A locked nucleic acid deoxyribozyme modified by rela cleavage and tlr7 activation motif for targeted therapy of tuberculosis and its application

A targeted therapy and deoxyribozyme technology, applied in gene therapy, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as single function, susceptibility to DNase degradation, and insufficient half-life of 10-23 DNAzyme, etc., to achieve strong Effects of specificity, good stability, good biosafety and development prospects

Active Publication Date: 2014-10-01
WEIFANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] Although compared with RNA drugs such as antisense RNA and ribozyme, 10-23DNAzyme has better stability, but the chemically synthesized 10-23DNAzyme still faces problems such as not long enough half-life and being easily degraded by DNase; and 10-23DNAzyme When targeting drug-resistant Mycobacterium tuberculosis, especially L-type Mycobacterium tuberculosis, there is a limitation of single function, so new breakthroughs are needed in this area

Method used

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  • A locked nucleic acid deoxyribozyme modified by rela cleavage and tlr7 activation motif for targeted therapy of tuberculosis and its application
  • A locked nucleic acid deoxyribozyme modified by rela cleavage and tlr7 activation motif for targeted therapy of tuberculosis and its application
  • A locked nucleic acid deoxyribozyme modified by rela cleavage and tlr7 activation motif for targeted therapy of tuberculosis and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: the design, synthesis of 3 locked nucleic acid deoxyribozymes of the present invention

[0054] 1. Using locked nucleic acid as the synthetic raw material for the alternative part of DNAzyme, using the 10-23 DNAzyme structure as the basic framework, using computer software to simulate the secondary structure of RelA-mRNA ( figure 1 ), select a suitable target to be cut according to the simulated structure diagram and design a specific locked nucleic acid deoxyribozyme for the corresponding target. Perform performance analysis, screening and synthesis, hybridization and modification of RNA components composed of specific motifs. A hybrid modification was performed with the "U"-rich RNA sequence of the TLR7-activating RNA motif at the end of the 10-23 deoxyribozyme-specific recognition binding arm.

[0055] 2. According to the cleavage pattern of 10-23 DNAzyme, design a series of corresponding 10-23 locked nucleic acid DNAzyme sequences with different subst...

Embodiment 2

[0060] Example 2: Obtaining Mycobacterium tuberculosis RelA full-length mRNA with an in vitro transcription system

[0061] Mycobacterium tuberculosis H was extracted according to the instructions of the bacterial genome DNA extraction kit (Dalian TaKaRa Company) 37 Genomic DNA of Rv.

[0062] The upstream and downstream primers were designed according to the RelA mRNA coding sequence registered in GenBank, and synthesized by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd.

[0063] Upstream: 5'-GGGATCCGATATCATGGCCGAGGACCAGC-3'

[0064] Downstream: 5'-GCGGCCGC AAGCTTCTACGCGGCCGAGGT-3'

[0065] Mycobacterium tuberculosis H 37 Genomic DNA of Rv was used as a template to carry out PCR reaction to obtain RelA gene.

[0066] After the PCR product was recovered, it was double-digested with endonuclease EcoRV / Hind III (purchased from Dalian TaKaRa Company, Shenzhen Jingmei Company, British NEB Company, and American Promega Company) and cloned into the vector pET-32a(+) (purchased f...

Embodiment 3

[0069] Embodiment 3: Observation of the cutting effect of each locked nucleic acid deoxyribozyme on Rel A mRNA extracellularly

[0070] Add the corresponding reagents (μL) to the 7 reaction tubes according to the table below:

[0071]

[0072] Each reaction tube was incubated at 37°C for 60 min, then taken out, and separated by electrophoresis on a 3.5% denaturing polyacrylamide gel. Then use the nucleic acid silver staining kit (purchased from Beijing Dingguo Company, and can also be purchased from Beijing Zhongshan Company and Shanghai Shenggong Company) for staining, and then use the gel imaging system to collect images, measure the optical density of each band, and calculate cut percentage. Cleavage percentage (%) = total optical density value of cleaved product bands / (optical density value of uncleaved substrate bands + total optical density value of cleaved product bands) × 100%.

[0073] Results: The cleavage systems of LDZ4 and LDZ5 had obvious specific cleavage p...

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Abstract

The invention discloses RelA cut and TLR7 active sequence modified locked nucleic acid deoxyribozyme for targeted therapy of tuberculosis. A nucleotide sequence is one of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3. The invention simultaneously discloses use of the RelA cut and TLR7 active sequence modified locked nucleic acid deoxyribozyme for targeted therapy of tuberculosis in a drug for treating latent infection caused by L-type mycobacterium tuberculosis. By adopting the RelA cut and TLR7 active sequence modified locked nucleic acid deoxyribozyme, the advantages of effects of gene therapy and immunological therapy are integrated; and the therapeutical effect is played specifically aiming at drug-resistant tuberculosis of infection in the body, especially the L-type mycobacterium tuberculosis.

Description

technical field [0001] The present invention relates to a locked nucleic acid deoxyribozyme, in particular to a locked nucleic acid deoxyribozyme capable of targeting treatment of tuberculosis by cutting RelA and modifying a TLR7 activation motif, and at the same time relating to the use of the locked nucleic acid deoxyribozyme in targeted treatment of normal and drug-resistant drugs Use in the field of infection caused by Mycobacterium tuberculosis, especially latent and persistent infection caused by L-type drug-resistant Mycobacterium tuberculosis. Background technique [0002] Tuberculosis ranks first among serious infectious diseases that cause death due to a single pathogen. More than 2 million people die from tuberculosis every year, and nearly 8.7 million new cases occur. The incidence of tuberculosis has continued to increase over the past decade, but no specific anti-TB drugs have been developed in the last 40 years. It is particularly worth pointing out that at l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K48/00A61P31/06
Inventor 付玉荣伊正君伊星昊伊鑫
Owner WEIFANG MEDICAL UNIV
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