Isolation and characterization of thermodynamically stable and unstable RNA hairpins from a triloop combinatorial library

Hairpins are the most common elements of RNA secondary structure, playing i mportant roles in RNA tertiary architecture and forming protein binding sit es. Triloops are common in a variety of naturally occurring RNA hairpins, b ut little is known about their thermodynamic stability. Reported here are t he sequences and thermodynamic parameters for a variety of stable and unsta ble triloop hairpins. Temperature gradient gel electrophoresis (TGGE) can b e used to separate a simple RNA combinatorial library based on thermal stab ility [Bevilacqua, J. M., and Bevilacqua, P. C, (1998) Biochemistry 45, 158 77-15884]. Here we introduce the application of TGGE to separating and anal yzing a complex RNA combinatorial library based on thermal stability, using an RNA triloop library. Several rounds of in vitro selection of an RNA tri loop library were carried out using TGGE, and preferences for exceptionally stable and unstable closing base pairs and loop sequences were identified. For stable hairpins, the most common closing base pair is CG, and U-rich l oop sequences are preferred. Closing base pairs of GC and UA result in mode rately stable hairpins when combined with a stable loop sequence. For unsta ble hairpins, the most common closing base pairs are AU and UG, and U-rich loop sequences are no longer preferred. In general, the contributions of th e closing base pair and loop sequence to overall hairpin stability appear t o be additive. Thermodynamic parameters for individual hairpins determined by UV melting are generally consistent with outcomes from selection experim ents, with hairpins containing a CG closing base pair having a Delta Delta G degrees(37) 2.1-2.5 kcal/mol more favorable than hairpins with other clos ing base pairs. Sequences and thermodynamic rules for triloop hairpins shou ld aid in RNA structure prediction and determination of whether naturally o ccurring triloop hairpins are thermodynamically stable.