Abstract：
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 E. coli 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.
摘要：
鉴于RNA三级结构的实验测定的挑战，探测溶剂可及性对于获得功能洞察力变得越来越重要。在开发的各种化学探针中，主链切割羟基自由基是唯一可以提供所有可接近核苷酸的无偏倚检测的自由基。然而,读数是基于在切割位点停止的逆转录(RT)，由于PCR扩增偏差，它们容易出现假阳性，逆转录酶的早期下降，以及随机引物在RT反应中的应用。这里,我们引入了一种称为RL-Seq的固定引物方法,即在高通量测序之前,在固定的5'-OH-末端接头和羟基自由基裂解的独特3'-P-末端片段之间进行RtcB连接(RL)。该方法对大肠杆菌核糖体的应用证实了其在单核苷酸分辨率下以高灵敏度(低要求的测序深度)和准确度(与结构衍生值的强相关性)准确探测溶剂可及性的能力。此外，在使用和不使用独特的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。讨论了RL-Seq的进一步改进及其潜在的转录组应用。
