Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, PO Box 210172, Cincinnati, OH 45221-0172, USA.
Analyst. 2013 Mar 7;138(5):1386-94. doi: 10.1039/c2an36515d.
The comparative analysis of ribonucleic acid digests (CARD) approach for sequencing of transfer ribonucleic acids (tRNAs) is described. This method is enabled by the differential labeling of two tRNA populations. A set of reference tRNAs, whose complete sequences including modifications are known, are labeled with (16)O during enzymatic digestion. The second (candidate) set of tRNAs, whose sequence information is desired, is labeled with (18)O. By combining the two digests, digestion products that share the same sequence between the reference and candidate will appear as doublets separated by 2 Da. Sequence or modification differences between the two will generate singlets that can be further characterized to identify how the candidate sequence differs from the reference. Using CARD, ca. 80% of the tRNAs from the bacterium Citrobacter koseri can be sequenced using ribonuclease T1 with Escherichia coli tRNAs as the reference. During these studies, we also discovered a sequence error for Escherichia coli tRNA-Thr1, and use this method to confirm the correct sequence for that tRNA.
本文描述了用于转移 RNA(tRNA)测序的核糖核酸消化物（CARD）比较分析方法。该方法通过两种 tRNA 群体的差异标记来实现。一组参考 tRNA 用（16）O 进行酶消化标记，其完整序列包括修饰序列已知。第二组（候选）tRNA 用（18）O 标记，其序列信息是所需的。通过将两个消化物组合在一起，在参考和候选物之间具有相同序列的消化产物将以相差 2 Da 的双峰形式出现。两者之间的序列或修饰差异将生成单峰，可进一步对其进行表征以确定候选序列与参考序列的差异。使用 CARD，可使用核糖核酸酶 T1 对来自柠檬酸杆菌的约 80%的 tRNA 进行测序，其中大肠杆菌 tRNA 用作参考。在这些研究中，我们还发现了大肠杆菌 tRNA-Thr1 的一个序列错误，并使用该方法确认了该 tRNA 的正确序列。
