Thermodynamic investigation of kissing-loop interactions

Abstract

Kissing loop interactions (KLIs) are a common motif that is critical in retroviral dimerization, viroid replication, mRNA, and riboswitches. In addition, KLIs are currently used in a variety of biotechnology applications, such as in aptamer sensors, RNA scaffolds and to stabilize vaccines for therapeutics. Here we describe the thermodynamics of a basic intramolecular DNA capable of engaging in a KLI, consisting of two hairpins connected by a flexible linker. Each hairpin loop has a five-nucleotide complementary sequence theoretically capable of engaging in a KLI. On either side of each loop is two thymines which will not engage in kissing but are present to provide more flexibility and optimal KLI positioning. Our results suggest that the KLI occurs even at physiological salt levels, and that the KLI does not alter the thermodynamics and stability of the two stem structures. The KLI does not involve all five nucleotides, or at least each base-pair stack is not making full contact. Adding a second strand complementary to the bottom of the kissing complex removes flexibility and causes destabilization of the stems. The KLI of this less flexible complex is maintained but the T_M is reduced, indicating an entopic penalty to its formation.

Keywords: Bimolecular interactions; Hairpins; Kissing-loop interactions; Sterics; Thermodynamics.