CN116694638A - Circular nucleic acid application-miRNA inhibitors - Google Patents
Circular nucleic acid application-miRNA inhibitors Download PDFInfo
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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Abstract
The invention discloses a circular nucleic acid application-miRNA inhibitor, which relates to the technical field of nucleic acids, wherein miRNA is a single-stranded non-coding RNA with the length of about 20 bases, and the miRNA is used for regulating and controlling biological processes involved in various diseases by combining with mRNA after transcription. The existing miRNA inhibitor is mainly antisense oligonucleotide (ASO), and is a single-stranded DNA molecule reversely complementary to miRNA. However, single-chain forms of ASO are easily degraded in the blood, resulting in low inhibition and bioavailability. The invention provides a method for controlling diseases by inhibiting the expression of miRNA by using artificially synthesized fully closed single-stranded circular nucleic acid (DNA or RNA). Due to its unique circular structure, the fully closed single-stranded circular nucleic acid can resist the degradation of exonuclease in blood and continuously exert the inhibition effect on miRNA. The fully closed single-stranded circular nucleic acid is hopeful to be developed into a new generation of nucleic acid medicaments taking miRNA as a target point.
Description
Technical Field
The invention relates to the technical field of nucleic acid, in particular to a circular nucleic acid application-miRNA inhibitor.
Background
microRNA (miRNA) is a class of non-coding small RNA molecules that play a central role in cell differentiation, proliferation and survival by binding to target mRNA resulting in inhibition or degradation of mRNA translation. The initial concept of mirnas as developmental regulators has now greatly expanded, and mirnas are found to be deregulated in many diseases, including cancer, hepatitis and cardiovascular disease. Mirnas are often altered in disease due to genomic events such as mutations, deletion amplifications or transcriptional changes, or defective biogenesis caused by down-regulation of enzymes that regulate the biogenesis of mirnas. In the initial studies, the goal of regulating miRNA expression was achieved by targeting tissue sites or local injection of miRNA mimics or miRNA antisense oligonucleotides (ASOs). However, initial studies have been rarely successful clinically due to degradation in blood, non-ideal target site delivery efficiency of miRNA mimics of delivery systems, etc.
MicroRNA (miRNA) -based therapies can be divided into miRNA mimics and miRNA inhibitors. miRNA mimics are synthetic double-stranded small RNA molecules that match the corresponding miRNA sequences and thus are functionally intended to complement lost miRNA expression in disease. In contrast, miRNA inhibitors are single-stranded, based on first generation antisense oligonucleotides (ASOs), designed to target binding to mirnas. These synthetic small RNA molecules have complementary sequences to the miRNA to be inhibited and block their function by strongly binding to the corresponding miRNA. One of the challenges in RNA-based therapies is the possibility of nuclease degradation of oligonucleotides in serum or endocytosis. For many years, by chemically modifying the nucleotide backbone, miRNA mimics and miRNA inhibitors have significantly improved binding affinity, stability and targeted regulatory effects. To avoid intracellular degradation, increasing the stability of ASO, two different strategies were investigated. The first generation ASOs were modified by substitution of sulfur for non-bridging oxygens in the phosphate groups to produce phosphorothioate nucleotides. Such modifications increase the stability of the intracellular ASOs (by making the internucleotide linkages resistant to nuclease degradation) while retaining sufficient rnase H activity to effect mRNA target cleavage and inhibit target gene expression. Other modifications of the ASO that have been tested include the addition of methyl groups at different positions of the RNA backbone. The addition of 2' -O-methyl to phosphorothioate nucleotides results in increased binding affinity to target mRNA, significant nuclease resistance and higher in vivo stability. The 2' -O-methoxyethyl modification also increases nuclease resistance and binding affinity. Currently, some ASOs have entered into various stages of clinical trials, including commercial ASOs for the treatment of homozygous familial hypercholesterolemia.
Despite numerous preclinical studies involving miRNA therapy over the years, only a few have entered clinical development to date. One of the challenges in developing miRNA-based therapies is to determine the best targeted miRNA candidates for each disease type and how to avoid degradation of miRNA inhibitors. Therefore, the development of a stable degradation-resistant nucleic acid drug molecule targeting miRNA is imperative.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a circular nucleic acid application-miRNA inhibitor, and solves the problems in the background art.
In order to achieve the above purpose, the invention is realized by the following technical scheme: circular nucleic acids, the structure of which is fully closed.
Preferably, the circular nucleic acid may have a single-ring structure or a multi-ring structure.
Preferably, the nucleic acid sequence comprises a single stranded nucleic acid sequence complementary to a miRNA.
Preferably, the nucleic acid sequence comprises, in addition to the miRNA complement, other helper nucleic acid sequences as well.
Preferably, the nucleic acid sequence wherein the nucleic acid sequence complementary to the miRNA is fully complementary to the miRNA or partially complementary to the miRNA.
Preferably, the nucleic acid sequence wherein the nucleic acid sequence is complementary to a miRNA may comprise one miRNA binding sequence or may comprise multiple miRNA binding sequences.
Preferably, the nucleic acid sequence may bind one miRNA, but may also bind multiple mirnas.
Preferably, the helper nucleic acid sequence may comprise a complementary mating sequence.
Preferably, the auxiliary nucleic acid sequence may be single-stranded, double-stranded or nucleic acid higher-order structure.
Preferably, the nucleic acid sequence comprises both a DNA sequence and an RNA sequence.
Preferably, the nucleic acid sequence may be chemically modified.
Use of circular nucleic acids: the application of the nucleic acid with similar characteristics to the single-stranded circular nucleic acid in the development of tumor medicaments.
The invention provides a circular nucleic acid application-miRNA inhibitor, which has the following beneficial effects:
the circular nucleic acid is applied to a miRNA inhibitor, and the method is simple, convenient, efficient, low in cost, strong in operability and suitable for large-scale engineering production.
Drawings
FIG. 1 is a synthetic scheme of a fully closed single stranded nucleic acid of the present invention;
FIG. 2 is a gel electrophoresis identification chart before and after annealing and ligation of single-stranded nucleic acids according to the present invention;
FIG. 3 is a gel electrophoresis diagram of the joined single-stranded circular nucleic acids of the present invention after treatment with T5 exonuclease and serum to identify their resistance to degradation;
FIG. 4 is a schematic diagram of the difference between the adsorption efficiency of the miRNA by the PCR method and the adsorption efficiency of the single-stranded circular nucleic acid by the ASO by taking miR-21 as an example;
FIG. 5 is a schematic diagram showing the effect of single-stranded circular nucleic acid and ASO of the present invention on tumor cell function after inhibition of miR-21 by adsorption;
FIG. 6 is a PCR graph showing the inhibitory effect of single-stranded circular nucleic acids of the present invention on different miRNAs;
FIG. 7 is a schematic representation of cell invasion according to the present invention;
FIG. 8 is a schematic representation of the ability of the present invention to inhibit cell migration;
FIG. 9 is a graph showing the inhibitory effect of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments.
Example 1
Synthesis of closed circular Single-stranded nucleic acid (for example miR-21)
Step one: the primary nucleotide sequence required for synthesizing the circular nucleic acid is designed. Each nucleotide chain designed comprises the following two components: 1. the 5 'end and the 3' end need to comprise self-complementary pairing sequences of more than 5 nucleotides, 2, nucleic acid sequences complementary to miR-21. The design roadmap is shown in fig. 1.
Step two: the synthesized nucleic acid was placed in an annealing buffer, and the PCR instrument was programmed to drop 0.1℃every 8 seconds to 25℃at an initial temperature of 95 ℃. The product obtained after the reaction is preserved at 4 ℃.
Step three: taking equimolar products obtained in the step two, and incubating at 16 ℃ for 1-18h or incubating at 20-25 ℃ for 1-2h under the action of ligase.
Step four: non-circular nucleic acids were removed using T5 exonuclease. Mixing the connected product with an exonuclease buffer solution, uniformly mixing the reaction system, and adding T5 exonuclease. The reaction system was properly mixed and incubated at 37℃for 30min. Immediately after the completion of the reaction, the reaction was terminated by ice-bath and addition of EDTA to a final concentration of 11 mM.
Step five: agarose gel electrophoresis was used for DNA and RNA circularization. The results of gel electrophoresis are shown in FIG. 2.
Step six: purification of circular nucleic acids: after incubating the circular nucleic acid with the binding buffer, binding magnetic beads are added to the solution. After 10 minutes of reaction, the circular nucleic acid was adsorbed by a magnetic rack. After removing impurities by centrifugation, the purified circular nucleic acid is dissolved and stored at low temperature.
Step seven: the obtained circular nucleic acid was subjected to T5 exonuclease and serum treatment to confirm its degradation resistance. The results are shown in FIG. 3.
Example 2
Verification of anti-tumor Effect of closed circular Single-stranded nucleic acid (taking humanized miR-21 as an example)
The miR-21-inhibited closed circular nucleic acid (CCN-21) obtained in example 1 and ASO-21 (antisense oligonucleotide of miR-21) were transfected into HCT-116, A549 and PLC/PRF/5 cells, and after 48 hours, the expression of miR-21 was detected by PCR. The results show that compared with ASO-21, CCN-21 has stronger inhibition effect on miR-21. The results are shown in FIG. 4.
CNN-21 was transfected into PLC/PRF/5 cells. After 48 hours, the effect of MTT assay on cell proliferation was measured. As shown in fig. 5, CNN-21 can significantly reduce proliferation ability of cells after adsorbing miRNA 21.
CNN-21 was transfected into HCT-116, A549, PLC/PRF/5 cells. After 48 hours, the morphological changes of the cells are observed by using a scanning electron microscope, as shown in fig. 6, the morphological changes of the transfected Hela and A549 cells are obvious, and the cells die in a large quantity, so that the inhibition effect of CNN-21 on the cell growth is obvious.
CNN-21 was transfected into PLC/PRF/5 cells. After 24 hours, cells were digested with pancreatin and added to a plated Transwell chamber. After 16 hours, the number of cells passing through the chamber was examined, and the results are shown in FIG. 7, and it can be seen that CNN-21 can significantly inhibit the invasive ability of cells after specifically adsorbing miRNA-21.
CNN-21 was transfected into PLC/PRF/5 cells. After 24 hours, cells digested with pancreatin were seeded into 24-well plates. After the cell density reached 80%, uniform scratches were made. Scratch width was measured for 48 hours, and as shown in FIG. 8, CNN-21 specifically adsorbed miRNA-21, and then the migration ability of cells was inhibited.
Example 3
And verifying the adsorption effect of the closed single-stranded circular nucleic acid on various miRNA designs.
Reference example 1, closed single stranded circular nucleic acids (CCN-221, CCN-155, CCN-9, CCN-10a, respectively) were designed and prepared according to miRNA-221, miRNA-155, miRNA-9, miRNA-10 a. These 4 mirnas are only exemplified here, but are not limited to these 4 mirnas.
The closed circular nucleic acid was transfected into HCT-116 cells and 48 hours later the expression of miRNA-221, miRNA-155, miRNA-9, miRNA-10a was detected by PCR. The results are shown in FIG. 9, which shows that the inhibition effect of CCN-221, CCN-155, CCN-9, CCN-10a on miRNA is stronger than ASO.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (12)
1. A single-stranded circular nucleic acid, characterized in that: the structure is completely closed.
2. The single stranded circular nucleic acid of claim 1, wherein: the circular nucleic acid may have a single-ring structure or a multi-ring structure.
3. The single stranded circular nucleic acid of claim 1, wherein: the nucleic acid sequence comprises a single stranded nucleic acid sequence complementary to a miRNA.
4. A single stranded circular nucleic acid according to claim 2, wherein: the nucleic acid sequence may comprise other auxiliary nucleic acid sequences in addition to the miRNA complement.
5. A single stranded circular nucleic acid according to claim 3, wherein: the nucleic acid sequence complementary to the miRNA can be completely complementary to the miRNA or can be partially complementary to the miRNA.
6. A single stranded circular nucleic acid according to claim 3, wherein: the nucleic acid sequence wherein the nucleic acid sequence complementary to the miRNA may comprise one miRNA binding sequence or may comprise multiple miRNA binding sequences.
7. A single stranded circular nucleic acid according to claim 3, wherein: the nucleic acid sequence may bind to one miRNA, or may bind to multiple miRNAs.
8. The single stranded circular nucleic acid of claim 4, wherein: the helper nucleic acid sequence may comprise a complementary pairing sequence.
9. The single stranded circular nucleic acid of claim 4, wherein: the auxiliary nucleic acid sequence can be single-stranded, double-stranded or nucleic acid higher-order structure.
10. The single stranded circular nucleic acid of claim 1, wherein: the nucleic acid sequence comprises both a DNA sequence and an RNA sequence.
11. The single stranded circular nucleic acid of claim 1, wherein: the nucleic acid sequence may be chemically modified.
12. Use of a single stranded circular nucleic acid according to any one of claims 1-8: the method is characterized in that: the application of the nucleic acid with similar characteristics to the single-stranded circular nucleic acid in the development of tumor medicaments.
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| US20150299702A1 (en) * | 2012-11-30 | 2015-10-22 | Aarhus Universitet | Circular rna for inhibition of microrna |
| CN108251424A (en) * | 2017-12-19 | 2018-07-06 | 天利康（天津）科技有限公司 | A kind of single stranded circle RNA and DNA and its preparation method and application |
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| US20150299702A1 (en) * | 2012-11-30 | 2015-10-22 | Aarhus Universitet | Circular rna for inhibition of microrna |
| CN108251424A (en) * | 2017-12-19 | 2018-07-06 | 天利康（天津）科技有限公司 | A kind of single stranded circle RNA and DNA and its preparation method and application |
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