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首页> 外文期刊>Environmental Science & Technology >Direct and Indirect Photochemical Reactions in Viral RNA Measured with RT-qPCR and Mass Spectrometry
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Direct and Indirect Photochemical Reactions in Viral RNA Measured with RT-qPCR and Mass Spectrometry

机译：RT-qPCR和质谱法测定病毒RNA中的直接和间接光化学反应

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RNA carries the genetic instructions for many viruses to replicate in their host cells. The photochemical reactions that take place in RNA and affect viral infectivity in natural and engineered environments, however, remain poorly understood We exposed RNA oligomer segments from the genome of bacteriophage MS2 to UV_(254), simulated sunlight, and singlet oxygen (~10_2) and analyzed the oligomer reaction kinetics with reverse transcription quantitative PCR (RT-qPCR) and quantitative matrix-assisted laser desorp-tion-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Following UV_(254) exposure, quantitative MALDI-TOF-MS detected significantly more RNA modifications than did RT-qPCR, suggesting that certain chemical modifications in the RNA were not detected by the reverse transcriptase enzyme. In contrast, MALDI-TOF-MS tracked as much ~10_2-induced RNA damage as RT-qPCR After 5 h of simulated sunlight exposure (5100 J/m~2 UVB and 1.2 X 10~5 J/m~2 UVA), neither MALDI-TOF-MS nor RT-qPCR detected significant decreases in the oligomer concentrations. High-resolution electrospray ionization (ESI)-Orbitrap MS analyses identified pyrimidine photohydrates as the major UV_(254) products, which likely contributed to the discrepancy between the MS- and RT-qPCR-based results. Reactions between RNA oligomers and ~10_2 resulted in an unidentified major product with a mass change of +6 Da. These results shed light on the photochemical reactions that take place in RNA and suggest that the analytical techniques used to detect RNA reactivity could bias the observed reaction kinetics.
机译：RNA具有许多病毒在其宿主细胞中复制的遗传指令。 RNA中发生的光化学反应会影响自然和工程环境中的病毒感染性，但人们对此知之甚少。我们将噬菌体MS2基因组中的RNA低聚物片段暴露于UV_（254），模拟阳光和单线态氧（〜10_2）并通过逆转录定量PCR（RT-qPCR）和定量基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱（MS）分析了低聚物的反应动力学。在UV_（254）暴露后，定量MALDI-TOF-MS检测到的RNA修饰比RT-qPCR显着多，这表明逆转录酶未检测到RNA中的某些化学修饰。相比之下，MALDI-TOF-MS在模拟阳光照射5小时（5100 J / m〜2 UVB和1.2 X 10〜5 J / m〜2 UVA）后，追踪到约10_2诱导的RNA损伤与RT-qPCR一样多， MALDI-TOF-MS和RT-qPCR均未检测到低聚物浓度的显着降低。高分辨率电喷雾电离（ESI）-Orbitrap MS分析确定嘧啶光水合物为主要的UV_（254）产物，这可能导致基于MS和RT-qPCR的结果之间存在差异。 RNA寡聚物与〜10_2之间的反应导致质量变化为+6 Da的未确定的主要产物。这些结果揭示了RNA中发生的光化学反应，并表明用于检测RNA反应性的分析技术可能会偏向观察到的反应动力学。

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  • 来源
    《Environmental Science & Technology》 |2016年第24期|13371-13379|共9页
  • 作者

    Zhong Qiao; Krista R. Wigginton;

  • 作者单位

    Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States;

    Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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