Is a Closing “GA Pair” a Rule for Stable Loop-Loop RNA Complexes?

Frédéric Ducongé,Carmelo Di Primo,Jean-Jacques Toulmé

doi:10.1074/jbc.m002694200

Abstract

RNA hairpin aptamers specific for the trans-activation-responsive (TAR) RNA element of human immunodeficiency virus type 1 were identified by in vitro selection (Ducongé, F., and Toulmé, J. J. (1999) RNA 5, 1605-1614). The high affinity sequences selected at physiological magnesium concentration (3 mm) were shown to form a loop-loop complex with the targeted TAR RNA. The stability of this complex depends on the aptamer loop closing "GA pair" as characterized by preliminary electrophoretic mobility shift assays. Thermal denaturation monitored by UV-absorption spectroscopy and binding kinetics determined by surface plasmon resonance show that the GA pair is crucial for the formation of the TAR-RNA aptamer complex. Both thermal denaturation and surface plasmon resonance experiments show that any other "pairs" leads to complexes whose stability decreases in the order AG > GG > GU > AA > GC > UA >> CA, CU. The binding kinetics indicate that stability is controlled by the off-rate rather than by the on-rate. Comparison with the complex formed with the TAR* hairpin, a rationally designed TAR RNA ligand (Chang, K. Y., and Tinoco, I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8705-8709), demonstrates that the GA pair is a key determinant which accounts for the 50-fold increased stability of the TAR-aptamer complex (K(d) = 2.0 nm) over the TAR-TAR* one (K(d) = 92. 5 nm) at physiological concentration of magnesium. Replacement of the wild-type GC pair next to the loop of RNA I' by a GA pair stabilizes the RNA I'-RNA II' loop-loop complex derived from the one involved in the control of the ColE1 plasmid replication. Thus, the GA pair might be the preferred one for stable loop-loop interactions.

Highlights

Intermolecular interactions between structured RNA play key roles in the regulation of gene expression
Thermal denaturation monitored by UV-absorption spectroscopy and binding kinetics determined by surface plasmon resonance show that the GA pair is crucial for the formation of the TAR-RNA aptamer complex
The contribution of the GA pair to the thermodynamics and the kinetics of the loop-loop interaction was investigated by using UV-absorption spectroscopy and surface plasmon resonance

Summary

Introduction

Intermolecular interactions between structured RNA play key roles in the regulation of gene expression. Non-Watson-Crick interactions in RNA molecules have been reported in the viral RNA element bound by the Rev protein of HIV-1 [9], in GRNA tetraloops [10, 11], tRNAs [12,13,14], and tandem mismatches within duplexes [15,16,17]. All these results indicate that interactions other than canonical base pairs contribute to the structural diversity displayed by RNAs, which, as a matter of fact, is crucial for activity. Several mutations of the GA pair that closes the loop of the identified RNA aptamers decrease the stability of the TAR-aptamer complex, as shown by preliminary electrophoretic mobility shift assays (EMSA)

Methods

Results

Conclusion