RNAs tend to be highly flexible and are better characterized as a dynamic ensemble containing multiple interconverting conformations as opposed to a single static structure. In addition to the commonly occupied ground state conformations, these ensembles also contain high-energy, lowly populated “excited” states that have only recently started to be characterized by NMR spectroscopy. Recent functional characterization of these states in specific systems, notably in bacterial riboswitches and human microRNAs, has found that they can have important biological roles. In addition, recent advancements in proton chemical exchange saturation transfer (HCEST) experiments have made it possible to search for these states in higher throughput. We aim to apply these HCEST methods, along with other structure prediction methodologies, to the HIV 5’ untranslated region (UTR), an essential region of the HIV genome that has roles in several different biological processes. While this RNA has been studied extensively, there are still open questions regarding its folding pathway as well as protein recognition events. We hypothesize that short-lived excited states may be involved in one or more of these processes. Here, we investigate several important RNA motifs of the 5’-UTR, using NMR spectroscopy to determine if exchange between a ground state and excited state is taking place at key residues. Specifically, we are including data for the dimerization-initiation sequence and the psi loop, both involved in viral packaging.
Li et al. (Sun,) studied this question.