Effects of Base Substitutions in an RNA Hairpin from Molecular Dynamics and Free Energy Simulations

Abstract

Contributions of individual interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using base substitutions. The G in the first tetraloop position was replaced by inosine (I) or adenosine (A), and the G in the C-G basepair closing the tetraloop was replaced by I. These substitutions eliminate particular hydrogen bonds proposed in the nuclear magnetic resonance model of the GCAA tetraloop. Molecular dynamics simulations of the GCAA tetraloop in aqueous solvent displayed a well-defined hydrogen pattern between the first and last loop nucleotides (G and A) stabilized by a bridging water molecule. Substitution of G-->I in the basepair closing the tetraloop did not significantly influence the loop structure and dynamics. The ICAA loop maintained the overall structure, but displayed variation in the hydrogen-bond network within the tetraloop itself. Molecular dynamics simulations of the ACAA loop led to conformational heterogeneity of the resulting structures. Changes of hairpin formation free energy associated with substitutions of individual bases were calculated by the free energy perturbation method. The calculated decrease of the hairpin stability upon G-->I substitution in the C-G basepair closing the tetraloop was in good agreement with experimental thermodynamic data. Our theoretical estimates for G-->I and G-->A mutations located in the tetraloop suggest larger loop destabilization than corresponding experimental results. The extent of conformational sampling of the structures resulting from base substitutions and its impact on the calculated free energy was discussed.

Nucleotide modifications and tRNA anticodon–mRNA codon interactions on the ribosome

“…The energy of the transformation is described by EðlÞ = lE C + ð1 À lÞE A , where l is a coupling parameter ranging from 0 (product state) to 1 (reactant state) in small steps. The free energy difference is calculated by summing the contributions from the simulations at all intermediate l-values: DG = + 1 l=0 ÀRT ln AEe (Sarzynska et al 2003) with virtually identical results, and we, therefore, used a reference system containing triplets, with the nucleotide being transformed in the middle, free in solution.…”

Structural and functional characterization of the 5′ region of subgenomic RNA5 of cucumber mosaic virus

“…thermodynamically stable UNCG tetraloop, a type that has been shown to be involved in tertiary RNA contacts acting as a nucleation site for folding (Molinaro & Tinoco, 1995;Sarzynska et al, 2003). We then went on to analyse full-length RNA3 sequences on the plus and minus strands using Mfold with standard default settings (Zuker, 2003).…”

CHARMM force field parameters for simulation of reactive intermediates in native and thio‐substituted ribozymes

“…Molecular simulation force fields for nucleic acids continue to improve [26][27][28][29][30][31] and a variety of simulations involving ribozymes have been carried out in recent years. [32][33][34][35][36][37][38][39][40] In order to study the structure and dynamics of different catalytic states along the reaction path of a ribozyme, however, reliable empirical force fields parameters must be developed for the transition states and reactive intermediates of these reactions. Furthermore, in order to use molecular simulation to aid in the interpretation of experimentally measured thio effects, parameters for thiosubstituted phosphate and phosphorane models and their interactions with metal ions are required.…”