Publikationsdatum:
2015-09-19
Beschreibung:
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.
Print ISSN:
0305-1048
Digitale ISSN:
1362-4962
Thema:
Biologie
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