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.

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To facilitate rigorous analysis of molecular motions in proteins, DNA, and RNA, we present a new version of ROTDIF, a program for determining the overall rotational diffusion tensor from single-or multiple-field Nuclear Magnetic Resonance (NMR) relaxation data. We introduce four major features that expand the program’s versatility and usability. The first feature is the ability to analyze, separately or together, 13C and/or 15N relaxation data collected at a single or multiple fields. A significant improvement in the accuracy compared to direct analysis of R2/R1 ratios, especially critical for analysis of 13C relaxation data, is achieved by subtracting high-frequency contributions to relaxation rates. The second new feature is an improved method for computing the rotational diffusion tensor in the presence of biased errors, such as large conformational exchange contributions, that significantly enhances the accuracy of the computation. The third new feature is the integration of the domain alignment and docking module for relaxation-based structure determination of multi-domain systems. Finally, to improve accessibility to all the program features, we introduced a graphical user interface (GUI) that simplifies and speeds up the analysis of the data. Written in Java, the new ROTDIF can run on virtually any computer platform. In addition, the new ROTDIF achieves an order of magnitude speedup over the previous version by implementing a more efficient deterministic minimization algorithm. We not only demonstrate the improvement in accuracy and speed of the new algorithm for synthetic and experimental 13C and 15N relaxation data for several proteins and nucleic acids, but also show that careful analysis required especially for characterizing RNA dynamics allowed us to uncover subtle conformational changes in RNA as a function of temperature that were opaque to previous analysis.

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.

RNAs are an important class of cellular regulatory elements, and they are well characterized by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy in their folded or bound states. However, the apo or unfolded states are more difficult to characterize by either method. Particularly, effective NMR spectroscopy studies of RNAs in the past were hampered by chemical shift overlap of resonances and associated rapid signal loss due to line broadening for RNAs larger than the median size found in the PDB (∼25 nt); most functional riboswitches are bigger than this median size. Incorporation of selective site-specific 13C/15N-labeled nucleotides into RNAs promises to overcome this NMR size limitation. Unlike previous isotopic enrichment methods such as phosphoramidite, de novo, uniform-labeling and selective biomass approaches, this newer chemical-enzymatic selective method presents a number of advantages for producing labeled nucleotides over these other methods. For example total chemical synthesis of nucleotides, followed by solid-phase synthesis of RNA using phosphoramidite chemistry, while versatile in incorporating isotope labels into RNA at any desired position, faces problems of low yields (<10 %) that drop precipitously for oligonucleotides larger than 50 nt; de novo pyrimidine biosynthesis of NTPs, also a robust technique with modest yields of up to 45%, comes at the cost of using 16 enzymes, expensive substrates, and difficulty in making some needed labeling patterns such as selective labeling of the ribose C1′ and C5′ and the pyrimidine nucleobase C2, C4, C5 or C6; the method of biomass-produced uniformly- or selectively-labeled NTPs suffers from low overall yield per labeled input metabolite and isotopic scrambling with only modest suppression of 13C-13C couplings. In contrast, our current chemo-enzymatic approach overcomes most of these shortcomings and allows for the synthesis of gram quantities of nucleotides with >80% yields while using a limited number of enzymes, six at most. The unavailability of selectively labeled ribose and base precursors had prevented the effective use of this versatile method until now. Recently, we combined an improved organic synthetic approach that selectively places 13C/15N labels in the pyrimidine nucleobase (either 15N1, 15N3, 13C2, 13C4, 13C5, or 13C6 or any combination) with a very efficient enzymatic method to couple ribose with uracil to produce previously unattainable labeling patterns (Alvarado et al 2014). Herein we provide detailed steps of both our chemo-enzymatic synthesis of custom nucleotides and their incorporation into RNAs with sizes ranging from 29 to 155 nt, and showcase the dramatic improvement in spectral quality of reduced crowding and narrow linewidths. Applications of this selective labeling technology should prove valuable in overcoming two major obstacles, chemical shift overlap of resonances and associated rapid signal loss due to line broadening, that have impeded studying the structure and dynamics of large RNAs such as full l...

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments are invaluable for probing sparsely and transiently populated biomolecular states that cannot be directly detected by traditional NMR experiments and that are invisible by other biophysical approaches. A notable gap for RNA is the absence of CPMG experiments for measurement of methine base H and methylene C5' chemical shifts of ribose moieties in the excited state, partly because of complications from homonuclearC-C scalar couplings. Here we present site-specific C labeling that makes possible the design of pulse sequences for recording accurateH-C MQ and SQ CPMG experiments for ribose methine H1'-C1' and H2'-C2', base and ribose H CPMG, as well as a newH-C TROSY-detected methylene (CH) C5' CPMG relaxation pulse schemes. We demonstrate the utility of these experiments for two RNAs, the A-Site RNA known to undergo exchange and the IRE RNA suspected of undergoing exchange on microseconds to millisecond time-scale. We anticipate the new labeling approaches will facilitate obtaining structures of invisible states and provide insights into the relevance of such states for RNA-drug interactions.

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