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HYPER-THERMOSTABLE LYSINE-MUTANT ssDNA/RNA LIGASES

A technology of RNA ligase and ligase, which is applied in the field of ultra-thermally stable lysine-mutant ssDNA/RNA ligase, which can solve the problems of low efficiency

Inactive Publication Date: 2019-03-15
RGENE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, T4 RNA ligase 1 (T4Rnl1) has long been known and used to ligate single-stranded RNA (ssRNA) and ssDNA, with less efficiency (Lau et al., 2001)

Method used

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  • HYPER-THERMOSTABLE LYSINE-MUTANT ssDNA/RNA LIGASES

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Experimental program
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Embodiment 1

[0055] Mutant hyperthermostable ssDNA / RNA ligase

[0056] First, a series of hyperthermostable RNA ligases were identified in a search of the field. This list in Table 2 below includes hyperthermophilic archaeal species, some of which tolerate environments in excess of 100°C.

[0057] Table 2

[0058]

[0059] *Pab1020 has been previously studied and shown to have ssRNA ligation activity, but not ssDNA ligation activity (Brooks et al., Protein Science, 2008).

[0060]Three of the ten ligases from Table 2 (PhoRnl2, PfuRnl2, HbuRnl2) were synthesized, expressed and purified using optimized E. figure 1 90°C) to check their activity on single-stranded RNA and DNA substrates. like figure 1 Enzyme activity was measured under ATP concentration titration using 5'-phosphorylated ssRNA and 5'-phosphorylated ssDNA oligonucleotides as substrates, as indicated in . like figure 1 As shown in A, 1C and 1E, all enzymes tested can convert 5'-phosphorylated ssRNA to 5'-adenylated ssRNA...

Embodiment 2

[0078] single-stranded adapter ligation

[0079] Hyperthermostable ssDNA / RNA ligases (eg, as described in SEQ ID NOs: 1 to 11) can be used in the library preparation process for next-generation high-throughput sequencing. Currently, the main library construction methods include a ligation step in which double-stranded library fragments are ligated to double-stranded adapters. There is a method based on single-strand ligation, but by using CircLigase, its optimal reaction temperature is about 65°C (Gansauge MT, Nat. Protol., 2013).

[0080] like Figure 7As shown in A, large DNA is first fragmented, eg, by using enzymatic methods or by mechanical / sonic shearing. Depending on the fragmentation method, the ends of the fragments may require a repair step using, for example, T4 polynucleotide kinase. The fragmented DNA was then ligated into DNA with 5'-adenylated ends and 3'-NH 2 The first single-stranded DNA adapter at the end. This intermolecular ligation is catalyzed by one...

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Abstract

Provided herein are compositions, systems, and methods employing hyper-thermostable lysine-mutant ssDNA / RNA ligases that possesses both ssRNA ligase and ssDNA ligase activity. In certain embodiments,such hyper-thermostable lysine-mutant ssDNA / RNA ligases are used to ligate an first single stranded nucleic acid sequence with a 5' adenylated end to a second single stranded nucleic acid sequence (e.g., at a temperature of at least 75 DEG C) to form a ligated nucleic acid sequence. In further embodiments, the ligated nucleic acid sequence is sequenced.

Description

[0001] This application claims priority to US Provisional Application Serial No. 62 / 307,658, filed March 14, 2016, which is hereby incorporated by reference in its entirety. technical field [0002] Provided herein are compositions, systems and methods for using hyperthermostable lysine-mutant ssDNA / RNA ligases having both ssRNA ligase and ssDNA ligase activities. In certain embodiments, such hyperthermostable lysine-mutant ssDNA / RNA ligases are used to ligate a first single-stranded nucleic acid sequence having a 5' adenylated end to a second single-stranded nucleic acid sequence (e.g. , at a temperature of at least 75° C.) to form linked nucleic acid sequences. In other embodiments, the linked nucleic acid sequences are amplified and / or sequenced. Background technique [0003] DNA or RNA ligases are divalent metal ion-dependent enzymes that utilize ATP or NAD+ to catalyze phosphodiester bond formation between adjacent polynucleotide ends with 3'-hydroxyl and 5'-phosphate ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30C12P19/34C12Q1/6869
CPCC07H21/04C12Q1/6862C12N15/66C12N9/93C12Y600/00C12Q2521/501C12Q1/686C12Q1/6869
Inventor 郑钰洪曼青
Owner RGENE INC
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