Collateral nuclease activity of TnpB triggered by high temperature enables fast and sensitive nucleic acid detection

期刊论文
出版模式：

开放获取
发表日期：

2024年11月20日

查看全文

Ying Xu,
Wen Yin,
Yibin Cheng,
Wei Zeng,
Wenqiang Li,
Wanping Chen,
Fei Wang,
Nan Peng,
Lixin Ma,
Tao Liu

摘要

TnpB proteins encoded in the IS200/IS605 family are RNA-guided endonuclease which can be harnessed in genome editing. However, the collateral nuclease activity of TnpB remains poorly understood, which limits the development of TnpB-based diagnostic tools. Here we showed that TnpB from a thermophilic archaeon exhibits enhanced collateral ssDNA cleavage activity (trans-cleavage) activated by high temperature. Mutations either in the TAM or seed sequences of the target DNA impair the trans-cleavage activity, which indicates its potential to be employed in molecular diagnostic. Importantly, by optimizing the length and the sequences of the collateral substrates, we have developed a new nucleic acid detection method based on TnpB with a sensitivity of 29 cp μl⁻¹ in 30 min, which we name it TESD (TnpB Enable fast and Sensitive Detection). In summary, our findings illustrate the collateral nuclease activity of a TnpB from thermophiles and provide a novel platform for molecular diagnostics.