Laboratory of Rna Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
Faculty of Biology, Institute of Genetics and Biotechnology, University of Warsaw, Warsaw, Poland.
RNA Biol. 2021 Nov 12;18(sup2):623-639. doi: 10.1080/15476286.2021.1999104.
Despite the development of non-radioactive DNA/RNA labelling methods, radiolabelled nucleic acids are commonly used in studies focused on the determination of RNA fate. Nucleic acid fragments with radioactive nucleotide analoguesincorporated into the body or at the 5' or 3' terminus of the molecule can serve as probes in hybridization-based analyses of degradation and processing of transcripts. Radiolabelled oligoribonucleotides are utilized as substrates in biochemical assays of various RNA metabolic enzymes, such as exo- and endoribonucleases, nucleotidyltransferases or helicases. In some applications, the placement of the label is not a concern, while in other cases it is required that the radioactive mark is located at the 5'- or 3'-end of the molecule. An unsurpassed method for 5'-end RNA labelling employs T4 polynucleotide kinase (PNK) and [γ-P]ATP. In the case of 3'-end labelling, several different possibilities exist. However, they require the use of costly radionucleotide analogues. Previously, we characterized an untypical nucleotidyltransferase named CutA, which preferentially incorporates cytidines at the 3'-end of RNA substrates. Here, we demonstrate that this unusual feature can be used for the development of a novel, efficient, reproducible and economical method of RNA 3'-end labelling by CutA-mediated cytidine tailing. The labelling efficiency is comparable to that achieved with the most common method applied to date, . [5'-P]pCp ligation to the RNA 3'-terminus catalysed by T4 RNA ligase I. We show the utility of RNA substrates labelled using our new method in exemplary biochemical assays assessing directionality of two well-known eukaryotic exoribonucleases, namely Dis3 and Xrn1.
尽管已经开发出了非放射性 DNA/RNA 标记方法，但放射性标记的核酸仍常用于研究 RNA 命运的实验中。将放射性核苷酸类似物掺入到核酸片段中，或者将其置于分子的 5' 或 3' 末端，可作为杂交分析中研究转录物降解和加工的探针。放射性标记的寡核糖核苷酸可用作各种 RNA 代谢酶（如外切和内切核酸酶、核苷酸转移酶或解旋酶）的生化测定中的底物。在某些应用中，标记的位置不是问题，而在其他情况下，需要将放射性标记位于分子的 5' 或 3' 末端。5' 端 RNA 标记的一种无与伦比的方法是使用 T4 多核苷酸激酶（PNK）和 [γ-P]ATP。对于 3' 端标记，存在几种不同的可能性。然而，它们需要使用昂贵的放射性核苷酸类似物。以前，我们鉴定了一种名为 CutA 的非典型核苷酸转移酶，它优先在 RNA 底物的 3' 端掺入胞嘧啶。在这里，我们证明这个不寻常的特性可用于开发一种新的、高效的、可重复的和经济的通过 CutA 介导的胞嘧啶加尾进行 RNA 3' 端标记的方法。标记效率可与迄今为止最常用的方法相媲美，即 T4 RNA 连接酶 I 催化的 5'-P]pCp 连接到 RNA 3' 末端。我们展示了使用我们的新方法标记的 RNA 底物在评估两种著名的真核外切核酸酶 Dis3 和 Xrn1 的方向性的典型生化测定中的实用性。
