R-loop stability as a function of RNA structure and size.
The sequence-specific formation of R-loops can be assayed using RNAs which overlap a HindIII cleavage site in a 3.5 kb plasmid. Chemical modification of the displaced DNA strand has permitted stabilization of these R-loops and allowed a systematic investigation of the dependence of these triple-stranded structures on the chain length and structure of the input RNA. RNAs as short as 50 nt form stable R-loops if 5-allylamine uridines (Uaa-RNA) are used in place of normal uridines; normal RNAs must be 100 nt long to form R-loops quantitatively. Since acetic anhydride decreases the hybridization efficiency of Uaa-RNAs, the positive charge of the RNAs must diminish the electrostatic repulsion of the three negatively charged phosphodiester backbones. The dependence of R-loop stability on the length of RNA can be stimulated with a random walk model, which also applies to strand migration within Holiday junctions. R-loop hybridization provides a versatile method to generate single-stranded DNA in a sequence-selective manner.
Landgraf
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Department of Biological Chemistry, School of Medicine, University of California-Los Angeles 90024-1570, USA.
Chen
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CH
Sigman
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D004247:DNA / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry; D006037:Glyoxal / Q000737:chemistry; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry; D012313:RNA / Q000737:chemistry*; D015183:Restriction Mapping; D013329:Structure-Activity Relationship
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D006037:Glyoxal; D012313:RNA; D004247:DNA