Double-stranded asymmetric small nucleic-acid-interference-molecule asiRNA inhibiting tumour apoptosis suppressor specifically and application thereof

A technology of small nucleic acid interference and apoptosis inhibition, which is applied in the field of double-stranded asymmetric small nucleic acid interference molecule asiRNA and its application, to achieve the effect of strong activity and less toxic side effects

Active Publication Date: 2012-10-10
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Generally speaking, there are still some technical bottlenecks in the stability and drug delivery syst

Method used

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  • Double-stranded asymmetric small nucleic-acid-interference-molecule asiRNA inhibiting tumour apoptosis suppressor specifically and application thereof
  • Double-stranded asymmetric small nucleic-acid-interference-molecule asiRNA inhibiting tumour apoptosis suppressor specifically and application thereof
  • Double-stranded asymmetric small nucleic-acid-interference-molecule asiRNA inhibiting tumour apoptosis suppressor specifically and application thereof

Examples

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Embodiment 1

[0066] In the present invention, a group of siRNA targeting tumor apoptosis suppressor gene Bcl2 mRNA is searched for in a gene bank (the sequence is: sense strand 5'CGGAGGCUGGGAUGCCUUU 3'(SEQ ID NO.1), antisense strand 3'GCCUCCGACCCUACGGAAA 5'(SEQ ID NO. .2)) based on the 5' or 3' end of the siRNA sense strand or antisense strand cut 1, or 2, or 3, or 4, or 5 bases to form two bases The length of the base is inconsistent, and there are two deoxyribonucleic acid dTdT at the 3' end of each strand, which is a single-strand suspension, and has a 14-21nt double-stranded asymmetric small nucleic acid interference molecule asiRNA that can induce RNA interference. Or on this basis and using cholesterol, or / and thio, or / and methylation, or / and phosphorylation, or / and fluorinated bases to chemically modify the bases of the sense strand, or / and the antisense strand, Phosphate backbone, ribose, form two strands with different base lengths, each strand has two deoxyribonucleic acid dTdT h...

Embodiment 2

[0072] The present invention uses LipofectAMINE TM 2000 (Lipo 2000) liposomes (Life Techonolobies, product number: 11668-019, trademark Inivitrogen) were used to transfect the Bcl2-asiRNA fragments in parts B-F of Table 1, and the silencing effect was determined 72 hours after transfection. All operations are carried out according to the instructions. Synthetic conventional 21 / 21siRNA (sequence is sense strand 5'CGGAGGCUGGGAUGCCUUUdTdT 3'antisense strand 3'dTdTGCCUCCGACCCUACGGAAA 5', that is, at the 3'end of both strands of SEQ ID NO.1 and SEQ ID NO.2, add dTdT is a single-strand suspension, consistent with the 3' end of asiRNA) as a positive control, siRNA and asiRNA powders were dissolved in DEPC water to prepare a 20 nM stock solution. The day before the transfection operation, inoculate the human cervical cancer cell HeLaB2 cells with high expression of Bcl2 (purchased from the Institute of Tumor Research, Chinese Academy of Medical Sciences, China Union Medical College) ...

Embodiment 3

[0078] The transfection operation was carried out according to the experimental method of Example 2, and the synthetic 21 / 21 siRNA was used as a positive control (sequence is the same as that of Example 2), and the asiRNA fragments of B-G described in Table 1 were transfected, and the concentration was also 33nM, and HeLaB2 was transfected After 72 hours, the original culture medium was discarded, washed once with PBS, and the cells were digested with trypsin, transferred to an Eppendoff tube, centrifuged at 1200rpm for 5 minutes, and the supernatant was discarded. Add 1ml of PBS, mix well, centrifuge at 1200rpm for 5 minutes, and discard the supernatant. Count the cells and adjust the cell concentration so that the number of cells per sample is 10 5 indivual. Add 500 μl of 4% paraformaldehyde to each sample, mix well, and place at room temperature for 20 minutes to fix the cells. Centrifuge at 1200rpm for 5 minutes, discard paraformaldehyde. Add 1ml PBS to wash once. Add ...

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Abstract

The invention belongs to the biomedical field, discloses a double-stranded asymmetric small nucleic-acid-interference-molecule asiRNA inhibiting a tumour apoptosis suppressor specifically and an application thereof. The asiRNA is designed by a following method and obtained by chemical synthesis, starting from a Bcl 2 gene symmetry small nucleic-acid-interference-molecule siRNA, by cutting 1-5 nt base off a 5 ' end or a 3 ' end of an siRNA sense strand or an siRNA antisense strand, and forming the double-stranded asymmetric small nucleic-acid-interference-molecule asiRNA wherein base lengths are different and the 3' end of each strand is equipped with two deoxyribonucleic acids dTdT in a single strand suspension and RNA interference can be induced, by using two nucleotides with abutting deoxyribonucleic acid dTdT on the 3' end of each strand. The asiRNA of the invention is more stable than the routine siRNA, activity to inhibit expression of Bcl 2 is stronger, and can be applied in antitumor drug preparation.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a double-stranded asymmetric small nucleic acid interference molecule asiRNA that specifically inhibits tumor apoptosis inhibitory gene Bcl2 and an application thereof. Background technique [0002] Cancer is the second biggest killer of human health. At present, drug resistance often occurs during the clinical use of chemotherapy drugs. Cancer cells are often not killed, and tumors often recur and metastasize, leading to death. One of the most important reasons is that the apoptosis suppressor gene (Bcl2 gene) in these cancer cells is highly expressed, which makes the tumor cells resist apoptosis and not die, so that tumor recurrence and metastasis often occur. Therefore, inhibiting the expression of tumor Bcl2 gene is one of the important ways to improve the efficacy of chemotherapy drugs, completely kill cancer cells and prevent tumor recurrence and metastasis. Abroad, an innovative ...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K48/00A61P35/00
Inventor 徐根兴殷妍陈旭王庆晓郭佳佳张昌栋傅更锋樊燕蓉吴稚伟侯亚义王建军
Owner NANJING UNIV
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