Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids
Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids
复制标题
硫酯核酸的无热、化学触发 RNA 释放
DOI：
10.1002/anie.202110741
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Bowman, Christopher N.
中科院分区：
文献类型：
--
作者：
Mavila, Sudheendran;Culver, Heidi R.;Anderson, Alex J.;Prieto, Tania R.;Bowman, Christopher N.
描述了一种使用自焚硫酯连接核酸 (TENA) 从总 RNA 中富集 mRNA 的无热方法。寡聚（胸腺嘧啶）（oT）TENA 碱基之间有 6 个原子间距，这使得 TENA 能够选择性地与聚腺嘌呤 RNA 碱基配对。由于 TENA 的中性主链和辛硫醇端基的疏水性，oT TENA 不溶于水，并且在浓度为 10 ng μL−1 时可有效下拉 93±2 % 的 EGFP mRNA。在环境温度下暴露于亲核缓冲液成分（Tris、DTT）时，TENA 发生自降解，可从 3.1 μg 总 RNA 中回收 55±27ng mRNA，这与使用 Dynabeads® mRNA DIRECT 试剂盒回收的量 (89±24ng) 没有统计学差异。通过 RT-qPCR 测量的基因表达在两种富集方法中具有可比性，这表明使用 oT TENA 富集 mRNA 所需的温和条件与 RT-qPCR 和其他下游分子生物学应用兼容。
An athermal approach to mRNA enrichment from total RNA using a self‐immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six‐atom spacing between bases which allowed TENA to selectively base‐pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93±2 % of EGFP mRNA at a concentration of 10 ng μL−1. Self‐immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55±27 ng of mRNA from 3.1 μg of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89±24 ng). Gene expression as measured by RT‐qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT‐qPCR and other downstream molecular biology applications.