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.

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.

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 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.

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.

Stable isotope-labeled RNA phosphoramidites to facilitate dynamics by NMR.

Given that Ribonucleic acids (RNAs) are a central hub of various cellular processes, methods to synthesize these RNAs for biophysical studies are much needed. Here, we showcase the applicability of 6-(13)C-pyrimidine phosphoramidites to introduce isolated (13)C-(1)H spin pairs into RNAs up to 40 nucleotides long. The method allows the incorporation of 6-(13)C-uridine and -cytidine residues at any desired position within a target RNA. By site-specific positioning of the (13)C-label using RNA solid phase synthesis, these stable isotope-labeling patterns are especially well suited to resolve resonance assignment ambiguities. Of even greater importance, the labeling pattern affords accurate quantification of important functional transitions of biologically relevant RNAs (e.g., riboswitch aptamer domains, viral RNAs, or ribozymes) in the µs- to ms time regime and beyond without complications of one bond carbon scalar couplings. We outline the chemical synthesis of the 6-(13)C-pyrimidine building blocks and their use in RNA solid phase synthesis and demonstrate their utility in Carr Purcell Meiboom Gill relaxation dispersion, ZZ exchange, and chemical exchange saturation transfer NMR experiments.

In vivo, large-scale preparation of uniformly (15)N- and site-specifically (13)C-labeled homogeneous, recombinant RNA for NMR studies.

Knowledge of how ribonucleic acid (RNA) structures fold to form intricate, three-dimensional structures has provided fundamental insights into understanding the biological functions of RNA. Nuclear magnetic resonance (NMR) spectroscopy is a particularly useful high-resolution technique to investigate the dynamic structure of RNA. Effective study of RNA by NMR requires enrichment with isotopes of (13)C or (15)N or both. Here, we present a method to produce milligram quantities of uniformly (15)N- and site-specifically (13)C-labeled RNAs using wild-type K12 and mutant tktA Escherichia coli in combination with a tRNA-scaffold approach. The method includes a double selection protocol to obtain an E. coli clone with consistently high expression of the recombinant tRNA-scaffold. We also present protocols for the purification of the tRNA-scaffold from a total cellular RNA extract and the excision of the RNA of interest from the tRNA-scaffold using DNAzymes. Finally, we showcase NMR applications to demonstrate the benefit of using in vivo site-specifically (13)C-labeled RNA.

Synthesis of a biotinylated photocleavable nucleotide monophosphate for the preparation of natively folded RNAs.

RNAs are involved in many functional roles in the cell, and this functional diversity is predicated on RNAs adopting requisite three-dimensional architectures. Preparing such "natively folded" RNAs with a homogeneous population is sometimes problematic for structural or enzymatic studies. Yet, standard methods for RNA preparations denature the RNA and create a heterogeneous population of conformers. Therefore, preparation of "natively folded" RNAs without going through the process of denaturing and refolding is important to obtain maximal biological function. 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 is readily accepted by T7 RNA polymerase to transcribe "natively folded" RNAs ranging in size from 27 to 493 nucleotides. This facile strategy allows efficient biotinylation of RNA and provides a traceless means to remove the biotin after the purification. Such preparation of natively folded RNAs should benefit biophysical and therapeutic applications.

Chemo-enzymatic synthesis of selectively ¹³C/¹5N-labeled RNA for NMR structural and dynamics studies.

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 (13)C/(15)N-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. The alternative method of de novo pyrimidine biosynthesis of NTPs is also a robust technique, with modest yields of up to 45%, but it 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. Biomass-produced, uniformly or selectively labeled NTPs offer a third method, but suffer from low overall yield per labeled input metabolite and isotopic scrambling with only modest suppression of (13)C-(13)C couplings. In contrast to these four methods, 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 (13)C/(15)N labels in the pyrimidine nucleobase (either (15)N1, (15)N3, (13)C2, (13)C4, (13)C5, or (13)C6 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-length riboswitches larger than the ~25 nt median size of RNA NMR structures found in the PDB.

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).

E88, a new cyclic-di-GMP class I riboswitch aptamer from Clostridium tetani, has a similar fold to the prototypical class I riboswitch, Vc2, but differentially binds to c-di-GMP analogs.

C-di-GMP has emerged as a ubiquitous second messenger, which regulates the transition between sessile and motile lifestyles and virulence factor expression in many pathogenic bacteria using both RNA riboswitches and protein effectors. We recently showed that two additional class I c-di-GMP riboswitch aptamers (Ct-E88 and Cb-17B) bind c-di-GMP with nanomolar affinity, and that Ct-E88 RNA binds 2'-F-c-di-GMP 422 times less tightly than class I Vc2 RNA. Based on sequence comparison, it was concluded that the global folds of Ct-E88 and Vc2 RNAs were similar and that differences in ligand binding were probably due to differences in binding site architectures. Herein, we utilized EMSA, aptamer sensing spinach modules, SAXS and 1D NMR titration to study the conformational transitions of Ct-E88. We conclude that whereas the global folds of the bound states of Vc2 and Ct-E88 RNAs are similar, the unbound states are different and this could explain differences in ligand affinities between these class I c-di-GMP riboswitches.

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.

Selective binding of 2'-F-c-di-GMP to Ct-E88 and Cb-E43, new class I riboswitches from Clostridium tetani and Clostridium botulinum respectively.

C-di-GMP is a second messenger in bacteria and partly regulates bacterial physiology by binding to class I and II riboswitches. Four class I c-di-GMP riboswitch aptamer candidates, Ct-E88, Cb-17B, Cb-E43 and Cd-630 RNAs, selected from a GEMM RNA sequence motif in the Rfam database, were expressed and experimentally verified to bind to c-di-GMP. The two newly characterized c-di-GMP riboswitches, Ct-E88 and Cb-E43, bound c-di-GMP with nanomolar Kd whereas the affinities of Cb-17B and Cd-630 for c-di-GMP were at least a 100-fold weaker. Interestingly, whereas the three riboswitches (Vc2, Et-E88 and Cb-E43) bound c-di-GMP with similar Kd values, 2'-modified analogs of c-di-GMP differentially bound to these three class I aptamers. For example, 2'-F-c-di-GMP bound Vc2 with a Kd value of 102 nM whereas the Kd value of 2'-F-c-di-GMP-Ct-E88 is 43 µM (422× higher than that for Vc2 RNA), revealing that there are differences in the binding sites of functional class I c-di-GMP riboswitches.

Nanomolar fluorescent detection of c-di-GMP using a modular aptamer strategy.

C-di-GMP regulates important processes involved in biofilm formation and virulence factors production in several bacteria. Herein we report a simple fluorescent strategy that allows for the detection of c-di-GMP (as low as 320 nM) using a Vc2 class I riboswitch domain as the sensing region and spinach as the fluorescent reporting module.

Differential binding of 2'-biotinylated analogs of c-di-GMP with c-di-GMP riboswitches and binding proteins.

C-di-GMP has emerged as a signalling molecule that regulates a variety of processes in several bacteria; therefore there is interest in the development of biotinylated analogs for the identification of binding partners. No detailed study has been done to evaluate if biotinylated analogs of c-di-GMP are capable of binding to c-di-GMP receptors. Herein, we evaluate the binding of commercially available 2'-biotinylated c-di-GMP and phosphorothioate 2'-biotinylated c-di-GMP, prepared via a facile solid-phase synthesis, to several c-di-GMP receptors. Docking, using Autodock vina software, as well as experimental studies of these analogs, with c-di-GMP class I and II riboswitches and binding proteins, reveal that some, but not all, c-di-GMP receptors can tolerate the 2'-modification of c-di-GMP with biotin.

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.

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.