Real time kinetic studies of the interaction between folded antisense and target RNAs using surface plasmon resonance

Antisense RNAs interact with their complementary target RNAs as folded stru ctures. 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 (antise nse/target RNA pair of plasmid R1), recent studies have identified a four-w ay junction structure as the major binding intermediate. Previously, the ki netics 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 develope d that permitted the determination of association and dissociation rate con stants 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 tempera ture dependence for dissociation rate constants. This may be indicative of the complexity of the steps required for interacting folded RNAs; intramole cular structure competes with intermolecular helix progression during compl ex formation. The association rate constants were not significantly depende nt on temperature. The analysis presented shows that the stability of a kis sing complex is not the primary determinant of the rate of stable CopA/CopT complex formation. (C) 2001 Academic Press.