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Kit and method for modifying vitro synthesized RNA

An in vitro synthesis and kit technology, applied in the field of nucleic acid research, to achieve mild conditions, high yield and good modification effect

Active Publication Date: 2009-10-07
GUANGZHOU RIBOBIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The biggest problem facing RNA modification by liquid-phase molecular ligation is the choice of ligation reaction

Method used

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  • Kit and method for modifying vitro synthesized RNA
  • Kit and method for modifying vitro synthesized RNA
  • Kit and method for modifying vitro synthesized RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Construction of a kit for modifying RNA synthesized in vitro

[0059] (1) Synthesis of phosphoramidite monomers carrying 1,3-dipolar groups and dipolar-philic groups

[0060] The present invention has synthesized a series of phosphoramidite monomers carrying 1,3-dipolar groups and dipolar-philic groups, including alkynyl glycerol phosphoramidites, alkynyl triethylene glycol phosphoramidites and Figure (3)-( 6) The phosphoramidite monomer corresponding to the nucleoside. These monomers are synthesized by reacting corresponding substrates with 2-cyanoethyl N, N-diisopropyl phosphoramidite chloride, so only alkynyl glycerol phosphoramidite and alkynyl triethylene glycol phosphoramidite , 5-ethynyl uridine phosphoramidite synthesis as an example.

[0061] Synthesis of Alkyglycerol Phosphoramidites (2)

[0062]

[0063] 0.5g (2.66mmol) of alkynyl triethylene glycol and 0.75ml (5.31mmol) of diisopropylamine were dissolved in 2ml of anhydrous dichloromethane, ...

Embodiment 2

[0111] Example 2: RNA is modified during different stages of synthesis

[0112] Use one or more of the following methods to obtain functional molecularly modified RNA.

[0113] "Click" reaction during abasic protection in RNA synthesis:

[0114] RNA carrying a dipolar group: In chemical solid-phase synthesis, alkynyl phosphoramidites (such as alkynyl glycerol phosphoramidites, alkynyl triethylene glycol phosphoramidites) are used as substrates to modify and synthesize RNA to introduce alkynyl groups;

[0115] Biomolecular probes carrying 1,3-dipolar groups: 1,1,1',1'-tetramethyl-3-ethyl-3'-azidopropyl-benzindole trimethine cyanine;

[0116] Catalyst: Cu(CH 3 EN) 4 PF 6 ;

[0117] Solvent: ethanol ammonia solution;

[0118] 100nmol of unprotected dipolar group-carrying RNA was dissolved in ethanol ammonia solution, 200nmol of 1,3-dipolar group-carrying biomolecular probe was added, and 100nmol of catalyst was heated overnight. After the ammonia water was spin-dried, it ...

Embodiment 3

[0152] Example 3: Cholesterol linked to RNA

[0153] RNA carrying a dipolar group: In chemical solid-phase synthesis, the RNA is modified with an alkynyl phosphoramidite to introduce an alkynyl group;

[0154] Biomolecular probes carrying 1,3-dipolar groups: azidocholesterol;

[0155] Catalyst: C 54 h 45 BrP 4 Cu;

[0156] Solvent: tetrahydrofuran.

[0157] 100nmol of undeprotected 2'-position RNA carrying dipolar group was dissolved in 100μl tetrahydrofuran, and 100nmol C was added 54 h 45 BrP 4 Cu, 500nmol azidocholesterol, and 1000nmol tetrabutylammonium fluoride were reacted for 4-12 hours, and the reaction solution was evaporated to dryness, followed by standard RNA post-processing methods to finally obtain 76nmol RNA linked to cholesterol molecules.

[0158] The connection method of other long-chain alkanes and non-polar active molecules (such as: liposome molecules, hydrophobic drugs, etc.) is the same as that of cholesterol.

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Abstract

The present invention discloses a kit and method for modifying vitro synthesized RNA, particular: (1) in chemical synthesis of RNA, using phosphoramidite monomer with 1, 3-di-dipole group or pro-dipole group to synthesis ribonucleic acid; (2) the after or during the synthetic process, modified ribonucleic acid and corresponding pio-dipole group or biomolecular probe of the 1, 3-di-dipole group performing 'click' reaction at the present of cuprous ion compounds catalyst, the ribonucleic acid is modified. The present invention provided kit and method for modifying RNA avoid shortcomings of traditional molecule connecting method active molecule decomposition and inactivation in RNA synthesis or doff protecting group process. The invention is provided with advantages of low cost, simple operation, mild condition, good modify impression and will not affect biological activity of any RNA.

Description

technical field [0001] The invention relates to a kit and method for modifying in vitro synthetic ribonucleic acid (RNA), belonging to the technical field of nucleic acid research. technical background [0002] In recent years, with the in-depth understanding of the structure and function of ribonucleic acid (RNA), research in the field of RNA has developed rapidly and changed with each passing day. RNA interference (RNAi), siRNA, microRNA, and piRNA have been discovered one after another. In-depth study of the structure of these RNAs and the analysis of RNA functions have put forward higher requirements for RNA synthesis and modification. [0003] Natural unmodified RNAs limit RNA research due to their enzymatic instability, non-targeted transport, and low cell penetration. Chemical modification of RNA can effectively improve the stability, targeting and cell penetration of RNA, and has become one of the most important methods in the field of RNA research. [0004] Molec...

Claims

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Application Information

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IPC IPC(8): C07H21/02C07J9/00C07K1/107
CPCY02P20/55
Inventor 张必良王玮渠德忠
Owner GUANGZHOU RIBOBIO
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