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New function and application of nucleoside transferase

A transferase and nucleotide technology, applied in the field of genetic engineering, can solve the problems of low DNA polymerase activity and difficult synthesis of enzymatic oligonucleotides, and achieve the effect of high substrate specificity

Active Publication Date: 2021-05-04
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the activity of existing DNA polymerases is low, and how to achieve efficient and controllable enzymatic oligonucleotide synthesis is still a big problem

Method used

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  • New function and application of nucleoside transferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Obtaining of TDT protein

[0065] 1. Amino acid sequences of TDT proteins from different species

[0066] The amino acid sequences are as follows: SEQ ID NO:1 (mouse), SEQ ID NO:2 (platypus), SEQ ID NO:3 (myotis), SEQ ID NO:4 (naked zokor), SEQ ID NO: 5 (chimpanzee), SEQ ID NO: 6 (gecko), SEQ ID NO: 7 (emu), SEQ ID NO: 8 (junga chicken), SEQ ID NO: 9 (white-throated finch), SEQ ID NO: 10 ( Peregrine Falcon), SEQ ID NO: 11 (Golden Eagle). The homology is shown in Table 1:

[0067] Table 1 Homology analysis of amino acid sequences of TDT proteins from different species

[0068]

[0069] 2. Construction of expression vector

[0070] All gene sequences that can synthesize the amino acid sequences shown in the sequence listing can be used in the construction of expression vectors. In this embodiment, the gene sequence encoding TDT protein in the above-mentioned species recorded in NCBI is constructed into the expression vector pET-28a (Novagen, Kan. + ,See...

Embodiment 2

[0086] Example 2 Function Verification

[0087] The described adopting substrate of the embodiment of the present invention is:

[0088] (1) Deoxyribonucleotides (dNTPs), including adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymine deoxyribonucleotides;

[0089] (2) Based on the deoxyribonucleotide in (1), the 3'-OH end is changed to a reversible blocking group, preferably, the modification group at the 3' end is an amino group.

[0090] In vitro pure enzyme reaction, the reaction schematic diagram see figure 2 .

[0091] Reaction system: 100mM NaCl, 0.25mM CoCl 2 , 50mM KAc, 10mM Mg(Ac) 2 , pH 6.8. Substrate: 1 μM starting sequence 1 (14bp), 100 μM deoxyribonucleotides or 100 μM deoxyribonucleotides with amino groups at the 3’ end. Enzyme: 50 μM TDT proteins from different species prepared in Example 1. Starting sequence 1: TAATACGACTCACT

[0092] Reaction conditions: react at 30°C for 30s, inactivate the protein at...

Embodiment 3

[0094] Embodiment 3 function verification

[0095] The described adopting substrate of the embodiment of the present invention is:

[0096] (1) Deoxyribonucleotides (dNTPs), including adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymine deoxyribonucleotides;

[0097] (2) Based on the deoxyribonucleotides in (1), the 3' end is changed to a reversible blocking group. Preferably, the modification group at the 3' end is an oxygen amino group; preferably, the modification group at the 3' end is an oxyallyl group; preferably, the modification group at the 3' end is an oxygen azido group; preferably, the modification group at the 3' end is an oxygen phosphoric acid group.

[0098]

[0099] The 3' deoxyribonucleotide with a reversible blocking group provided above is an example of thymine deoxyribonucleotide, and the difference between the other three deoxyribonucleotides and this example is only at the base end, 3 The reversible ...

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Abstract

The invention provides a new function and application of nucleoside transferase. The invention provides terminal deoxyribonucleoside transferase from various species sources, and the terminal deoxyribonucleoside transferase has excellent catalytic activity when catalyzing the polymerization reaction of nucleotides modified by blocking groups, and is beneficial to realizing efficient and controllable nucleic acid molecule synthesis.

Description

[0001] Cross References to Related Applications [0002] This application claims the priority and rights of the Chinese invention patent application with application number 201911034444.6 filed on October 29, 2019, the entire contents of which are incorporated herein by reference. technical field [0003] The invention belongs to the field of genetic engineering and relates to a new function and application of a terminal deoxyribonucleoside transferase. Background technique [0004] DNA is the carrier of life information, and obtaining DNA is the first step in researching, transforming and creating life. DNA synthesis technology is the most important common basic support technology in the field of life sciences. There are two main methods for de novo synthesis of oligonucleotides: chemical synthesis (solid-phase phosphoramidite triester synthesis) and biosynthesis (enzymatic synthesis). Since the 1950s, chemical methods and enzymatic methods have developed separately, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12P19/34
CPCC12N9/1264C12Y207/07031C12P19/34
Inventor 江会锋逯晓云卢丽娜
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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