, in conventional siRNA design, in order to facilitate the loading of the guide strand (antisense strand) into RISC, designers introduce AMP or UMP at the 5' end of the guide strand (antisense strand) to form a U:A pairing with lower thermal stability, and introduce GMP or CMP at the 5' end of the follower strand (sense strand) to form a G:C pairing with higher thermal stability (see Figure 3This siRNA design configuration, combined with factors such as the A / U ratio of the 5′-end seed region of the guide strand and parameters such as the GC content ratio, effectively improves the success rate of designing highly active siRNAs (see Figure 3 ), all currently marketed siRNA drugs use this siRNA design configuration. However, the 5′-terminal nucleotides of the antisense strand of the siRNA are preferably A / U, while the 5′-terminal nucleotides of the sense strand are preferably G / C. This siRNA design scheme also limits the siRNA design space ("Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2", Filipp Frank et al., Nature, vol. 465, pp. 818-822, June 10, 2010). Summary of the Invention

[0005] To solve the above problems, the inventors have conducted long and in-depth research and found that modifying pseudouracil and N1-methyl pseudouracil at the 5' end of the sense strand of siRNA can significantly reduce the binding of the sense strand itself to the AGO2 protein (Strand selection), and the gene silencing ability of siRNA mediated by the antisense strand is also significantly improved. Modifying pseudouracil and N1-methyl pseudouracil at the 5' end can make the design of siRNA sense strand more effective. At the same time, in double-stranded RNA, pseudouracil and N1-methyl pseudouracil are paired with adenine to form a U:A pairing. Based on this, when designing siRNA, unlike the common G:C pairing, the 5' end of the sense strand can also choose U:A pairing. During chemical synthesis, this uracil is replaced with pseudouracil or N1-methyl pseudouracil to achieve pU:A or NpU:A pairing, thereby significantly reducing the necessity of selecting G or C at the 5' end of the sense strand, directly increasing the siRNA design space.

[0006] The present application provides a small interfering RNA, comprising an antisense strand and a sense strand, wherein the base at the 5′ end of the sense strand is a pseudouracil nucleotide or a derivative of a pseudouracil nucleotide.

[0007] The present application also provides the use of pseudouridine nucleotides or derivatives of pseudouridine nucleotides in the preparation of small interfering RNA.

[0008] The present application also provides a method for enhancing the gene silencing ability of small interfering RNA, wherein the 5′-end base of the sense strand of the small interfering RNA is replaced with a pseudouridine nucleotide or a derivative of a pseudouridine nucleotide.

[0009] The present application also provides a method for reducing the binding of the small interfering RNA sense chain to the AGO2 protein, wherein the 5'-end base of the small interfering RNA sense chain is replaced with a pseudouracil nucleotide or a derivative of a pseudouracil nucleotide.

[0010] The present application also provides a biomaterial selected from any of the following:

[0011] a. a polynucleotide encoding the above-mentioned small interfering RNA;

[0012] b. a nucleic acid construct comprising the polynucleotide described in claim 1;

[0013] c. a lentiviral system containing the nucleic acid construct described in b;

[0014] d. A cell comprising the nucleic acid construct described in b or the polynucleotide described in a integrated into its genome.

[0015] The present application also provides uses of the aforementioned small interfering RNA and biological materials, selected from one or more of the following:

[0016] Use for preparing gene silencing products;

[0017] and / or, the use of a product for preventing and / or treating any one or more of cardiovascular disease, hypercholesterolemia, mixed dyslipidemia, hyperlipidemia, fatty liver, hepatitis B, hypertension, immune disease, tumor disease, inflammatory disease, metabolic disease, diabetes, central nervous system or ophthalmology-related disease;

[0018] and / or, use for preparing a product that improves gene silencing ability;

[0019] And / or, use for preparing a product for inhibiting target gene expression.

[0020] The beneficial effects brought about by this specification include but are not limited to: the present application provides an oligonucleotide with a pseudouracil nucleotide residue at the 5' end of the sense chain of siRNA. This design scheme can significantly reduce the binding of the sense chain itself to the AGO2 protein, and the gene silencing ability of the siRNA mediated by the antisense chain is also significantly improved. The 5' end modification of pseudouracil and N1-methyl pseudouracil can make the siRNA sense chain design more effective. At the same time, in double-stranded RNA, pseudouracil and N1-methyl pseudouracil are paired with adenine to form a U:A pairing. Based on this, when designing siRNA, unlike the common G:C pairing, the 5' end of the sense chain can also choose U:A pairing. During chemical synthesis, this uracil is replaced with pseudouracil or N1-methyl pseudouracil to achieve pU:A or NpU:A pairing, thereby significantly reducing the necessity of selecting G or C at the 5' end of the sense chain, directly increasing the siRNA design space. Furthermore, the oligonucleotide can be used to prepare drugs for preventing and / or treating cardiovascular diseases, hypercholesterolemia, mixed dyslipidemia, hyperlipidemia, fatty liver, hepatitis B, hypertension, immune diseases, tumor diseases, inflammatory-related diseases, metabolic diseases, diabetes, and central nervous system or ophthalmology-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present application will be further described in terms of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are non-limiting, and include:

[0022] Figure 1 This is the effect of pseudouracil modification at the 5′ end of the antisense strand on siRNA activity as shown in some examples of the present application.

[0023] Figure 2 This is the effect of pseudouracil modification at the 5′ end of the sense strand on siRNA activity as shown in some examples of the present application.

[0024] Figure 3 Schematic diagram of siRNA design according to some embodiments of the present application. DETAILED DESCRIPTION

[0025] To more clearly illustrate the technical solutions of the embodiments of this specification, the following briefly describes the drawings required for describing the embodiments. Obviously, the drawings described below are merely examples or embodiments of this specification. Those skilled in the art can apply this specification to other similar scenarios based on these drawings without inventive effort. Unless otherwise apparent from the context or otherwise noted, the same reference numerals in the figures represent the same structure or operation.

[0026] As used in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not refer to the singular but also include the plural. Generally speaking, the terms "comprises" and "include" only indicate the inclusion of the steps and elements specifically identified, and these steps and elements do not constitute an exclusive list. A method or apparatus may also include other steps or elements.

[0027] Flowcharts are used throughout this specification to illustrate the operations performed by systems according to embodiments of this specification. It should be understood that preceding or following operations do not necessarily need to be performed in exact order. Instead, the steps may be processed in reverse order or simultaneously. Furthermore, other operations may be added to these processes, or one or more operations may be removed from these processes.

[0028] The present application provides a small interfering RNA, comprising an antisense strand and a sense strand, wherein the base at the 5′ end of the sense strand is a pseudouracil nucleotide or a derivative of a pseudouracil nucleotide.

[0029] In some embodiments, the derivative of pseudouracil may be N1-methylpseudouridine nucleotide.

[0030] In some embodiments, the pseudouracil nucleotide or derivative of the pseudouracil nucleotide may be modified with 2′-OMe.

[0031] In some embodiments, the small interfering RNA may have 2′-OMe and / or 2-F modifications.

[0032] In some embodiments, the 5' end of the antisense strand may be a uracil nucleotide or an adenine nucleotide.

[0033] In some embodiments, the antisense strand of the small interfering RNA can completely match the target gene mRNA.

[0034] In some embodiments, the length of the small interfering RNA may be 19 to 23 nucleotides. In some embodiments, preferably, the length of the small interfering RNA may be 21 nucleotides.

[0035] In some embodiments, the nucleotide sequence of the sense strand of the small interfering RNA may be as shown in SEQ ID NOs. 1-9.

[0036] In some embodiments, the nucleotide sequence of the antisense strand of the small interfering RNA may be as shown in SEQ ID NOs. 10-18.

[0037] The present application also provides the use of pseudouridine nucleotides or derivatives of pseudouridine nucleotides in the preparation of small interfering RNA.

[0038] The present application also provides a method for enhancing the gene silencing ability of small interfering RNA, wherein the 5′-end base of the sense strand of the small interfering RNA is replaced with a pseudouridine nucleotide or a derivative of a pseudouridine nucleotide.

[0039] Gene silencing refers to the process of suppressing gene expression or downregulating gene expression in cells through specific mechanisms, thereby inhibiting its function. Gene silencing is an important biological phenomenon that is widely present in processes such as development, cell differentiation, gene regulation, and disease.

[0040] The present application also provides a method for reducing the binding of the small interfering RNA sense chain to the AGO2 protein, wherein the 5'-end base of the small interfering RNA sense chain is replaced with a pseudouracil nucleotide or a derivative of a pseudouracil nucleotide.

[0041] In some embodiments, the pseudouracil derivative may be an N1-methyl pseudouracil nucleotide;

[0042] In some embodiments, the pseudouracil nucleotide or derivative of the pseudouracil nucleotide may be modified with 2′-OMe;

[0043] In some embodiments, the small interfering RNA may have 2′-OMe and / or 2-F modifications;

[0044] In some embodiments, the 5′ end of the antisense strand of the small interfering RNA may be a uracil nucleotide or an adenine nucleotide;

[0045] In some embodiments, the antisense strand of the small interfering RNA can completely match the target gene mRNA.

[0046] The present application also provides a biomaterial selected from any of the following:

[0047] a. a polynucleotide encoding the above-mentioned small interfering RNA;

[0048] b. a nucleic acid construct comprising the polynucleotide described in claim 1;

[0049] c. a lentiviral system containing the nucleic acid construct described in b;

[0050] d. A cell comprising the nucleic acid construct described in b or the polynucleotide described in a integrated into its genome.

[0051] The present application also provides uses of the aforementioned small interfering RNA and biological materials, selected from one or more of the following:

[0052] Use for preparing gene silencing products;

[0053] and / or, the use of a product for preventing and / or treating any one or more of cardiovascular disease, hypercholesterolemia, mixed dyslipidemia, hyperlipidemia, fatty liver, hepatitis B, hypertension, immune disease, tumor disease, inflammatory disease, metabolic disease, diabetes, central nervous system or ophthalmology-related disease;

[0054] and / or, use for preparing a product that improves gene silencing ability;

[0055] And / or, use for preparing a product for inhibiting target gene expression.

[0056] The term "prevention and / or treatment" (and grammatical variations thereof) refers to an attempt to alter the natural course of a disease in a treated individual and can be a clinical intervention performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and eliminating or improving prognosis.

[0057] In some embodiments, the target gene may be ANGPTL3 or ASGR1.

[0058] In some embodiments, the cardiovascular disease is atherosclerotic cardiovascular disease.

[0059] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0060] Example 1: AGO2-MID domain affinity data summary

[0061] According to the disclosed test method for the affinity of the AGO2 MID domain, the method is performed by methods familiar to those skilled in the art or according to the methods described in detail in the prior art (such as RNA (2021) 27:163–173).

[0062] Table 1: AGO2 MID domain affinity

[0063] Compound number Content in the body Kd(μM) UMP high 315.5 AMP high 652.1 GMP high 7243.7 CMP high 2633.3 pUMP Low 2977 NpUMP Low 7849.3 IMP Low 3433.8

[0064] Conclusion: The affinity detection test results (Table 1) show that the Kd values ​​of pseudouridine monophosphate (pUMP) and inosine monophosphate (IMP) are similar to cytosine monophosphate (CMP); the Kd of N1-methylpseudouridine monophosphate (NpUMP) is close to that of guanine monophosphate (GMP); the larger the Kd value, the lower the affinity with the AGO2-MID domain.

[0065] Example 2: In vitro experiments

[0066] Test substance: modified siRNA sequence shown in Table 2

[0067] In vitro experimental process:

[0068] (1) Cell culture and transfection

[0069] HepG2 cells (Cell Bank, Chinese Academy of Sciences) were cultured in high-glucose DMEM (10% fetal bovine serum, penicillin-streptomycin) in a 37°C, 5% CO2 incubator. After trypsinization, the cells were harvested and counted. The siRNA sequence transfection protocol was as follows: 0.2 μl Lipofectamine RNAiMax (ThermoFisher) was added to 14.8 μl Opti-MEM, followed by 5 μl of siRNA sequence solution. After mixing, the cells were added to a 24-well plate and incubated at room temperature for 15 minutes. The cells were then plated at 2x10 5 The cell suspension was added to a 24-well plate at a concentration of 100 μl / well. Culture was continued for 48 hours. Unless otherwise stated, the final siRNA concentrations were 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, and 3 nM.

[0070] (2) RNA extraction and reverse transcription

[0071] Total RNA was isolated using Trizol total RNA isolation reagent (Thermofisher). Trizol solution was added to a 24-well plate at a volume of 250 μl per well. After shaking at 1000 rpm for 9 minutes, the cell lysate was transferred to a 1.5 ml centrifuge tube. For every 1 ml of Trizol cell lysate, 0.2 ml of chloroform was added, shaken vigorously for 1 minute, allowed to stand at room temperature for 2 minutes, and then centrifuged at 17,000 rpm for 15 minutes. The supernatant obtained by centrifugation was transferred to a new 1.5 ml centrifuge tube, an equal volume of isopropanol was added, inverted to mix, and allowed to stand at room temperature for 20 minutes. The entire volume of the above mixture was added to the top of the RNA adsorption column, centrifuged at 17,000 rpm for 2 minutes, and the filtrate was discarded. 700 μl of wash buffer was added to the top of the RNA adsorption column, centrifuged at 17,000 rpm for 2 minutes, and the filtrate was discarded. The wash operation was repeated once. Finally, 50 μl of elution buffer was added to the top of the RNA adsorption column, and the column was centrifuged at 17,000 rpm for 2 minutes. The filtrate was collected, quantified for nucleic acid, and stored in a -80°C refrigerator.

[0072] cDNA synthesis was performed using a Takara cDNA reverse transcription kit (Takara). To 10 μl of total RNA solution, 10 μl of reverse transcription mix (2 μl of 10x buffer, 0.8 μl of 25x dNTPs, 2 μl of random primers, 1 μl of reverse transcriptase, 1 μl of RNase inhibitor, and 3.2 μl of water) was added. cDNA reverse transcription was performed using an Eppendorf PCR instrument: 16°C for 10 minutes, 42°C for 30 minutes, 85°C for 2 minutes, and then held at 10°C.

[0073] (3) Implementation of Fluorescence Quantitative PCR

[0074] 5ul of cDNA was added to 15ul of qPCR mix (Fuji Biotechnology, catalog number DRT-01021-C2), each well containing 0.5ul of GAPDH TaqMan probe (Shanghai Biotechnology) and 0.5ul of ASGR1 or ANGPTL3 TaqMan probe (Shanghai Biotechnology). Real-time PCR was performed using the ΔΔCt (RQ) assay in an ABI7500 Fast Real-Time PCR System (ThermoFisher). Each siRNA sequence was tested for 2-3 independent transfections, and qPCR analysis was performed in duplicate for each transfection. (4) AGO2RISC-loading analysis

[0075] 5x10 5 HepG2 cells were seeded at 1:100 / well in a 6-well plate. 24 hours later, lipofectamine was used to transfect the siRNAs listed in Table 2 at a final concentration of 10 nM, along with 10 nM internal control RNA (mA12, sense strand 5-mC*mC*mUmCfAmUfUfUfAmUmAmUmCmCmUmUmA*mA*mU-3, SEQ ID NO. 19; antisense strand 5-mA*fU*mUmAmAfGmGfAfUmAmUmAmAfAmUfGmAmGmG*mG*mG-3, SEQ ID NO. 20). AGO2 immunoprecipitation was then performed according to the following method, and the binding ability of the modified siRNAs to the AGO2 protein, i.e., RISC-loading ability, was analyzed. Immunoprecipitation was performed using 500 μg of cell lysate, 3.4 μg of AGO2 antibody (Clone 11A9, Sigma-Aldrich) pre-bound to 25 μL Dynabeads Protein G (10004D, Thermo Fisher) for precipitation, and 200 μg of glycogen (R0551, Thermo Fisher), a carrier for small RNA fragments. AGO2-bound RNA was purified using the Directzol RNA purification kit (R2051, Zymo Research) according to the manufacturer's instructions.

[0076] The RNA samples were heated at 95°C in a PCR instrument for 10 minutes and immediately reverse-transcribed. Reverse transcription was performed using the TaqMan Micro RNA Reverse Transcription Kit (Thermofisher, catalog #4366596). Reverse transcription wells were set up on a separate PCR instrument, and 10 μL of RT reaction mix was added to each well, maintained at 4°C. Subsequently, 5 μL of the RNA sample, previously heated to 95°C, was added directly to the 4°C RT reaction mix, and the reverse transcription process was initiated (16°C for 30 minutes, 42°C for 30 minutes, and 85°C for 5 minutes). Finally, 2 μL of the reverse-transcribed product (cDNA) was added to the PCR reaction mix (0.2 μM TaqMan probe, 1.5 μM forward primer, 0.7 μM reverse primer, TaqMan 2x Universal PCR Master Mix; ABI catalog #4366596). PCR reactions were performed using an ABI7500 Fast PCR instrument (ABI, Thermofisher).

[0077] Example 3: Effect of pseudouracil modification at the 5-terminus of the antisense strand on siRNA activity

[0078] The siRNAs aB10-U, aB10-pU, and aB10-NpU listed in Table 2 were analyzed for their RISC-loading ability and their ability to inhibit the target gene human ANGPTL3 in HepG2 cells using the in vitro assay described above. The experiment found that the modification of pseudouracil (pU, mpU) and N1-methyl pseudouracil (NpU, mNpU) in the antisense strand of the siRNA gene silencing effect significantly inhibited the binding of the siRNA antisense strand to the AGO2 protein, indicating that the RISC loading ability was significantly reduced (see Appendix). Figure 1 A); Further gene silencing ability tests also found that the ability of the siRNA antisense strand carrying the above modification to inhibit the target gene was also significantly reduced (Appendix Figure 1 B, Table 2).

[0079] Table 2 Effects of pseudouracil modification at the 5-terminal end of the positive chain on siRNA gene silencing activity

[0080]

[0081] Wherein, mC, mG, mA, and mU represent monomeric nucleotides modified with 2′-O-methyl (2′-OMe); fC, fG, fA, and fU represent monomeric nucleotides modified with 2′-deoxy-2′-fluoro (2′-F); mpU represents a monomeric pseudouracil nucleotide modified with 2′-OMe; and mNpU represents a monomeric N1-methyl pseudouracil nucleotide modified with 2′-OMe. * represents a phosphorothioate bond.

[0082] After the siRNA conjugate is prepared, it is lyophilized to a solid powder and stored for later use. When used, it can be redissolved in a solution of desired concentration using, for example, water for injection, physiological saline, phosphate buffer or phosphate buffer.

[0083] In this study, pseudouracil and N1-methyl pseudouracil were modified at the 5' end of the sense strand of siRNA hA36 targeting the human ASGR1 gene and siRNA aB628 targeting the human ANGPTL3 gene, respectively. The siRNAs hA36-U, hA36-pU, and hA36-NpU, as well as aB628-U, aB628-pU, and aB628-NpU (Table 2), were analyzed for their target gene silencing ability (IC50) using the aforementioned in vitro assay. The experiments revealed that pseudouracil modification at the 5' end of the sense strand slightly enhanced the ability of the siRNA antisense strand to inhibit target gene mRNA expression (Table 2).

[0084] Example 4: Effect of pseudouracil modification at the 5-terminus of the sense strand on the RISC-loading ability of the siRNA sense and antisense strands

[0085] The siRNAs hA36-U, hA36-pU, and hA36-NpU, as well as aB628-U, aB628-pU, and aB628-NpU in Table 2, were analyzed for their RISC-loading abilities using the in vitro assay described above. The results showed that, compared to the sense strands of the 5-terminal mU-modified siRNAs (mU-S), the sense strands of the siRNAs modified with pseudouracil (mpU-S) and N1-methylpseudouracil (mNpU-S) exhibited significantly decreased binding abilities to AGO2, with only less than 20% of the binding ability of the mU-S sense strands (see Appendix). Figure 2 A and B).

[0086] In the antisense chains of the two siRNAs hA36 and aB628, compared with the siRNA antisense chain with mU modified at the 5th end of the sense chain (mU-AS, the 5th end of the antisense chain is mU), the siRNA antisense chain with pseudouracil modified at the 5th end of the sense chain (mpU-AS, the 5th end of the antisense chain is mU), and the siRNA antisense chain with N1-methylpseudouracil modified at the 5th end of the sense chain (mNpU-AS, the 5th end of the antisense chain is mU) have slightly enhanced affinity for AGO2 protein, which is consistent with the IC50 data in Table 2 above.

[0087] In summary, the modification of pseudouracil and N1-methyl pseudouracil at the 5' end of the sense strand of siRNA significantly reduced the selection of the sense strand itself by the AGO2 protein (Strand selection), and the gene silencing ability of siRNA mediated by the antisense strand was significantly improved. The modification of pseudouracil and N1-methyl pseudouracil at the 5' end can make the design of siRNA sense strand more effective and reduce the off-target effect of the 5' end mU modified sense strand. At the same time, in double-stranded RNA, pseudouracil and N1-methyl pseudouracil both pair with adenine to form a U:A-like pairing.

[0088] Based on this, when designing siRNA, unlike the common G:C pairing, the 5-end of the sense chain can choose U:A pairing. During chemical synthesis, this uracil is replaced with pseudouracil and N1-methylpseudouracil to achieve pU:A or NpU:A pairing, avoiding the need to select guanine or cytosine at the 5-end of the sense chain, directly increasing the siRNA design space.

[0089] While the basic concepts have been described above, it will be apparent to those skilled in the art that the detailed disclosure is merely illustrative and does not limit this specification. Although not explicitly stated herein, various modifications, improvements, and revisions to this specification may be made by those skilled in the art. Such modifications, improvements, and revisions are suggested in this specification and remain within the spirit and scope of the exemplary embodiments of this specification.

[0090] This specification also uses specific terms to describe the embodiments of this specification. For example, "one embodiment," "an embodiment," and / or "some embodiments" refer to a feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "one embodiment," "an embodiment," or "an alternative embodiment" two or more times in different locations in this specification do not necessarily refer to the same embodiment. Furthermore, certain features, structures, or characteristics of one or more embodiments of this specification may be appropriately combined.

[0091] In some embodiments, numbers are used to describe the quantity of components and attributes. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise stated, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the description and claims are approximate values, which may change according to the required characteristics of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the settings of such numerical values ​​are as accurate as possible within the feasible range.

[0092] Finally, it should be understood that the embodiments described in this specification are intended only to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, by way of example and not limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly described and illustrated in this specification.

Claims

1. A small interfering RNA comprising an antisense strand and a sense strand, wherein the base at the 5' end of the sense strand is a pseudouridine nucleotide or a derivative of a pseudouridine nucleotide.

2. The small interfering RNA according to claim 1, wherein The pseudouracil derivative is N1-methyl pseudouracil nucleotide; and / or, the pseudouracil nucleotide or the derivative of the pseudouracil nucleotide is modified with 2′-OMe; And / or, the small interfering RNA carries 2′-OMe and / or 2-F modifications.

3. The small interfering RNA according to claim 1, wherein The 5′ end of the antisense strand is a uracil nucleotide or an adenine nucleotide; And / or, the antisense strand of the small interfering RNA completely matches the target gene mRNA.

4. Use of pseudouridine nucleotides or derivatives of pseudouridine nucleotides in the preparation of small interfering RNA.

5. A method for enhancing the gene silencing ability of small interfering RNA, characterized in that: The 5'-end base of the positive chain of the small interfering RNA is replaced with a pseudouridine nucleotide or a derivative of a pseudouridine nucleotide.

6. A method for reducing the binding of the sense strand of small interfering RNA to AGO2 protein, characterized in that: The 5'-end base of the positive chain of the small interfering RNA is replaced with a pseudouridine nucleotide or a derivative of a pseudouridine nucleotide.

7. The use according to claim 4, or the method according to claim 5 or 6, characterized in that The pseudouracil derivative is N1-methyl pseudouracil nucleotide; and / or, the pseudouracil nucleotide or the derivative of the pseudouracil nucleotide is modified with 2′-OMe; and / or, the small interfering RNA carries 2′-OMe and / or 2-F modifications; and / or, the 5′ end of the antisense strand of the small interfering RNA is a uracil nucleotide or an adenine nucleotide; And / or, the antisense strand of the small interfering RNA completely matches the target gene mRNA.

8. A biomaterial selected from any of the following: a. A polynucleotide encoding the small interfering RNA according to any one of claims 1 to 3; b. a nucleic acid construct comprising the polynucleotide described in claim 1; c. a lentiviral system containing the nucleic acid construct described in b; d. A cell comprising the nucleic acid construct described in b or the polynucleotide described in a integrated into its genome.

9. Use of the small interfering RNA according to any one of claims 1 to 3 or the biomaterial according to claim 8, selected from one or more of the following: Use for preparing gene silencing products; and / or, the use of a product for preventing and / or treating any one or more of cardiovascular disease, hypercholesterolemia, mixed dyslipidemia, hyperlipidemia, fatty liver, hepatitis B, hypertension, immune disease, tumor disease, inflammatory disease, metabolic disease, diabetes, central nervous system or ophthalmology-related disease; and / or, use for preparing a product that improves gene silencing ability; And / or, use for preparing a product for inhibiting target gene expression.

10. The use according to claim 9, characterized in that The cardiovascular disease is atherosclerotic cardiovascular disease.

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