Sharma Ashwani K, Plant Joshua J, Rangel Alexandra E, Meek Kirsten N, Anamisis April J, Hollien Julie, Heemstra Jennifer M
Department of Chemistry and the Center for Cell and Genome Science and ‡Department of Biology and the Center for Cell and Genome Science, University of Utah , Salt Lake City, Utah 84112, United States.
ACS Chem Biol. 2014 Aug 15;9(8):1680-4. doi: 10.1021/cb5002119. Epub 2014 Jun 11.
The ability to fluorescently label specific RNA sequences is of significant utility for both in vitro and live cell applications. Currently, most RNA labeling methods utilize RNA-nucleic acid or RNA-protein molecular recognition. However, in the search for improved RNA labeling methods, harnessing the small-molecule recognition capabilities of RNA is rapidly emerging as a promising alternative. Along these lines, we propose a novel strategy in which a ribozyme acts to promote self-alkylation with a fluorophore, providing a robust, covalent linkage between the RNA and the fluorophore. Here we describe the selection and characterization of ribozymes that promote self-labeling with fluorescein iodoacetamide (FIA). Kinetic studies reveal a second-order rate constant that is on par with those of other reactions used for biomolecular labeling. Additionally, we demonstrate that labeling is specific to the ribozyme sequences, as FIA does not react nonspecifically with RNA.
对特定RNA序列进行荧光标记的能力在体外和活细胞应用中都具有重要用途。目前，大多数RNA标记方法利用RNA-核酸或RNA-蛋白质分子识别。然而，在寻找改进的RNA标记方法时，利用RNA的小分子识别能力正迅速成为一种有前途的替代方法。沿着这些思路，我们提出了一种新策略，其中核酶作用于促进与荧光团的自烷基化，在RNA和荧光团之间提供牢固的共价连接。在这里，我们描述了促进与荧光素碘乙酰胺（FIA）自标记的核酶的筛选和表征。动力学研究揭示了一个二级速率常数，该常数与用于生物分子标记的其他反应的速率常数相当。此外，我们证明标记对核酶序列具有特异性，因为FIA不会与RNA发生非特异性反应。
