Isotope labeling revolutionized NMR studies of small nucleic acids, but to extend this technology to larger RNAs requires site-specific labeling tools to expedite NMR structural and dynamics studies. Using enzymes from the pentose phosphate pathway, we couple chemically synthesized uracil nucleobase with specifically 13C-labeled ribose to synthesize both UTP and CTP with nearly quantitative yields. This chemo-enzymatic method affords a cost-effective preparation of labels that are unattainable by current methods. The methodology generates versatile 13C and 15N labeling patterns which, when employed with relaxation-optimized NMR spectroscopy, effectively mitigates problems of rapid relaxation that result in low resolution and sensitivity. The methodology is demonstrated with RNAs of various sizes, complexity, and importance: the exon splicing silencer 3 (27 nt), iron responsive element (29 nt), Pro-tRNA (76 nt), and HIV-1 core encapsidation signal (155 nt).

Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis (48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure μs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.

Conformational equilibria are increasingly recognized as pivotal for biological function. Traditional structural analyses provide a static image of conformers in solution that sometimes present conflicting views. From (13) C and (1) H chemical exchange saturation transfer experiments, in concert with ligation and selective labeling strategies, we show that in the absence of metabolite, a Mg(2+) (0-0.5 mm)-bound apo SAM-II riboswitch RNA exists in a minor (≈10 %) partially closed state that rapidly exchanges with a predominantly (≈90 %) open form with a lifetime of ≈32 ms. The base and sugar (H6,C6, H1',C1') chemical shifts of C43 for the dominant conformer are similar to those of a free CMP, but those of the minor apo species are comparable to shifts of CMPs in helical RNA regions. Our results suggest that these transient, low populated states stabilized by Mg(2+) will likely enhance rapid ligand recognition and, we anticipate, will play potentially ubiquitous roles in RNA signaling.

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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