2‘-Mercaptonucleotide Interference Reveals Regions of Close Packing within Folded RNA Molecules

Abstract

The 2'-hydroxyl group makes essential contributions to RNA structure and function. As an approach to assess the ability of a mercapto group to serve as a functional analogue for the 2'-hydroxyl group, we synthesized 2'-mercaptonucleotides for use in nucleotide analogue interference mapping. To correlate the observed interference effects with tertiary structure, we used the independently folding DeltaC209 P4-P6 domain from the Tetrahymena group I intron. We generated populations of DeltaC209 P4-P6 molecules containing 2'-mercaptonucleotides located randomly throughout the domain and separated the folded molecules from the unfolded molecules by nondenaturing gel electrophoresis. Iodine-induced cleavage of the RNA molecules revealed the sites at which 2'-mercaptonucleotides interfere with folding. These interferences cluster in the most densely packed regions of the tertiary structure, occurring only at sites that lack the space and flexibility to accommodate a sulfur atom. Interference mapping with 2'-mercaptonucleotides therefore provides a method by which to identify structurally rigid and densely packed regions within folded RNA molecules.

2′-Fluoro Substituents Can Mimic Native 2′-Hydroxyls within Structured RNA

“…Previous work has demonstrated that 15 sites of 2′-deoxynucleotide interference within this domain identify residues bearing important 2′-OH groups, and that these 15 sites coincide with all of the crystallographically inferred 2′-OH interactions (Schwans et al, 2003; Schwans et al, 2004). An additional site, A151, shows weak 2′-H interference, although structural models provide no evidence for a hydrogen bond.…”

“…Crystallographic analyses have defined the P4-P6 structure at atomic resolution (Cate et al, 1996; Juneau et al, 2001; Ye et al, 2008), and biochemical analyses have defined its folding thermodynamics and kinetics (Battle and Doudna, 2002; Matsumura et al, 2003; Sattin et al, 2008; Schlatterer et al, 2008; Takamoto et al, 2004; Young and Silverman, 2002). Changes in global compaction of the domain upon folding allow separation of folded and unfolded molecules by non-denaturing gel electrophoresis (Murphy et al, 1994), which provides a convenient assay for folding interference (Basu and Strobel, 1999; Schwans et al, 2003). …”

“…We determined the sites of 2′-fluoro/2′-deoxynucleotide interference using non-denaturing gel electrophoresis to separate folded from unfolded molecules as previously described (Basu and Strobel, 1999; Schwans et al, 2003). To achieve maximal sensitivity to folding interference without compromising signal we conducted the folding reactions and non-denaturing gel electrophoresis at the [Mg 2+ ] ½ for folding of ΔC209 P4-P6 (0.45 mM MgCl 2 ).…”

“…Full details are given in the Supplemental Information. 2′-Fluorocytidine and 2′-fluorouridine were purchased from ChemGenes and 2′-fluoroguanosine was synthesized as described by Kawaskai et al (Kawasaki et al, 1993) The nucleosides were converted to the α-thiotriphosphates and purified as previously described for 2′-mercaptonucleoside-α-thiotriphosphates (Schwans et al, 2003). Interference mapping experiments were conducted as previously described by Schwans et al and references therein (Schwans et al, 2003).…”

A Packing‐Density Metric for Exploring the Interior of Folded RNA Molecules

“…We synthesized 2′‐chloro, 2′‐methyl, and 2′‐sulfanylnucleoside analogues of adenosine, cytidine, guanosine, and uridine and converted these analogues into the corresponding α‐thiotriphosphates. We have reported the synthesis of 2′‐sulfanylnucleotide analogues previously 6. The 2′‐deoxy‐2′‐methylnucleoside analogues were synthesized by glycosylation of methyl 2‐α‐acetoxymethyl‐3,5‐di‐ O ‐(4‐chlorobenzyl)‐2‐deoxy‐α‐ D ‐ribofuranoside, followed by deoxygenation and deprotection 7.…”