NMR probing of invisible excited states using selectively labeled RNAs.

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 1H and methylene C5' chemical shifts of ribose moieties in the excited state, partly because of complications from homonuclear 13C-13C scalar couplings. Here we present site-specific 13C labeling that makes possible the design of pulse sequences for recording accurate 1H-13C MQ and SQ CPMG experiments for ribose methine H1'-C1' and H2'-C2', base and ribose 1H CPMG, as well as a new 1H-13C TROSY-detected methylene (CH2) 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.

CCR5 RNA Pseudoknots: Residue and Site-Specific Labeling correlate Internal Motions with microRNA Binding.

Conformational dynamics of RNA molecules play a critical role in governing their biological functions. Measurements of RNA dynamic behavior sheds important light on sites that interact with their binding partners or cellular stimulators. However, such measurements using solution-state NMR are difficult for large RNA molecules (>70 nt; nt=nucleotides) owing to severe spectral overlap, homonuclear 13 C scalar couplings, and line broadening. Herein, a strategic combination of solid-phase synthesis, site-specific isotopic labeled phosphoramidites, and enzymatic ligation is introduced. This approach allowed the position-specific insertion of isotopic probes into a 96 nt CCR5 RNA fragment. Accurate measurements of functional dynamics using the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD) experiments enabled extraction of the exchange rates and populations of this RNA. NMR chemical shift perturbation analysis of the RNA/microRNA-1224 complex indicated that A90-C1' of the pseudoknot exhibits similar changes in chemical shift observed in the excited state. This work demonstrates the general applicability of a NMR-labeling strategy to probe functional RNA structural dynamics.

A magnesium-induced triplex pre-organizes the SAM-II riboswitch.

Our 13C- and 1H-chemical exchange saturation transfer (CEST) experiments previously revealed a dynamic exchange between partially closed and open conformations of the SAM-II riboswitch in the absence of ligand. Here, all-atom structure-based molecular simulations, with the electrostatic effects of Manning counter-ion condensation and explicit magnesium ions are employed to calculate the folding free energy landscape of the SAM-II riboswitch. We use this analysis to predict that magnesium ions remodel the landscape, shifting the equilibrium away from the extended, partially unfolded state towards a compact, pre-organized conformation that resembles the ligand-bound state. Our CEST and SAXS experiments, at different magnesium ion concentrations, quantitatively confirm our simulation results, demonstrating that magnesium ions induce collapse and pre-organization. Agreement between theory and experiment bolsters microscopic interpretation of our simulations, which shows that triplex formation between helix P2b and loop L1 is highly sensitive to magnesium and plays a key role in pre-organization. Pre-organization of the SAM-II riboswitch allows rapid detection of ligand with high selectivity, which is important for biological function.

Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations.

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.

Chemo-enzymatic labeling for rapid assignment of RNA molecules.

Even though Nuclear Magnetic Resonance (NMR) spectroscopy is one of the few techniques capable of determining atomic resolution structures of RNA, it is constrained by two major problems of chemical shift overlap of resonances and rapid signal loss due to line broadening. Emerging tools to tackle these problems include synthesis of atom specifically labeled or chemically modified nucleotides. Herein we review the synthesis of these nucleotides, the design and production of appropriate RNA samples, and the application and analysis of the NMR experiments that take advantage of these labels.

SAM-II Riboswitch Samples at least Two Conformations in Solution in the Absence of Ligand: Implications for Recognition.

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.

Regio-selective chemical-enzymatic synthesis of pyrimidine nucleotides facilitates RNA structure and dynamics studies.

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

Multiple conformations of SAM-II riboswitch detected with SAXS and NMR spectroscopy.

Riboswitches are a newly discovered large family of structured functional RNA elements that specifically bind small molecule targets out of a myriad of cellular metabolites to modulate gene expression. Structural studies of ligand-bound riboswitches by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy have provided insights into detailed RNA-ligand recognition and interactions. However, the structures of ligand-free riboswitches remain poorly characterized. In this study, we have used a variety of biochemical, biophysical and computational techniques including small-angle X-ray scattering and NMR spectroscopy to characterize the ligand-free and ligand-bound forms of SAM-II riboswitch. Our data demonstrate that the RNA adopts multiple conformations along its folding pathway and suggest that the RNA undergoes marked conformational changes upon Mg(2+) compaction and S-adenosylmethionine (SAM) metabolite binding. Further studies indicated that Mg(2+) ion is not essential for the ligand binding but can stabilize the complex by facilitating loop/stem interactions. In the presence of millimolar concentration of Mg(2+) ion, the RNA samples a more compact conformation. This conformation is near to, but distinct from, the native fold and competent to bind the metabolite. We conclude that the formation of various secondary and tertiary structural elements, including a pseudoknot, occur to sequester the putative Shine-Dalgarno sequence of the RNA only after metabolite binding.

Deriving quantitative dynamics information for proteins and RNAs using ROTDIF with a graphical user interface.

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 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, (13)C and/or (15)N 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 (13)C 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 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 (13)C and (15)N 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.

RNAs synthesized using photocleavable biotinylated nucleotides have dramatically improved catalytic efficiency.

Obtaining homogeneous population of natively folded RNAs is a crippling problem encountered when preparing RNAs for structural or enzymatic studies. Most of the traditional methods that are employed to prepare large quantities of RNAs involve procedures that partially denature the RNA. Here, we present a simple strategy using 'click' chemistry to couple biotin to a 'caged' photocleavable (PC) guanosine monophosphate (GMP) in high yield. This biotin-PC GMP, accepted by T7 RNA polymerase, has been used to transcribe RNAs ranging in size from 27 to 527 nt. Furthermore we show, using an in-gel fluorescence assay, that natively prepared 160 and 175 kDa minimal group II intron ribozymes have enhanced catalytic activity over the same RNAs, purified via denaturing conditions and refolded. We conclude that large complex RNAs prepared by non-denaturing means form a homogeneous population and are catalytically more active than those prepared by denaturing methods and subsequent refolding; this facile approach for native RNA preparation should benefit synthesis of RNAs for biophysical and therapeutic applications.

Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy.

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of (13)C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using Escherichia coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-(13)C]-pyruvate affords ribonucleotides with site specific labeling at C5' (~95%) and C1' (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-(13)C]-glycerol for which the ribose ring is labeled in all but the C4' carbon position, leading to multiplet splitting of the C1', C2' and C3' carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.

Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR.

Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here an E. coli strain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at different carbon sites is shown to facilitate cost-effective and large scale production of useful nucleotides. These nucleotides are site specifically labeled in C1' and C5' with minimal scrambling within the ribose ring. To demonstrate the utility of this labeling approach, the new site-specific labeled and the uniformly labeled nucleotides were used to synthesize a 36-nt RNA containing the catalytically essential domain 5 (D5) of the brown algae group II intron self-splicing ribozyme. The D5 RNA was used in binding and relaxation studies probed by NMR spectroscopy. Key nucleotides in the D5 RNA that are implicated in binding Mg(2+) ions are well resolved. As a result, spectra obtained using selectively labeled nucleotides have higher signal-to-noise ratio compared to those obtained using uniformly labeled nucleotides. Thus, compared to the uniformly (13)C/(15)N-labeled nucleotides, these specifically labeled nucleotides eliminate the extensive (13)C-(13)C coupling within the nitrogenous base and ribose ring, give rise to less crowded and more resolved NMR spectra, and accurate relaxation rates without the need for constant-time or band-selective decoupled NMR experiments. These position selective labeled nucleotides should, therefore, find wide use in NMR analysis of biologically interesting RNA molecules.

Synthesis of [7-15N]-GTPs for RNA structure and dynamics by NMR spectroscopy.

Several isotope-labeling strategies have been developed for the study of RNA by nuclear magnetic resonance (NMR) spectroscopy. Here, we report a combined chemical and enzymatic synthesis of [7-15N]-guanosine-5'-triphosphates for incorporation into RNA via T7 RNA polymerase-based in vitro transcription. We showcase the utility of these labels to probe both structure and dynamics in two biologically important RNAs. Supplementary Information: The online version contains supplementary material available at 10.1007/s00706-022-02892-1.

Asymmetry of (13)C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy.

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of (13)C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using E. coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-(13)C]-pyruvate affords ribonucleotides with site specific labeling at C5' (~95%) and C1' (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-(13)C]-glycerol for which the ribose ring is labeled in all but the C4' carbon position, leading to multiplet splitting of the C1', C2' and C3' carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.

Expression, purification and analysis of the activity of enzymes from the pentose phosphate pathway.

RNAs, more than ever before, are increasingly viewed as biomolecules of the future, in the versatility of their functions and intricate three-dimensional folding. To effectively study them by nuclear magnetic resonance (NMR) spectroscopy, structural biologists need to tackle two critical challenges of spectral overcrowding and fast signal decay for large RNAs. Stable-isotope nucleotide labeling is one attractive solution to the overlap problem. Hence, developing effective methods for nucleotide labeling is highly desirable. In this work, we have developed a facile and streamlined source of recombinant enzymes from the pentose phosphate pathway for making such labeled nucleotides. The Escherichia coli (E. coli) genes encoding ribokinase (RK), adenine phosphoribosyltransferase (APRT), xanthine/guanine phosphoribosyltransferase (XGPRT), and uracil phosphoribosyltransferase (UPRT) were sub-cloned into pET15b vectors. All four constructs together with cytidine triphosphate synthetase (CTPS) and human phosphoribosyl pyrophosphate synthetase isoform 1 (PRPPS) were transformed into the E. coli BL21(AI) strain for protein over-expression. The enzyme preparations were purified to >90% homogeneity by a one-step Ni-NTA affinity chromatography, without the need of a further size-exclusion chromatography step. We obtained yields of 1530, 22, 482, 3120, 2120 and 2280 units of activity per liter of culture for RK, PRPPS, APRT, XGPRT, UPRT and CTPS, respectively; the specific activities were found to be 70, 22, 21, 128, 144 and 113 U/mg, respectively. These specific activities of these enzyme constructs are comparable to or higher than those previously reported. In addition, both the growth conditions and purification protocols have been streamlined so that all the recombinant proteins can be expressed, purified and characterized in at most 2 days. The availability and reliability of these constructs should make production of fully and site-specific labeled nucleotides for making labeled RNA accessible and straightforward, to facilitate high-resolution NMR spectroscopic and other biophysical studies.

Probing adenine rings and backbone linkages using base specific isotope-edited Raman spectroscopy: application to group II intron ribozyme domain V.

Raman difference spectroscopy is used to probe the properties of a 36-nt RNA molecule, "D5", which lies at the heart of the catalytic apparatus in group II introns. For D5 that has all of its adenine residues labeled with (13)C and (15)N and utilizing Raman difference spectroscopy, we identify the conformationally sensitive -C-O-P-O-C- stretching modes of the unlabeled bonds adjacent to adenine bases, as well as the adenine ring modes themselves. The phosphodiester modes can be assigned to individual adenine residues based on earlier NMR data. The effect of Mg(2+) binding was explored by analyzing the Raman difference spectra for [D5 + Mg(2+)] minus [D5 no Mg(2+)], for D5 unlabeled, or D5 labeled with (13)C/(15)N-enriched adenine. In both sets of data we assign differential features to G ring modes perturbed by Mg(2+) binding at the N7 position. In the A-labeled spectra we attribute a Raman differential near 1450 cm(-1) and changes of intensity at 1296 cm(-1) to Mg binding at the N7 position of adenine bases. The A and G bases involved in Mg(2+) binding again can be identified using earlier NMR results. For the unlabeled D5, a change in the C-O-P-O-C stretch profile at 811 cm(-1) upon magnesium binding is due to a "tightening up" (in the sense of a more rigid molecule with less dynamic interchange among competing ribose conformers) of the D5 structure. For adenine-labeled D5, small changes in the adenine backbone bond signatures in the 810-830 cm(-1) region suggest that small conformational changes occur in the tetraloop and bulge regions upon binding of Mg(2+). The PO(2)(-) stretching vibration, near 1100 cm(-1), from the nonbridging phosphate groups, probes the effect of Mg(2+)-hydrate inner-sphere interactions that cause an upshift. In turn, the upshift is modulated by the presence of monovalent cations since in the presence of Na(+) and Li(+) the upshift is 23 +/- 2 cm(-1) while in the presence of K(+) and Cs(+) it is 13 +/- 3 cm(-1), a finding that correlates with the differences in hydration radii. These subtle differences in electrostatic interactions may be related to observed variations in catalytic activity. For a reconstructed ribozyme comprising domains 1-3 (D123) connected in cis plus domain 5 (D5) supplied in trans, cleavage of spliced exon substrates in the presence of magnesium and K(+) or Cs(+) is more efficient than that in the presence of magnesium with Na(+) or Li(+).

Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an E. coli strain disabled in the oxidative pentose phosphate pathway.

Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes ((13)C, (15)N, and/or (2)H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-(13)C-glycerol and (15)N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, (13)C-sodium formate combined with (13)C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and C6 positions of the pyrimidine rings are labeled with minimal multiplet splitting due to (1)J(C5C6) scalar coupling. However, the C2/C8 sites within purine rings and the C1'/C3'/C5' positions within the ribose rings have reduced labeling. Addition of (13)C-labeled formate leads to increased labeling at the base C2/C8 and the ribose C1'/C3'/C5' positions; these new specific labels result in two- to three-fold increase in the number of resolved resonances. This use of formate and (15)N-ammonium sulfate promises to extend further the utility of these alternate site specific labels to make labeled RNA for downstream biophysical applications such as structural, dynamics and functional studies of interesting biologically relevant RNAs.

Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle.

Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on (13)C-2-glycerol and (13)C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in (13)C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3' and C5' carbon positions. Consequently the C1', C2' and C4' positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with (13)C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a (13)C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4' carbon position, such that the C2' and C3' positions suffer from multiplet splitting but the C5' position remains singlet and the C1' position shows a small amount of residual C1'-C2' coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with (13)C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5' position (~90%) that makes it particularly attractive for NMR applications involving CH(2)-TROSY modules without the need for decoupling the C4' carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems.

Growth of wildtype and mutant E. coli strains in minimal media for optimal production of nucleic acids for preparing labeled nucleotides.

Since RNAs lie at the center of most cellular processes, there is a need for synthesizing large amounts of RNAs made from stable isotope-labeled nucleotides to advance the study of their structure and dynamics by nuclear magnetic resonance (NMR) spectroscopy. A particularly effective means of obtaining labeled nucleotides is to harvest these nucleotides from bacteria grown in defined minimal media supplemented with 15NH4Cl and various carbon sources. Given the high cost of carbon precursors required for labeling nucleic acids for NMR studies, it becomes important to evaluate the optimal growth for commonly used strains under standard minimal media conditions. Such information is lacking. In this study, we characterize the growth for Escherichia coli strains K12, K10zwf, and DL323 in three minimal media with isotopic-labeled carbon sources of acetate, glycerol, and glycerol combined with formate. Of the three media, the LeMaster-Richards and the Studier media outperform the commonly used M9 media and both support optimal growth of E. coli for the production of nucleotides. However, the growth of all three E. coli strains in acetate is reduced almost twofold compared to growth in glycerol. Analysis of the metabolic pathway and previous gene array studies help to explain this differential growth in glycerol and acetate. These studies should benefit efforts to make selective 13C-15N isotopic-labeled nucleotides for synthesizing biologically important RNAs.

Solution NMR readily reveals distinct structural folds and interactions in doubly 13C- and 19F-labeled RNAs.

RNAs form critical components of biological processes implicated in human diseases, making them attractive for small-molecule therapeutics. Expanding the sites accessible to nuclear magnetic resonance (NMR) spectroscopy will provide atomic-level insights into RNA interactions. Here, we present an efficient strategy to introduce 19F-13C spin pairs into RNA by using a 5-fluorouridine-5'-triphosphate and T7 RNA polymerase-based in vitro transcription. Incorporating the 19F-13C label in two model RNAs produces linewidths that are twice as sharp as the commonly used 1H-13C spin pair. Furthermore, the high sensitivity of the 19F nucleus allows for clear delineation of helical and nonhelical regions as well as GU wobble and Watson-Crick base pairs. Last, the 19F-13C label enables rapid identification of a small-molecule binding pocket within human hepatitis B virus encapsidation signal epsilon (hHBV ε) RNA. We anticipate that the methods described herein will expand the size limitations of RNA NMR and aid with RNA-drug discovery efforts.