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Methods. 2019 Mar 26. pii: S1046-2023(18)30279-2. doi: 10.1016/j.ymeth.2019.03.025. [Epub ahead of print]

Rapid enzymatic synthesis of long RNA polymers: A simple protocol to generate RNA libraries with random sequences.

Author information

1
Unit of Structural Dynamics of Biological Macromolecules and CNRS UMR 3528, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France. Electronic address: irina.gbalou@gmail.com.
2
Unit of Structural Dynamics of Biological Macromolecules and CNRS UMR 3528, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.

Abstract

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.

KEYWORDS:

2′-O-methyl library; C2′-modified oligonucleotides; DNA polymerase theta; Functional RNA synthesis; RNA; Randomized library

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