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Nucleic Acid Crystallography pp 11–20Cite as

Preparation of Short 5′-Triphosphorylated Oligoribonucleotides for Crystallographic and Biochemical Studies

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Part of the Methods in Molecular Biology book series (MIMB,volume 1320)

Abstract

RNA molecules participate in virtually all cellular processes ranging from transfer of hereditary information to gene expression control. In cells, many RNAs form specific interactions with proteins often using short nucleotide sequences for protein recognition. Biochemical and structural studies of such RNA–protein complexes demand preparation of short RNAs. Although short RNAs can be synthesized chemically, certain proteins require monophosphate or triphosphate moieties on the 5′ end of RNA. Given high cost of chemical triphosphorylation, broad application of such RNAs is impractical. In vitro transcription of RNA by DNA-dependent bacteriophage T7 RNA polymerase provides an alternative option to prepare short RNAs with different phosphorylation states as well as modifications on the 5′ terminus. Here we outline the in vitro transcription methodology employed to prepare ≤5-mer oligoribonucleotide for structural and biochemical applications. The chapter describes the principles of construct design, in vitro transcription and RNA purification applied for characterization of a protein that targets the 5′ end of RNA.

Key words

  • Triphosphorylated RNA
  • 5′ Modification
  • Crystallization
  • Ion-exchange chromato-graphy
  • In vitro transcription

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  • DOI: 10.1007/978-1-4939-2763-0_2
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Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, 10016, USA

    Nikita Vasilyev

  2. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, Medical Science Building, Room 393, 550 First Avenue, New York, NY, 10016, USA

    Alexander Serganov

Authors
  1. Nikita Vasilyev
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  2. Alexander Serganov
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Corresponding author

Correspondence to Alexander Serganov .

Editor information

Editors and Affiliations

  1. CNRS Inst de Biol Moléculaire et Cellulaire, Strasbourg, France

    Eric Ennifar

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Vasilyev, N., Serganov, A. (2016). Preparation of Short 5′-Triphosphorylated Oligoribonucleotides for Crystallographic and Biochemical Studies. In: Ennifar, E. (eds) Nucleic Acid Crystallography. Methods in Molecular Biology, vol 1320. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2763-0_2

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  • DOI: https://doi.org/10.1007/978-1-4939-2763-0_2

  • Publisher Name: Humana Press, New York, NY

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