Hydrogen-bonding modulation of RNA versus DNA hairpin folding pathways

The ribose 2'-hydroxyl (2'-OH) group is a key chemical feature distinguishing RNA from DNA, yet its precise role in RNA tetraloop folding dynamics remains unclear. Here, the folding processes of RNA UUCG and its counterpart quasi-DNA dUdUdCdG tetraloop hairpins are studied in detail using Gaussian accelerated molecular dynamics (GaMD) and minimum free energy pathways (MFEP). For the RNA UUCG, the folded state is achieved by overcoming ca. 2.0 times the thermal energy at T = 300 K from the most stable misfolded structure. Analysis of Markov state models (MSMs) indicates that the folding time of RNA UUCG is longer than that of the DNA dUdUdCdG. The rate constants for the initial interaction of the stem (optimal path) or the loop (suboptimal path) are almost equal. In contrast, there only exists a single misfolded state along the optimal folding path of DNA dUdUdCdG, giving a higher rate constant than that of RNA UUCG. Hydrogen-bonding and π-π interactions induced by the 2'-OH moieties in the RNA tetraloop lead to more misfolded states along the MFEP which have an impeding effect on the RNA folding kinetics. These findings highlight the kinetic influence of 2'-OH on RNA folding and provide mechanistic insights into the distinct folding behavior of RNA versus DNA hairpins, informing future studies of nucleic acid dynamics and design of RNA-targeted therapeutics.