Accurate curve fitting procedure for UV melting analysis of highly thermostable RNA hairpins.

Abstract

Thermostable RNA hairpins with specific loop sequences are critically important structural elements for proper folding of functional RNAs. Thermodynamic parameters of the structural elements are indispensable for secondary structure prediction. However when RNA folds into highly thermostable hairpin-loop structures, it is difficult to determine the melting temperature and other thermodynamic parameters from the normal UV melting analysis. In this study, we demonstrate a procedure to solve the problem using our software, Jfit. UV absorbance in the higher temperature region above 95 degrees C and the upper baseline were estimated using this analytical procedure. The ideal lines of -RlnK vs. 1/T were generated from the predicted thermodynamic parameters and the solution candidates were compared with the van't Hoff plot generated from the measured melting curve to judge their self-consistency. As a result, our analysis was able to reduce the number of the possible solution and to specify the best set of thermodynamic parameters for the stable hairpin formation.