Transcription, Reverse Transcription, and Amplification of Backbone‐Modified Nucleic Acids with Laboratory‐Evolved Thermophilic DNA Polymerases
Transcription, Reverse Transcription, and Amplification of Backbone‐Modified Nucleic Acids with Laboratory‐Evolved Thermophilic DNA Polymerases
复制标题
使用实验室进化的嗜热 DNA 聚合酶进行主链修饰核酸的转录、反转录和扩增
DOI：
10.1002/cpz1.188
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Tingjian Chen
中科院分区：
文献类型：
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作者：
Ping Song;Rujie Zhang;Chuanping He;Tingjian Chen
主链修饰的核酸通常比其天然对应物更稳定，使其能够作为潜在的诊断或治疗剂广泛应用。此外，具有非天然骨架的核酸的发展扩大了遗传信息载体的库，并为合成异生物学铺平了道路。然而，合成这些分子仍然非常具有挑战性，因为在传统的固相合成过程中需要苛刻的反应条件和低偶联效率。虽然酶促合成提供了一种有吸引力的替代方法，也允许这些分子的复制和人工进化，但它关键取决于能够合成这些骨架修饰的核苷酸的聚合酶的可用性。以前，通过基于噬菌体展示的聚合酶进化方法，已经进化出一系列可以有效合成甚至扩增骨架修饰的DNA或RNA的热稳定聚合酶。在此，我们总结了使用这些进化的聚合酶突变体来转录、逆转录和PCR扩增骨架修饰的核酸的方案。我们还概述了聚合酶链式转录方法，该方法后来发展用于使用这些进化的聚合酶之一SFM 4 - 3快速生产RNA或骨架修饰的RNA。© 2021 Wiley Periodicals LLC.
Backbone‐modified nucleic acids are usually more stable enzymatically than their natural counterparts, enabling their broad application as potential diagnostic or therapeutic agents. Moreover, the development of nucleic acids with unnatural backbones has expanded the pool of genetic information carriers and paved the way toward synthetic xenobiology. However, synthesizing these molecules remains very challenging due to the requirement for harsh reaction conditions and the low coupling efficiency during their traditional solid‐phase synthesis. Although enzymatic synthesis provides an attractive alternative that also allows the replication and artificial evolution of these molecules, it is crucially dependent on the availability of polymerases capable of synthesizing these backbone‐modified nucleotides. Previously, a series of thermostable polymerases that can efficiently synthesize or even amplify backbone‐modified DNA or RNA have been evolved through a polymerase evolution method based on phage display. Herein we summarize protocols to use these evolved polymerase mutants to transcribe, reverse transcribe, and PCR amplify backbone‐modified nucleic acids. We also outline the polymerase chain transcription method, developed later for the rapid production of RNA or backbone‐modified RNA with one of these evolved polymerases, SFM4‐3. © 2021 Wiley Periodicals LLC.
影响因子：
18.3
作者：
Kotikam V;Rozners E
通讯作者：
Rozners E
DOI：
10.3390/molecules25204659
发表时间：
2020-10-13
期刊：
Molecules (Basel, Switzerland)
影响因子：
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作者：
Ochoa S;Milam VT
通讯作者：
Milam VT