Ultraviolet shadowing of RNA can cause significant chemical damage in seconds.
Ultraviolet shadowing of RNA can cause significant chemical damage in seconds.
复制标题
DOI：
10.1038/srep00517
复制
发表时间：
2012
影响因子：
4.6
通讯作者：
Das, Rhiju
中科院分区：
文献类型：
--
作者：
Kladwang, Wipapat;Hum, Justine;Das, Rhiju
RNA样品的化学纯度对于RNA折叠和催化行为的高精度研究非常重要，但是在样品制备的紫外线（UV）遮蔽步骤中产生的光损伤会降低这种纯度。在这里，我们报告的定量紫外线诱导的损伤，使用逆转录和单核苷酸分辨率毛细管电泳。我们在十几种天然和人工RNA中发现了光损伤;在多个序列环境中，主要是但不限于嘧啶双峰;以及来自推荐用于UV遮蔽的多个灯。辐照时间过程显示，在距离0.5 mm厚凝胶5 - 10 cm的254 nm灯暴露数秒内可检测到损伤。 在这些条件下，200个核苷酸的RNA受到20秒的紫外线遮蔽，导致16-27%的分子受损;并且，由于“皮肤效应”，病变的分子-分子分布比泊松分布的方差高4倍。较厚的凝胶、较长波长的灯和较短的暴露时间减少了损伤，但没有消除损伤。这些结果表明，RNA生物物理学研究应报告采取的预防措施，以避免人为的异质性，从紫外阴影。
Chemical purity of RNA samples is important for high-precision studies of RNA folding and catalytic behavior, but photodamage accrued during ultraviolet (UV) shadowing steps of sample preparation can reduce this purity. Here, we report the quantitation of UV-induced damage by using reverse transcription and single-nucleotide-resolution capillary electrophoresis. We found photolesions in a dozen natural and artificial RNAs; across multiple sequence contexts, dominantly at but not limited to pyrimidine doublets; and from multiple lamps recommended for UV shadowing. Irradiation time-courses revealed detectable damage within a few seconds of exposure for 254 nm lamps held at a distance of 5 to 10 cm from 0.5-mm thickness gels. Under these conditions, 200-nucleotide RNAs subjected to 20 seconds of UV shadowing incurred damage to 16-27% of molecules; and, due to a ‘skin effect’, the molecule-by-molecule distribution of lesions gave 4-fold higher variance than a Poisson distribution. Thicker gels, longer wavelength lamps, and shorter exposure times reduced but did not eliminate damage. These results suggest that RNA biophysical studies should report precautions taken to avoid artifactual heterogeneity from UV shadowing.
登录
查看更多内容
影响因子：
21.8
作者：
通讯作者：
--
影响因子：
2.9
作者：
Kladwang, Wipapat;Das, Rhiju
通讯作者：
Das, Rhiju
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
2.9
作者：
Korennykh, Alexei V.;Plantinga, Matthew J.;Piccirilli, Joseph A.
通讯作者：
Piccirilli, Joseph A.
影响因子：
16.8
作者：
Mandal, M;Breaker, RR
通讯作者：
Breaker, RR