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Real time kinetic studies of the interaction between folded antisense and target RNAs using surface plasmon resonance
S Nordgren1, J G Slagter-Jäger, G H Wagner
1Institute of Cell and Molecular Biology, Uppsala University, Sweden.
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.
Insights
Antisense RNA binding kinetics were measured in real-time using surface plasmon resonance. The study reveals that intramolecular structure, not kissing complex stability, dictates the rate of stable CopA/CopT complex formation.
Area of Science:
- Molecular Biology
- Biophysics
Background:
- Antisense RNAs regulate gene expression by binding to complementary target RNAs.
- Formation of early binding intermediates is crucial for stable complex formation and regulatory efficiency.
- The CopA/CopT RNA pair from plasmid R1 utilizes a four-way junction as a key binding intermediate.
Purpose of the Study:
- To investigate the real-time kinetics of antisense-target RNA interaction.
- To determine association and dissociation rate constants for the CopI-CopT RNA pair.
- To elucidate the factors influencing the rate of stable complex formation between folded RNAs.
Main Methods:
- Surface plasmon resonance (SPR) was employed for real-time kinetic analysis.
- A novel SPR protocol was developed to measure rate constants.
- Wild-type and mutant CopI-CopT pairs were analyzed.
Main Results:
- Association and dissociation rate constants were determined for CopI-CopT interactions.
- Calculated dissociation constants (K(D)) aligned with previous indirect measurements.
- Dissociation rate constants exhibited an unusual temperature dependence, suggesting competition between intramolecular structures and helix progression.
Conclusions:
- The study provides the first real-time kinetic data for the CopA/CopT system.
- Complex folding and intramolecular structures significantly influence RNA-RNA interaction kinetics.
- Kissing complex stability is not the main driver for the rate of stable CopA/CopT complex formation.