A delayed decoupling methyl-TROSY pulse sequence for atomic resolution studies of folded proteins and RNAs in condensates.

Solution NMR spectroscopy has tremendous potential for providing atomic resolution insights into the interactions between proteins and nucleic acids partitioned into condensed phases of phase-separated systems. However, the highly viscous nature of the condensed phase challenges applications, and in particular, the extraction of quantitative, site-specific information. Here, we present a delayed decoupling-based HMQC pulse sequence for methyl-TROSY studies of 'client' proteins and nucleic acids partitioned into 'scaffold' proteinaceous phase-separated solvents. High sensitivity and excellent quality spectra are recorded of a nascent form of superoxide dismutase and of a small RNA fragment partitioned into CAPRIN1 condensates.

Dynamic basis for dA•dGTP and dA•d8OGTP misincorporation via Hoogsteen base pairs.

Replicative errors contribute to the genetic diversity needed for evolution but in high frequency can lead to genomic instability. Here, we show that DNA dynamics determine the frequency of misincorporating the A•G mismatch, and altered dynamics explain the high frequency of 8-oxoguanine (8OG) A•8OG misincorporation. NMR measurements revealed that Aanti•Ganti (population (pop.) of >91%) transiently forms sparsely populated and short-lived Aanti+•Gsyn (pop. of ~2% and kex = kforward + kreverse of ~137 s-1) and Asyn•Ganti (pop. of ~6% and kex of ~2,200 s-1) Hoogsteen conformations. 8OG redistributed the ensemble, rendering Aanti•8OGsyn the dominant state. A kinetic model in which Aanti+•Gsyn is misincorporated quantitatively predicted the dA•dGTP misincorporation kinetics by human polymerase ß, the pH dependence of misincorporation and the impact of the 8OG lesion. Thus, 8OG increases replicative errors relative to G because oxidation of guanine redistributes the ensemble in favor of the mutagenic Aanti•8OGsyn Hoogsteen state, which exists transiently and in low abundance in the A•G mismatch.

2'-O-Methylation can increase the abundance and lifetime of alternative RNA conformational states.

2'-O-Methyl (Nm) is a highly abundant post-transcriptional RNA modification that plays important biological roles through mechanisms that are not entirely understood. There is evidence that Nm can alter the biological activities of RNAs by biasing the ribose sugar pucker equilibrium toward the C3'-endo conformation formed in canonical duplexes. However, little is known about how Nm might more broadly alter the dynamic ensembles of flexible RNAs containing bulges and internal loops. Here, using NMR and the HIV-1 transactivation response (TAR) element as a model system, we show that Nm preferentially stabilizes alternative secondary structures in which the Nm-modified nucleotides are paired, increasing both the abundance and lifetime of low-populated short-lived excited states by up to 10-fold. The extent of stabilization increased with number of Nm modifications and was also dependent on Mg2+. Through phi-value analysis, the Nm modification also provided rare insights into the structure of the transition state for conformational exchange. Our results suggest that Nm could alter the biological activities of Nm-modified RNAs by modulating their secondary structural ensembles as well as establish the utility of Nm as a tool for the discovery and characterization of RNA excited state conformations.