Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral polymerase with a cis-acting regulatory signal, designated epsilon (e), located at the 5 0 -end of its pre-genomic RNA (pgRNA). Binding of polymerase to e is also necessary for pgRNA encapsidation. While the mechanistic basis of this interaction remains elusive, mutagenesis studies suggest its internal 6-nt "priming loop" provides an important structural contribution. e might therefore be considered a promising target for small molecule interventions to complement current nucleoside-analog based anti-HBV therapies. An ideal prerequisite to any RNA-directed small molecule strategy would be a detailed structural description of this important element. Herein, we present a solution NMR structure for HBV e which, in combination with molecular dynamics and docking simulations, reports on a flexible ligand "pocket", reminiscent of those observed in proteins. We also demonstrate the binding of the selective estrogen receptor modulators (SERMs) Raloxifene, Bazedoxifene, and a de novo derivative to the priming loop.

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RNA structure and dynamic studies by NMR spectroscopy suffer from chemical shift overlap and line broadening, both of which become worse as RNA size increases. Incorporation of stable isotope labels into RNA has provided several solutions to these limitations. Nevertheless, the only method to circumvent the problem of spectral overlap completely is the solid-phase chemical synthesis of RNA with labeled RNA phosphoramidites. In this review, we summarize the practical aspects of this methodology for NMR spectroscopy studies of RNA. These types of investigations lie at the intersection of chemistry and biophysics and highlight the need for collaborative efforts to tackle the integrative structural biology problems that exist in the RNA world. Finally, examples of RNA structure and dynamic studies using labeled phosphoramidites are highlighted.

RNA structural research lags behind that of proteins, preventing a robust understanding of RNA functions. NMR spectroscopy is an apt technique for probing the structures and dynamics of RNA molecules in solution at atomic resolution. Still, RNA analysis by NMR suffers from spectral overlap and line broadening, both of which worsen for larger RNAs. Incorporation of stable isotope labels into RNA has provided several solutions to these challenges. In this review, we summarize the benefits and limitations of various methods used to obtain isotope-labeled RNA building blocks and how they are used to prepare isotope-labeled RNA for NMR structure and dynamics studies.