RNA的紫外线遮蔽在几秒钟内造成大量非泊松化学损伤
Ultraviolet Shadowing of RNA Causes Substantial Non-Poissonian Chemical Damage in Seconds
摘要：
RNA样品的化学纯度对于RNA折叠和催化行为的高精度研究至关重要，但这种纯度可能会受到凝胶纯化样品紫外线（UV）可视化过程中产生的光损伤的影响。在这里，我们定量评估的广度和程度，这种损害，然后使用逆转录单核苷酸分辨率毛细管电泳。我们检测了十几种天然和人工RNA（包括核糖开关结构域、其他非编码RNA和人工序列）中的UV诱导病变;检测了多种序列背景下的UV诱导病变，主要是但不限于嘧啶双联体;以及文献中推荐用于UV遮蔽的多种灯。最引人注目的是，照射时间过程揭示了可检测到的损害在几秒钟内曝光，这些数据可以定量地适合的“趋肤效应”模型，该模型占辐射凝胶切片的顶部附近的分子的暴露增加。结果表明，200个核苷酸的RNA受到20秒或更短的紫外线遮蔽可以引起20%的分子损伤，并且这些损伤的分子-分子分布比泊松分布更不均匀。因此，来自UV遮蔽的光损伤可能是定量和单分子分辨率RNA生物物理测量中人为异质性的普遍但未被认识到的原因。
英文摘要：
Chemical purity of RNA samples is critical for high-precision studies of RNA folding and catalytic behavior, but such purity may be compromised by photodamage accrued during ultraviolet (UV) visualization of gel-purified samples. Here, we quantitatively assess the breadth and extent of such damage by using reverse transcription followed by single-nucleotide-resolution capillary electrophoresis. We detected UV-induced lesions across a dozen natural and artificial RNAs including riboswitch domains, other non-coding RNAs, and artificial sequences; across multiple sequence contexts, dominantly at but not limited to pyrimidine doublets; and from multiple lamps that are recommended for UV shadowing in the literature. Most strikingly, irradiation time-courses reveal detectable damage within a few seconds of exposure, and these data can be quantitatively fit to a 'skin effect' model that accounts for the increased exposure of molecules near the top of irradiated gel slices. The results indicate that 200-nucleotide RNAs subjected to 20 seconds or less of UV shadowing can incur damage to 20% of molecules, and the molecule-by-molecule distribution of these lesions is more heterogeneous than a Poisson distribution. Photodamage from UV shadowing is thus likely a widespread but unappreciated cause of artifactual heterogeneity in quantitative and single-molecule-resolution RNA biophysical measurements.
机构、总结、中文摘要均由AI分析而来，可能存在错误，请以论文正文为准。
