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New production process for large-scale synthesis of long-chain RNA drugs

A large-scale, new process technology, applied in the field of long-chain RNA drugs, can solve the problem of no solution for DNA template protein residues, unsolved separation of long-chain RNA complete transcripts, RNA transcripts, and RNA products that cannot meet the needs of RNA drug development, etc. problems, to achieve a reliable and stable synthesis method, reduced host DNA contamination, and low cost

Inactive Publication Date: 2014-08-20
BIOMICS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Lukavsky et al. use linear plasmids or PCR products as templates to transcribe and synthesize RNA products on a large scale, and then use gel filtration chromatography, affinity chromatography, anion exchange chromatography and other methods to purify RNA to obtain better separation effects, but these methods are obviously not effective. Solve how to separate the complete transcript (N) of long-chain RNA and the RNA transcript (N+) with extra nucleotides, and there are no complete and systematic solutions for DNA templates, endotoxins, and protein residues in these production processes , the purified RNA product cannot meet the needs of RNA drug development (PJ Lukavsky et al. RNA (2007), 2004, 10(5): 889-93)
More importantly, these production processes do not involve the large-scale preparation of long-chain RNA transcription templates with special secondary structures. For example, in the purification process of AC Anderson, RNA aptamers with secondary structures are chemically Synthetic (AC Anderson et al. RNA. 1996, 2(2): 110-7)

Method used

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  • New production process for large-scale synthesis of long-chain RNA drugs
  • New production process for large-scale synthesis of long-chain RNA drugs
  • New production process for large-scale synthesis of long-chain RNA drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Transcription synthesis and purification of miRNA precursor

[0055] 1. Reagents, materials and instruments

[0056] 1. Reagent materials: Pfu DNA Polymerase (purchased from Biomics Biotechnology Co., Ltd.), T7 RNA polymerase (endotoxin content is less than 10EU / mg, purchased from Biomics Biotechnology Co., Ltd.), RNase H-free 2 O, 70% ethanol aqueous solution (RNase-free), rNTP (100mM) (purchased from Biomics Biotechnology Co., Ltd., unmodified), isopropanol, 10×Reaction Buffer (PCR & Transcription, purchased from Biometics Biotechnology Co., Ltd.), Buffer A (5mmol / L EDTA, 5mmol / L Tris-HCl, pH7.5), Buffer B (1M NaCl, 5mmol / L EDTA, 5mmol / L Tris-HCl, pH7.5). All chemical reagents were of analytical grade and were purchased from Shanghai Sangon Bioengineering Co., Ltd. unless otherwise specified.

[0057] 2. Instruments: High pressure liquid chromatography system (Shimadzu LC-20AT), HPLC separation column HR16-Source 15Q (General Medical GE); nucleic acid anal...

Embodiment 2

[0102] Example 2 Preparation of DNA transcription template by PCR amplification and transcription synthesis of miRNA precursor

[0103] 1. Experimental materials: the same as in Example 1.

[0104] 2. Experimental steps:

[0105] 1) Design a pair of primers (chemically synthesized by Biomaike Biotechnology Co., Ltd.)

[0106] hsa-mir-21-F: 5'-CACTAATACGACTCACTATAGG-3' (SEQ ID NO: 6);

[0107] hsa-mir-21-R: 5'-TGTCAGACGACCCATCGACT-3' (SEQ ID NO: 7).

[0108] 2) Template preparation and purification

[0109] Using the miRNA precursor (has-miR-21) DNA transcription template obtained in Example 1 as a PCR template, follow the conventional PCR program (95°C 3min; 95°C 30sec, 55~68°C 30sec, 72°C 1min, 25Cycles; 72°C 5min) for PCR amplification, reaction system: DNA template (2.5ng / μl) 1μl, hsa-miR-21-F (10μM) 2μl, hsa-miR-21-R (10μM) 2μl, 10×PCR Buffer 5μl, dNTPs (2.5mM) 4μl, Taq DNA polymerase (2.5U / μl) 0.5μl, and ultrapure water to make up to 50μlL.

[0110] Template purific...

Embodiment 3

[0114] Example 3 Synthesis and purification of one-strand multi-target siRNA

[0115] 1. Experimental material: with embodiment 1.

[0116] 2. Experimental steps:

[0117] 1) Sequence design: one-strand multi-target siRNA (SUB) sequence, DNA transcription template sequence, DNA primer sequence design such as Figure 10 shown. Design T-SUB and T-SUB XS DNA transcription templates with different SUB sense strand RNAs. The difference between the two is that the first two bases at the 5' end of the antisense strand of the transcription template are modified with 2'-OMe, and the unmodified DNA transcription template is named For T-SUB, the DNA transcription template modified with 2'-OMe at the 5' end is named T-SUB XS. DNA primers were chemically synthesized by Biomaike Biotechnology Co., Ltd.

[0118] 2) Preparation and purification of transcription template and in vitro transcription reaction: the proportions of the components in the reaction system were the same as those in ...

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Abstract

The invention provides a new production process for large-scale synthesis of long-chain RNA drugs. The process is characterized by specifically including the steps of: DNA transcription template design; template preparation and purification; in-vitro transcription; and product purification, purity detection and the like. The production process provided by the invention is especially suitable for synthesis of long-chain RNA drugs with a stable secondary structure. With the characteristics of simple operation and low cost, the process provided by the invention meets the requirements of large-scale production, and the produced long-chain RNA drugs can be used for cell experiments, animal experiments and other preclinical RNA drugs' functional study.

Description

technical field [0001] The invention relates to a new production process for large-scale transcription and synthesis of long-chain RNA drugs, especially for long-chain RNA drugs with stable secondary structures, using RNA polymerase to transcribe methoxy-modified DNA templates for large-scale synthesis of long-chain RNA drugs Molecules, HPLC purification of RNA drugs to remove endotoxin, protein, DNA and other residues. Background technique [0002] In the past few decades, with the rapid development of RNA technologies such as siRNA, non-coding RNA, RNA aptamers, and ribozymes, RNA has received more and more attention in biology (John C et al. Chemistry & Biology.2012, 19( 1): 60-71), some previous synthesis methods have gradually failed to meet the needs of scientific research experiments and drug development for RNA, and more and more RNA biological research and drug development need to use milligram-level or even gram-level high Purity of RNA, meanwhile, higher requirem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10A61K48/00
Inventor 张平静李铁军周宋峰朱远源陈建新陆毅祥文锋
Owner BIOMICS BIOTECH
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