| The sequence-dependence of thermodynamics of RNA duplexes with Watson-Crick base pairs were studied by UV optical melting experiments and analyzed with various models. A novel approach is employed to provide direct evidence of the effect of base composition and terminal base pairs on duplex stability. Through statistical analysis, an improved nearest-neighbor model is proposed in which a term that accounts for the total number of hydrogen bonds, therefore base composition, is included. This model distinguishes different duplexes that have the same nearest-neighbor composition but different base pair compositions, and therefore different ends. Thus this model predicts thermodynamic properties of RNA duplexes better than previous models, and forms the foundation of RNA secondary structure prediction.; Thermodynamics and structures of non-symmetric tandem mismatches were studied by UV optical melting and NMR. It is found that stabilizing mismatches in a symmetric context may not be stabilizing in a non-symmetric context. If the two mismatches differ in size and flexibility, and their backbone distortions are not compatible, there is a very unfavorable factor. Quantitative measurements on the sequence-dependence of this factor have been conducted, and a working model is developed. The structural basis of these observations is investigated. The solution structure of one tandem mismatch motif, 5'GUGG3' 3'CUAC5' , is solved by two-dimensional NMR and simulated annealing. This provides a rationale for the observed thermodynamic pattern. This structure bears resemblance to, yet is different to the x-ray crystal structure of the related internal loops in a conserved domain of large subunit ribosomal RNA recognized by ribosomal protein L11. |
