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广泛适用的寡核苷酸质谱法用于体外分析 RNA 写入器和擦除器。

Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro.

机构信息

Department of Chemistry, Ludwig Maximilians University Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.

Department of Biology, Center for RNA Biology, University of Rochester, Rochester, NY 14627, USA.

出版信息

Nucleic Acids Res. 2020 Apr 17;48(7):e41. doi: 10.1093/nar/gkaa091.

DOI:10.1093/nar/gkaa091
PMID:32083657
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7144906/
Abstract

RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNAValAAC is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNAValIACin vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA.

摘要

RNAs 可被专门的写入器或擦除器酶通过添加或去除特定修饰来进行转录后修饰。RNA 的质谱（MS）是一种有用的工具，可用于以敏感的方式研究寡核苷酸（ON）的修饰状态。在这里，我们开发了一种无离子对试剂色谱法，用于通过低和高分辨率 MS 对 ONs 进行正离子检测，该方法不会干扰在同一仪器上进行的其他类型的小分子化合物分析。我们应用 ON-MS 来确定体外转录 tRNA 的 RNase T1 消化物中的 ONs，这些 ONs 在核酶融合转录后通过自动排阻色谱法进行纯化。由此产生的 tRNAValAAC 是人类 tRNA ADAT2/3 酶的底物，我们通过 ON-MS 确认了腺嘌呤的脱氨为肌苷和 tRNAValIAC 的形成。此外，低分辨率 ON-MS 用于监测体外含 1-甲基腺苷的 ONs 被细菌 AlkB 的去甲基化。高分辨率 ON-MS 的强大功能通过对用 RNase T1 消化的天然总 tRNA 进行修饰的 ONs 的检测和作图来证明。总的来说，我们提出了一种寡核苷酸 MS 方法，该方法广泛适用于监测体外 RNA（去）修饰过程和天然 RNA。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/b1beaede3584/gkaa091fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/38d8363ae6c9/gkaa091fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/aaeb195514d9/gkaa091fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/76b846f7a5eb/gkaa091fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/d3abe7dd2601/gkaa091fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/8948d42a73b7/gkaa091fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/d2738b994066/gkaa091fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/b1beaede3584/gkaa091fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/38d8363ae6c9/gkaa091fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/aaeb195514d9/gkaa091fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/76b846f7a5eb/gkaa091fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/d3abe7dd2601/gkaa091fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/8948d42a73b7/gkaa091fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/d2738b994066/gkaa091fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e3e0/7144906/b1beaede3584/gkaa091fig7.jpg

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