Thermodynamic studies of the sequence dependence of stability for interfaces of coaxially stacked helixes and tandem GA mismatches in RNA.

摘要

Thermodynamic parameters from optical melting studies are determined for two RNA motifs. Stability of helix-helix interfaces of coaxially stacked helixes is reported. The model system consists of a short oligomer binding to a four nucleotide overhang at the end of a hairpin stem, creating the helix-helix interface. Coaxial stacked interfaces involving Watson-Crick base pairs are approximately 1 kcal/mol more stable than the corresponding nearest neighbor interaction in an uninterrupted helix. The stability for these helix-helix interfaces has a similar sequence dependence as that for intact helixes. For coaxial stacked interfaces where unpaired nucleotides are appended to each side of the interface {dollar}(matrix{lcub}5spprime&rm C{lcub}-{rcub}Gcr&rm G/Ccr&rm X Z{rcub}){dollar} the free energy of coaxial stacking is approximately equal to the nearest neighbor free energy of an uninterrupted helix sequence, {dollar}matrix{lcub}5spprime&rm CGcr&rm GC{rcub}{dollar}. This experimental information has been used in a RNA secondary structure prediction computer algorithm by M. Zuker, and the predicted folding of RNA structures improved from 67 to 74% on average (J. Kim and D. H. Mathews, experimental results).; Thermodynamic results illustrate that the stability of tandem GA mismatches in RNA are dependent upon closing base pairs around these mismatches. Self-complementary oligoribonucleotides of eight nucleotides in length that form internal loops of the sequence {dollar}matrix{lcub}5spprime&rm XGAY3spprimecr3spprime&rm YAGX5spprime{rcub}{dollar} and {dollar}matrix{lcub}5spprime&rm XAGY3spprimecr3spprime&rm YGAX5spprime{rcub}{dollar} have a range of stabilities over 5 kcal/mol when XY is a Watson-Crick or GU base pair. The order of stabilities is: 5{dollar}spprime{dollar}GGAC3{dollar}spprime{dollar} {dollar}>{dollar} UGAG, CGAG {dollar}>{dollar} AGAU {dollar}>{dollar} UGAA {dollar}>{dollar} GGAU. The motifs GGAC, UGAG, and CGAG are stabilizing while the other GA motifs are destabilizing. The motif GAGC is more stable than CAGG and CGAG, but less stable than GGAC. These results illustrate the importance of the base pairs closing RNA internal loops. This work along with other information should help improve RNA structure prediction.