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Real time kinetic studies of the interaction between folded antisense and target RNAs using surface plasmon resonance.
- Source :
-
Journal of molecular biology [J Mol Biol] 2001 Jul 27; Vol. 310 (5), pp. 1125-34. - Publication Year :
- 2001
-
Abstract
- Antisense RNAs interact with their complementary target RNAs as folded structures. The formation of early binding intermediates is the most important step in determining the overall rates of stable complex formation in vitro and the efficiency of control in vivo. In the case of CopA and CopT (antisense/target RNA pair of plasmid R1), recent studies have identified a four-way junction structure as the major binding intermediate. Previously, the kinetics of antisense/target RNA interaction was studied by indirect methods. Here we have used surface plasmon resonance to follow the binding of CopI (a truncated variant of CopA) to CopT in real time. A protocol was developed that permitted the determination of association and dissociation rate constants for wild-type and mutant CopI-CopT pairs. The K(D)-values calculated from these rate constants were in good agreement with the results obtained by indirect methods. In comparison to earlier model studies of interactions between simple complementary nucleic acids, we observe a different temperature dependence for dissociation rate constants. This may be indicative of the complexity of the steps required for interacting folded RNAs; intramolecular structure competes with intermolecular helix progression during complex formation. The association rate constants were not significantly dependent on temperature. The analysis presented shows that the stability of a kissing complex is not the primary determinant of the rate of stable CopA/CopT complex formation.
- Subjects :
- Base Pairing
Base Sequence
Kinetics
Molecular Sequence Data
Mutation genetics
Oligoribonucleotides chemistry
Oligoribonucleotides genetics
Oligoribonucleotides metabolism
Plasmids genetics
RNA Probes chemistry
RNA Probes genetics
RNA Probes metabolism
RNA, Antisense genetics
RNA, Bacterial genetics
Substrate Specificity
Temperature
Thermodynamics
Nucleic Acid Conformation
RNA, Antisense chemistry
RNA, Antisense metabolism
RNA, Bacterial chemistry
RNA, Bacterial metabolism
Surface Plasmon Resonance
Subjects
Details
- Language :
- English
- ISSN :
- 0022-2836
- Volume :
- 310
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Journal of molecular biology
- Publication Type :
- Academic Journal
- Accession number :
- 11502000
- Full Text :
- https://doi.org/10.1006/jmbi.2001.4802