SUNY at Albany
June 19-23, 2001
Structure and Thermodynamics of Hairpins containing 2Õ,5Õ-Linked RNA Loops
Hairpin loops represent the dominant secondary structure in RNA, occurring frequently in mRNA, rRNA and in DNA cruciforms. The tetraloop sequences r(UNCG) and r(GNRA) are characterized by exceptionally high thermodynamic stability and can serve as termination sites for transcriptional processes, or as means of stabilizing mRNA in vivo. In the present study, we report on the thermodynamic and structural properties of RNA hairpins constructed with 2Õ,5Õ-linked RNA tetraloops, and containing the following stem combinations: RNA:RNA, DNA:DNA, DNA:RNA, 2',5'-RNA:RNA, 2',5'-RNA:DNA and 2',5'-RNA:2',5'-RNA. The sequence under study was the dodecanucleotide 5Õ-GGAC(UUCG)GUCC-3Õ.
Thermodynamic parameters were calculated from plots of lnK versus 1/T assuming a two-state (all-or-none) model using UV-Spectroscopy. Melting temperatures (TmÕs) were calculated according to the base line method and values determined from plots of fraction of single strands in the hairpin state versus temperature. The hairpin global conformational structures were probed by Circular Dichroism Spectroscopy.
We show that a 2Õ,5Õ-linked RNA (UUCG) tetraloop is as stable as the wild-type RNA tetraloop. Surprisingly, in hairpins with the stem compositions DNA:DNA, DNA:2Õ,5Õ-RNA, 2Õ,5Õ-RNA:2Õ,5Õ-RNA, the stability imparted by 2,Õ5Õ-r(UUCG) loop is higher than that of the 3,Õ5Õ-r(UUCG) counterpart which exerts the same order of stability only in hairpins with RNA:RNA and RNA:DNA stems. Our data suggest that substitution of 2Õ,5Õ-RNA for 3Õ,5Õ-RNA in the loop does not have a significant effect on the entropy change associated with hairpin formation. Rather, it appears that the stabilization brought about by 2Õ,5Õ-RNA tetraloops is enthalpic in origin. Furthermore, our studies show that the stability imparted by 2Õ,5'-RNA loops is dependent on loop sequence, a property that is shared with the regioisomeric 3Õ,5Õ-RNA loops. Hairpins containing 2Õ,5Õ-RNA have helical structures that fall within the A/B-type conformational families. Incorporation of 2Õ,5Õ-RNA in the loop increases notably the depth of the CD peak at 212 nm relative to the loop with normal 3',5'-RNA residues. Finally, we have demonstrated the effectiveness of 2Õ,5Õ-RNA loops in stabilizing hairpin structures against degradation by nucleases.
This work was supported by NSERC Canada.
Rami N. Hannoush and Masad J. Damha
Department of Chemistry, McGill University, Montreal, QC, CANADA H3A2K6