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Beren C, Liu KN, Dreesens LL, et al. Enzymatic Synthesis and Fractionation of Fluorescent PolyU RNAs. Bio Protoc. 2018;8(17):e2988doi: 10.21769/BioProtoc.2988.
Beren, C., Liu, K. N., Dreesens, L. L., Knobler, C. M., & Gelbart, W. M. (2018). Enzymatic Synthesis and Fractionation of Fluorescent PolyU RNAs. Bio-protocol, 8(17), e2988. https://doi.org/10.21769/BioProtoc.2988
Beren, Christian, et al. "Enzymatic Synthesis and Fractionation of Fluorescent PolyU RNAs." Bio-protocol vol. 8,17 (2018): e2988. doi: https://doi.org/10.21769/BioProtoc.2988
Beren C, Liu KN, Dreesens LL, Knobler CM, Gelbart WM. Enzymatic Synthesis and Fractionation of Fluorescent PolyU RNAs. Bio Protoc. 2018 Sep 05;8(17):e2988. doi: 10.21769/BioProtoc.2988. eCollection 2018 Sep 05. PMID: 34395788; PMCID: PMC8328661.
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Bio Protoc. 2018 Sep 05;8(17):e2988. doi: 10.21769/BioProtoc.2988. eCollection 2018 Sep 05.

Enzymatic Synthesis and Fractionation of Fluorescent PolyU RNAs.

Bio-protocol

Christian Beren, Katherine N Liu, Lisa L Dreesens, Charles M Knobler, William M Gelbart

Affiliations

  1. Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA, USA.

PMID: 34395788 PMCID: PMC8328661 DOI: 10.21769/BioProtoc.2988

Abstract

The physical properties of viral-length polyuridine (PolyU) RNAs, which cannot base-pair and form secondary structures, are compared with those of normal-composition RNAs, composed of comparable numbers of each of A, U, G and C nucleobases. In this protocol, we describe how to synthesize fluorescent polyU RNAs using the enzyme polynucleotide phosphorylase (PNPase) from Uridine diphosphate (UDP) monomers and how to fractionate the polydisperse synthesis mixture using gel electrophoresis, and, after electroelution, how to quantify the amount of polyU recovered with UV-Vis spectrophotometry. Dynamic light scattering was used to determine the hydrodynamic radii of normal-composition RNAs as compared to polyU. It showed that long polyU RNAs behave like linear polymers for which the radii scale with chain length as N

Copyright © 2018 The Authors; exclusive licensee Bio-protocol LLC.

Keywords: Dynamic light scattering; Gel electrophoresis electroelution; Homopolymer polyU RNA; RNA secondary structure; Viral RNA

Conflict of interest statement

Competing interestsThe authors declare no conflicts of interest.

References

  1. Proc Natl Acad Sci U S A. 2008 Oct 21;105(42):16153-8 - PubMed
  2. Small. 2011 Aug 8;7(15):2217-24 - PubMed
  3. RNA. 2012 Feb;18(2):284-99 - PubMed
  4. Proc Natl Acad Sci U S A. 1962 Dec;48(12):2171-8 - PubMed
  5. Biochim Biophys Acta. 1956 Apr;20(1):269-85 - PubMed
  6. J Mol Biol. 1970 Jun 14;50(2):373-89 - PubMed
  7. J Chem Phys. 2011 Oct 21;135(15):155105 - PubMed
  8. RNA. 2015 May;21(5):877-86 - PubMed
  9. Biochimie. 2007 Jun-Jul;89(6-7):779-88 - PubMed
  10. RNA. 2003 Oct;9(10):1174-9 - PubMed
  11. Biophys J. 2017 Jul 25;113(2):339-347 - PubMed

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