Ding Yu, Li Xianming, Zhang Xinfeng, Li Feng, Hou Xiandeng, Wu Peng
Analytical & Testing Center, College of Chemistry, Sichuan University, Chengdu 610064, China.
College of Materials and Chemistry & Chemical Engineering, Chengdu University of Technology, Chengdu 610059, China.
ACS Omega. 2019 Aug 6;4(8):13382-13387. doi: 10.1021/acsomega.9b01560. eCollection 2019 Aug 20.
Exonuclease III (Exo III) is an important enzymatic tool that is being widely used in molecular biology, biotechnology, and bioassay development. Exo III prefers to cleave double-stranded DNA (dsDNA) with blunt and recessed 3'-termini rather than their protruding counterpart. While it has been accepted that a short 3'-overhang (e.g., >4 nt) is necessary to protect a dsDNA from Exo III cleavage, critical roles of the length and sequence of this 3'-overhang remain unexplored. Herein, we develop a novel light-induced colorimetric assay allowing the systematic probe of the sequence selectivity of Exo III in a rapid and high-throughput manner. Our finding that Exo III is highly specific to 3'-overhang in terms of both length and sequence will be valuable for guiding the design of bioassays and DNA manipulating tools mediated by Exo III.
核酸外切酶III（Exo III）是一种重要的酶工具，在分子生物学、生物技术和生物测定开发中被广泛应用。Exo III更倾向于切割具有平端和凹陷3'末端的双链DNA（dsDNA），而不是其突出的对应物。虽然人们已经认识到短的3'突出端（例如，>4 nt）对于保护dsDNA免受Exo III切割是必要的，但这个3'突出端的长度和序列的关键作用仍未被探索。在此，我们开发了一种新型的光致比色测定法，能够以快速且高通量的方式系统地探究Exo III的序列选择性。我们发现Exo III在长度和序列方面对3'突出端具有高度特异性，这对于指导由Exo III介导的生物测定和DNA操作工具的设计将具有重要价值。
