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A quantitative study of the flexibility contributed to RNA structures by nicks and single-stranded gaps
1Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
RNA (New York, N.Y.)
|October 13, 1998
Summary
Disulfide crosslinking quantifies RNA flexibility. Nicks in RNA structures cause rigidity, while single-stranded gaps of one or more nucleotides allow significant flexibility in two-helix junctions.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- RNA structures feature two-helix junctions critical for function.
- Understanding the flexibility of these junctions is essential for RNA-based therapeutics and molecular mechanisms.
Purpose of the Study:
- To quantitatively assess the relative flexibility imparted by nicks versus single-stranded gaps in RNA structures.
- To investigate the impact of gap size on the flexibility of two-helix junctions.
Main Methods:
- Utilized disulfide crosslinking via thiol-disulfide interchange to probe RNA flexibility.
- Constructed a three-stranded RNA duplex model system with varying lengths of the third strand to create nicks or single-stranded gaps (1-3 nucleotides).
- Measured crosslinking rates to infer the relative flexibility of the resulting two-helix junctions.
Main Results:
- Crosslinking in a nicked duplex was two orders of magnitude slower than in a duplex with a 3-nucleotide gap, indicating significant rigidity.
- Crosslinking rates in duplexes with 2- and 3-nucleotide gaps showed minimal sequence dependence.
- Single-stranded nucleotides at a two-helix junction significantly increased flexibility compared to a nick.
Conclusions:
- Two-helix junctions with a nick are substantially rigid.
- The presence of one or more single-stranded nucleotides at a two-helix junction confers significant flexibility.
- Findings are relevant to understanding the dynamics of natural RNAs, including ribozymes.