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  3. Thermodynamic insights into 2-thiouridine-enhanced RNA hybridization

Thermodynamic insights into 2-thiouridine-enhanced RNA hybridization

  • Nucleic Acids Res. 2015 Sep 18;43(16):7675-87. doi: 10.1093/nar/gkv761.
Aaron T Larsen 1,  Albert C Fahrenbach 2,  Jia Sheng 3,  Julia Pian 1,  Jack W Szostak 4
Affiliations

Affiliations

  • 1 Howard Hughes Medical Institute, Center for Computational and Integrative Biology, and Department of Molecular Biology, Simches Research Center, Massachusetts General Hospital, Boston, MA 02114, USA.
  • 2 Howard Hughes Medical Institute, Center for Computational and Integrative Biology, and Department of Molecular Biology, Simches Research Center, Massachusetts General Hospital, Boston, MA 02114, USA Earth-Life Science Institute, Tokyo Institute of Technology, 2-12-1-IE-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan.
  • 3 University at Albany, State University of New York, Department of Chemistry, The RNA Institute, 1400 Washington Avenue, Albany, NY 12222, USA.
  • 4 Howard Hughes Medical Institute, Center for Computational and Integrative Biology, and Department of Molecular Biology, Simches Research Center, Massachusetts General Hospital, Boston, MA 02114, USA Earth-Life Science Institute, Tokyo Institute of Technology, 2-12-1-IE-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan szostak@molbio.mgh.harvard.edu.
PMID: 26240387 DOI: 10.1093/nar/gkv761
Abstract

Nucleobase modifications dramatically alter nucleic acid structure and thermodynamics. 2-thiouridine (s(2)U) is a modified nucleobase found in tRNAs and known to stabilize U:A base pairs and destabilize U:G wobble pairs. The recently reported crystal structures of s(2)U-containing RNA duplexes do not entirely explain the mechanisms responsible for the stabilizing effect of s(2)U or whether this effect is entropic or enthalpic in origin. We present here thermodynamic evaluations of duplex formation using ITC and UV thermal denaturation with RNA duplexes containing internal s(2)U:A and s(2)U:U pairs and their native counterparts. These results indicate that s(2)U stabilizes both duplexes. The stabilizing effect is entropic in origin and likely results from the s(2)U-induced preorganization of the single-stranded RNA prior to hybridization. The same preorganizing effect is likely responsible for structurally resolving the s(2)U:U pair-containing duplex into a single conformation with a well-defined H-bond geometry. We also evaluate the effect of s(2)U on single strand conformation using UV- and CD-monitored thermal denaturation and on nucleoside conformation using (1)H NMR spectroscopy, MD and umbrella sampling. These results provide insights into the effects that nucleobase modification has on RNA structure and thermodynamics and inform efforts toward improving both ribozyme-catalyzed and nonenzymatic RNA copying.

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