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Originally published In Press as doi:10.1074/jbc.M002694200 on May 4, 2000

J. Biol. Chem., Vol. 275, Issue 28, 21287-21294, July 14, 2000
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Is a Closing "GA Pair" a Rule for Stable Loop-Loop RNA Complexes?*

Frédéric Ducongé, Carmelo Di Primo, and Jean-Jacques ToulméDagger

From INSERM U386, Institut Fédératif de Recherche Pathologies Infectieuses, Université Victor Segalen, 146 rue Léo Saignat, 33076 Bordeaux cedex, France

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 (Kd = 2.0 nM) over the TAR-TAR* one (Kd = 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.

* This work was supported by the "Agence Nationale de Recherche contre le SIDA," by the "Fondation pour la Recherche Médicale," and by the " Conseil Régional d'Aquitaine."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: INSERM U386, IFR Pathologies Infectieuses, Université Victor Segalen, 146 rue Léo Saignat, 33076 Bordeaux cedex, France. Tel.: 33-5-57-57-10-14; Fax: 33-5-57-57-10-15; E-mail: jean-jacques.toulme@bordeaux.inserm.fr.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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