Rapid enzymatic synthesis of long RNA polymers: A simple protocol to generate RNA libraries with random sequences
| Autor: | Marc Delarue, Irina Randrianjatovo-Gbalou |
|---|---|
| Přispěvatelé: | Dynamique structurale des Macromolécules / Structural Dynamics of Macromolecules, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was funded by Action Incitative Concertée of Institut Pasteur granted to M.D. The authors declare that they have no conflict of interest. A provisional patent application no. 62/560693, titled DNA POLYMERASE THETA MUTANTS, THE METHODS OF PRODUCING THESE MUTANTS, AND THEIRS USES, has been filed and deposited to the UPSTO on 20th of September 2017., We would like to thank Sandrine Rosario, Institut Pasteur, for her help with the modified nucleotides incorporation experiments (Fig. 5, Fig. 6), and Dr. Michel Ryckelynck from IBMC of CNRS and Strasbourg University for his advices in the SELEX library preparation., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Polymers
Aptamer Computational biology Protein Structure Secondary General Biochemistry Genetics and Molecular Biology Functional RNA synthesis 03 medical and health sciences Randomized library DNA polymerase theta [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Molecular Biology Polymerase Gene Library 030304 developmental biology 0303 health sciences biology Sequence Analysis RNA Chemistry 030302 biochemistry & molecular biology Ribozyme RNA C2′-modified oligonucleotides Polynucleotide Nucleic acid biology.protein 2′-O-methyl library Nucleotidyltransferase activity Cell-Free Nucleic Acids Systematic evolution of ligands by exponential enrichment |
| Zdroj: | Methods Methods, Elsevier, 2019, 161, pp.83-90. ⟨10.1016/j.ymeth.2019.03.025⟩ Methods, 2019, 161, pp.83-90. ⟨10.1016/j.ymeth.2019.03.025⟩ |
| ISSN: | 1046-2023 1095-9130 |
| DOI: | 10.1016/j.ymeth.2019.03.025⟩ |
| Popis: | International audience; RNA aptamers have several advantages over DNA aptamers due to their propensity to fold into three-dimensional structures. However, the synthesis of large RNA libraries remains a challenge as it requires more precautions to conserve their functional integrity, especially when such libraries are intended for aptamers or ribozymes selection. Here, we present an enzymatic method that enables the rapid synthesis of RNA polymers thanks to the efficient incorporation of ribonucleotides (NTPs) as well as chemically modified ribonucleotides by human DNA polymerase Theta (θ) mutants. These mutants have the ability to generate long single-stranded RNA polynucleotides of random sequences due to their improved template-free terminal nucleotidyltransferase activity. Here we describe the detailed protocols to produce large and diverse libraries of RNA, to make them ready to use in repeated cycles of Systematic Evolution of Ligands by Exponential enrichment (SELEX) and to synthesize C2'-modified nucleic acids with higher nuclease resistance. |
| Databáze: | OpenAIRE |
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