Solayman Md, Litfin Thomas, Zhou Yaoqi, Zhan Jian
Institute for Glycomics, Griffith University, Parklands Dr, Southport, QLD, Australia.
Institute for Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen, China.
RNA Biol. 2022 Jan;19(1):1179-1189. doi: 10.1080/15476286.2022.2145098.
Given the challenges for the experimental determination of RNA tertiary structures, probing solvent accessibility has become increasingly important to gain functional insights. Among various chemical probes developed, backbone-cleaving hydroxyl radical is the only one that can provide unbiased detection of all accessible nucleotides. However, the readouts have been based on reverse transcription (RT) stop at the cleaving sites, which are prone to false positives due to PCR amplification bias, early drop-off of reverse transcriptase, and the use of random primers in RT reaction. Here, we introduced a fixed-primer method called RL-Seq by performing RtcB Ligation (RL) between a fixed 5'-OH-end linker and unique 3'-P-end fragments from hydroxyl radical cleavage prior to high-throughput sequencing. The application of this method to ribosomes confirmed its ability to accurately probe solvent accessibility with high sensitivity (low required sequencing depth) and accuracy (strong correlation to structure-derived values) at the single-nucleotide resolution. Moreover, a near-perfect correlation was found between the experiments with and without using unique molecular identifiers, indicating negligible PCR biases in RL-Seq. Further improvement of RL-Seq and its potential transcriptome-wide applications are discussed.
鉴于RNA三级结构实验测定面临的挑战，探测溶剂可及性对于获得功能见解变得越来越重要。在已开发的各种化学探针中，裂解骨架的羟基自由基是唯一能够对所有可及核苷酸进行无偏差检测的探针。然而，读数一直基于在裂解位点的逆转录（RT）终止，由于PCR扩增偏差、逆转录酶的早期脱落以及RT反应中使用随机引物，这些读数容易出现假阳性。在这里，我们通过在高通量测序之前，在固定的5'-OH末端接头与羟基自由基裂解产生的独特3'-P末端片段之间进行RtcB连接（RL），引入了一种称为RL-Seq的固定引物方法。将该方法应用于核糖体证实了其能够在单核苷酸分辨率下，以高灵敏度（所需测序深度低）和准确性（与结构推导值高度相关）准确探测溶剂可及性。此外，在使用和不使用独特分子标识符的实验之间发现了近乎完美的相关性，表明RL-Seq中的PCR偏差可忽略不计。文中还讨论了RL-Seq的进一步改进及其潜在的全转录组应用。
