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Application of dideoxynucleoside phosphoramidite monomer containing succinimide structure in recognition of mismatched bases

A deoxynucleoside phosphoramidite and succinamide technology, applied in the field of non-natural oligonucleotide applications, can solve the problem of high cost

Active Publication Date: 2020-06-12
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The introduction of metal complexes and some ligands with specific structures shows good detection sensitivity for some specific base mismatches, but for some special types of mismatch combinations, such as T-C base specific mismatches, it is rare. There are reports
The recognition of some specific base mismatch combinations can be realized by modifying the nucleic acid backbone, but the cost is relatively high

Method used

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  • Application of dideoxynucleoside phosphoramidite monomer containing succinimide structure in recognition of mismatched bases
  • Application of dideoxynucleoside phosphoramidite monomer containing succinimide structure in recognition of mismatched bases
  • Application of dideoxynucleoside phosphoramidite monomer containing succinimide structure in recognition of mismatched bases

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Experimental program
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Effect test

Embodiment 1

[0048] The present invention obtains a modification reagent of a non-natural oligonucleotide, that is, a deoxythymidine-deoxythymidine phosphoramidite monomer containing a succinamide structure, the chemical structure of which is as follows:

[0049]

[0050] The synthetic route of this monomer is as follows:

[0051]

[0052] Specific steps:

[0053] (a) Dissolve 10mmol zivudodine in 50mL anhydrous pyridine, add 10-20mmol 4,4-dimethoxytriphenylchloromethane in 10mL pyridine solution at room temperature, stir at room temperature for 1-12h; the reaction solution is concentrated After removing the solvent, add 50mL tetrahydrofuran, 1mL water and 1mL triethylamine, react at 85°C for 1-8h under the protection of nitrogen, concentrate to remove the organic solvent; Wash with water and dry over anhydrous sodium sulfate; the crude product can be directly used in the next step;

[0054] Dissolve 10mmol of the above-prepared product 5-(4,4-methoxytriphenylmethyl ether)-3-amino-...

Embodiment 2

[0070] Use of deoxythymidine-deoxythymidine phosphoramidite monomers containing succinamide structure for DNA synthesis

[0071] The synthetic route is as follows:

[0072]

[0073] Specific steps:

[0074] (d) 800 mg of double thymidine reagent (deoxythymidine-deoxythymidine phosphonamidite monomer containing succinamide structure) was dissolved in 3 mL of anhydrous acetonitrile, and transferred to the end group modification position of ABI394 nucleic acid synthesizer under nitrogen protection in the reagent bottle. Input nucleic acid sequence: 5'-TTC CAC TTA CCA GAT TGA TT-3', perform synthesis on the order of 1 μmol;

[0075] (e) After the synthesis is completed, the solid phase carrier is taken out, dispersed in 1mL 5-32% methylammonia aqueous solution, heated at 55-75°C for 1-4h, centrifuged to remove insoluble matter, and the solution is concentrated to obtain the crude product; the target product is obtained by Separation by HPLC and characterization of structural...

Embodiment 3

[0080] Application of Double Thymidine Reagent Containing Succinamide Structure in Construction of Resistant Enzyme Cutting Artificial Nucleic Acid

[0081] 1. A synthetic method for modifying the 5' end of an artificial nucleic acid with a double thymidine reagent containing a succinamide structure

[0082]

[0083] 2. A synthetic method for modifying the 3' end of an artificial nucleic acid with a double thymidine reagent containing a succinamide structure

[0084]

[0085] A method for synthesizing an enzyme-resistant artificial nucleic acid, the specific steps of which are:

[0086] Step a: Dissolve 800 mg of the reagent in 3 ml of anhydrous acetonitrile, and transfer it to the terminal modification site reagent bottle of the ABI 394 nucleic acid synthesizer under nitrogen protection. Input nucleic acid sequence: 5'-TTC CAC TTA CCA GAT TGA TT-3', perform synthesis on the order of 1 μmol.

[0087] Step b: After the synthesis is completed, the solid phase carrier is ...

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Abstract

The invention discloses an application of a dideoxynucleoside phosphoramidite monomer containing a succinimide structure in recognition of mismatched bases, and belongs to the technical field of application of non-natural oligonucleotides. The preparation method comprises the following steps: constructing the dideoxynucleoside phosphoramidite monomer containing the succinimide structure into artificial deoxyribonucleic acid; replacing part of phosphodiester bonds in natural nucleic acid with butanediamide bonds, the butanediamide bonds being used as a probe sequence to detect the base mismatchcondition in a complementary sequence. A melting point determination experiment is used for detecting a mismatch unit containing T-C, a good effect is achieved, and a new research thought is providedfor designing a novel detection unit.

Description

technical field [0001] The invention belongs to the technical field of application of non-natural oligonucleotides, and in particular relates to the application of a dideoxynucleoside phosphoramidite monomer containing a succinamide structure in mismatch base recognition. Background technique [0002] Single base mismatch is a kind of single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP), which is one of the important types of DNA mutation damage. How to realize the detection of single base mismatch has important theoretical and practical significance for elucidating the causes of various diseases at the molecular level and realizing the treatment at the gene level. The development of single-base mismatch detection methods with high sensitivity and high selectivity is a hot research field in recent years. Commonly used single-base mismatch detection methods include gel electrophoresis, fluorescence detection, SPR, and mass spectrometry. [0003] Non-natural ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6816C07H19/10C07H21/04C07H1/02C07H1/00
CPCC12Q1/6816C07H19/10C07H21/04C07H1/02C07H1/00
Inventor 孙亚伟吕环芳孙立梅王栋王继乾
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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