Amino-Functionalized 5' Cap Analogs as Tools for Site-Specific Sequence-Independent Labeling of mRNA
| Autor: | Pawel J. Sikorski, Marcin Warminski, Zofia Warminska, Anna Kropiwnicka, Maciej Lukaszewicz, Edward Darzynkiewicz, Joanna Kowalska, Joanna Zuberek, Jacek Jemielity |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
RNA Caps Five-prime cap Transcription Genetic Biomedical Engineering Pharmaceutical Science Succinimides Bioengineering 010402 general chemistry 01 natural sciences 03 medical and health sciences Transcription (biology) Protein biosynthesis MRNA transport Animals Humans RNA Messenger RNA Processing Post-Transcriptional Pharmacology Regulation of gene expression Messenger RNA Cell-Free System Staining and Labeling Chemistry Organic Chemistry RNA Translation (biology) 0104 chemical sciences 030104 developmental biology Biochemistry Protein Biosynthesis Rabbits Biotechnology HeLa Cells |
| Zdroj: | Bioconjugate chemistry. 28(7) |
| ISSN: | 1520-4812 |
| Popis: | mRNA is a template for protein biosynthesis, and consequently mRNA transport, translation, and turnover are key elements in the overall regulation of gene expression. Along with growing interest in the mechanisms regulating mRNA decay and localization, there is an increasing need for tools enabling convenient fluorescent labeling or affinity tagging of mRNA. We report new mRNA 5' cap analog-based tools that enable site-specific labeling of RNA within the cap using N-hydroxysuccinimide (NHS) chemistry. We explored two complementary methods: a co-transcriptional labeling method, in which the label is first attached to a cap analog and then incorporated into RNA by in vitro transcription, and a post-transcriptional labeling method, in which an amino-functionalized cap analog is incorporated into RNA followed by chemical labeling of the resulting transcript. After testing the biochemical properties of RNAs carrying the novel modified cap structures, we demonstrated the utility of fluorescently labeled RNAs in decapping assays, RNA decay assays, and RNA visualization in cells. Finally, we also demonstrated that mRNAs labeled by the reported method are translationally active. We envisage that the novel analogs will provide an alternative to radiolabeling of mRNA caps for in vitro studies and open possibilities for new applications related to the study of mRNA fates in vivo. |
| Databáze: | OpenAIRE |
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