Nanopore Dwell Time Analysis Permits Sequencing and Conformational Assignment of Pseudouridine in SARS-CoV-2.
Nanopore Dwell Time Analysis Permits Sequencing and Conformational Assignment of Pseudouridine in SARS-CoV-2.
复制标题
DOI：
10.1021/acscentsci.1c00788
复制
发表时间：
2021-10-27
影响因子：
18.2
通讯作者：
Burrows CJ
中科院分区：
文献类型：
--
作者：
Fleming AM;Mathewson NJ;Howpay Manage SA;Burrows CJ
Direct RNA sequencing for the epitranscriptomic modification pseudouridine (Ψ), an isomer of uridine (U), was conducted with a protein nanopore sensor using a helicase brake to slowly feed the RNA into the sensor. Synthetic RNAs with 100% Ψ or U in 20 different known human sequence contexts identified differences during sequencing in the base-calling, ionic current, and dwell time in the nanopore sensor; however, the signals were found to have a dependency on the context that would result in biases when sequencing unknown samples. A solution to the challenge was the identification that the passage of Ψ through the helicase brake produced a long-range dwell time impact with less context bias that was used for modification identification. The data analysis approach was employed to analyze publicly available direct RNA sequencing data for SARS-CoV-2 RNA taken from cell culture to locate five conserved Ψ sites in the genome. Two sites were found to be substrates for pseudouridine synthase 1 and 7 in an in vitro assay, providing validation of the analysis. Utilization of the helicase as an additional sensor in direct RNA nanopore sequencing provides greater confidence in calling RNA modifications. Sequencing RNA for the U isomer pseudouridine (Ψ) with a nanopore-helicase setup found Ψ-specific signals in the nanopore and helicase. Analysis of SARS-CoV-2 RNA sequencing data revealed conserved Ψs.
登录
查看更多内容
DOI：
10.1002/wrna.1586
发表时间：
2020-07
期刊：
Wiley interdisciplinary reviews. RNA
影响因子：
--
作者：
Jones JD;Monroe J;Koutmou KS
通讯作者：
Koutmou KS
影响因子：
7.2
作者：
Linder, Bastian;Jaffrey, Samie R.
通讯作者：
Jaffrey, Samie R.
影响因子：
14.9
作者：
Crain, PF;McCloskey, JA
通讯作者：
McCloskey, JA
影响因子：
16.8
作者：
Buettner, Katharina;Nehring, Sebastian;Hopfner, Karl-Peter
通讯作者：
Hopfner, Karl-Peter
影响因子：
64.8
作者：
Goyal, Parveen;Krasteva, Petya V.;Van Genven, Nani;Gubellini, Francesca;Van den Broeck, Imke;Troupiotis-Tsailaki, Anastassia;Jonckheere, Wim;Pehau-Arnaudet, Gerard;Pinkner, Jerome S.;Chapman, Matthew R.;Hultgren, Scott J.;Howorka, Stefan;Fronzes, Remi;Remaut, Han
通讯作者：
Remaut, Han