Hepatitis B Virus Epsilon (ε) RNA Element: Dynamic Regulator of Viral Replication and Attractive Therapeutic Target.

Hepatitis B virus (HBV) chronically infects millions of people worldwide, which underscores the importance of discovering and designing novel anti-HBV therapeutics to complement current treatment strategies. An underexploited but attractive therapeutic target is ε, a cis-acting regulatory stem-loop RNA situated within the HBV pregenomic RNA (pgRNA). The binding of ε to the viral polymerase protein (P) is pivotal, as it triggers the packaging of pgRNA and P, as well as the reverse transcription of the viral genome. Consequently, small molecules capable of disrupting this interaction hold the potential to inhibit the early stages of HBV replication. The rational design of such ligands necessitates high-resolution structural information for the ε-P complex or its individual components. While these data are currently unavailable for P, our recent structural elucidation of ε through solution nuclear magnetic resonance spectroscopy marks a significant advancement in this area. In this review, we provide a brief overview of HBV replication and some of the therapeutic strategies to combat chronic HBV infection. These descriptions are intended to contextualize our recent experimental efforts to characterize ε and identify ε-targeting ligands, with the ultimate goal of developing novel anti-HBV therapeutics.

Cross-correlated relaxation rates provide facile exchange signature in selectively labeled RNA.

Gerhard Wagner has made numerous contributions to NMR spectroscopy, particularly his developments in the field of spin-relaxation stand out in directly mapping the spectral density functions of proteins. He and his group developed experimental techniques to reveal the importance of dynamics to protein biological function and drug discovery. On his 75th birthday, we take this opportunity to highlight how some of those seminal ideas developed for proteins are being extended to RNAs. The role of dynamics in the structure and function of RNA has been a major interest in drug design and therapeutics. Here we present the use of cross-correlated relaxation rates (ηxy) from anti-TROSY (R2α) and TROSY (R2ß) to rapidly obtain qualitative information about the chemical exchange taking place within the bacterial and human A-site RNA system while reducing the sets of relaxation experiments required to map dynamics. We show that ηxy correlates with the order parameter which gives information on how flexible or rigid a residue is. We further show R2ß/ηxy can rapidly be used to probe chemical exchange as seen from its agreement with Rex. In addition, we report the ability of R2ß/ηxy to determine chemical exchange taking place within the bacterial A-site RNA during structural transitions at pH 6.2 and 6.5. Finally, comparison of the R2ß/ηxy ratios indicates bacterial A-site has greater R2ß/ηxy values for G19 (1.34 s-1), A20 (1.38 s-1), U23 (1.63 s-1) and C24 (1.51 s-1) than human A-site [A19 (0.76 s-1), A20 (1.01 s-1), U23 (0.74 s-1) and C24 (0.71 s-1)]. Taken together, we have shown that the chemical exchange can quickly be analyzed for RNA systems from cross-correlated relaxation rates.

Structural insights of the conserved "priming loop" of hepatitis B virus pre-genomic RNA.

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 (ε), located at the 5'-end of its pre-genomic RNA (pgRNA). Binding of polymerase to ε 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. ε 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 ε 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.Communicated by Ramaswamy H. Sarma.

Isotope-Labeled RNA Building Blocks for NMR Structure and Dynamics Studies.

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.

Deleterious effects of carbon-carbon dipolar coupling on RNA NMR dynamics.

Many regulatory RNAs undergo dynamic exchanges that are crucial for their biological functions and NMR spectroscopy is a versatile tool for monitoring dynamic motions of biomolecules. Meaningful information on biomolecular dynamics requires an accurate measurement of relaxation parameters such as longitudinal (R1) rates, transverse (R2) rates and heteronuclear Overhauser effect (hNOE). However, earlier studies have shown that the large 13C-13C interactions complicate analysis of the carbon relaxation parameters. To investigate the effect of 13C-13C interactions on RNA dynamic studies, we performed relaxation measurements on various RNA samples with different labeling patterns and compared these measurements with the computational simulations. For uniformly labeled samples, contributions of the neighboring carbon to R1 measurements were observed. These contributions increased with increasing magnetic field and overall correlation time ([Formula: see text]) for R1 rates, necessitating more careful analysis for uniformly labeled large RNAs. In addition, the hNOE measurements were also affected by the adjacent carbon nuclei. Unlike R1 rates, R1ρ rates showed relatively good agreement between uniformly- and site-selectively labeled samples, suggesting no dramatic effect from their attached carbon, in agreement with previous observations. Overall, having more accurate rate measurements avoids complex analysis and will be a key for interpreting 13C relaxation rates for molecular motion that can provide valuable insights into cellular molecular recognition events.

A drug discovery toolbox for Nuclear Magnetic Resonance (NMR) characterization of ligands and their targets.

Information about the structure, dynamics, and ligand-binding properties of biomolecules can be derived from Nuclear Magnetic Resonance (NMR) spectroscopy and provides valuable information for drug discovery. A multitude of experimental approaches provides a wealth of information that can be tailored to the system of interest. Methods to study the behavior of ligands upon target binding enable the identification of weak binders in a robust manner that is critical for the identification of truly novel binding interactions. This is particularly important for challenging targets. Observing the solution behavior of biomolecules yields information about their structure, dynamics, and interactions. This review describes the breadth of approaches that are available, many of which are under-utilized in a drug-discovery environment, and focuses on recent advances that continue to emerge.

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.

Combining asymmetric 13C-labeling and isotopic filter/edit NOESY: a novel strategy for rapid and logical RNA resonance assignment.

Although ∼98% of the human genomic output is transcribed as non-protein coding RNA, <2% of the protein data bank structures comprise RNA. This huge structural disparity stems from combined difficulties of crystallizing RNA for X-ray crystallography along with extensive chemical shift overlap and broadened linewidths associated with NMR of RNA. While half of the deposited RNA structures in the PDB were solved by NMR methods, the usefulness of NMR is still limited by the high cost of sample preparation and challenges of resonance assignment. Here we propose a novel strategy for resonance assignment that combines new strategic 13C labeling technologies with filter/edit type NOESY experiments to greatly reduce spectral complexity and crowding. This new strategy allowed us to assign important non-exchangeable resonances of proton and carbon (1', 2', 2, 5, 6 and 8) nuclei using only one sample and <24 h of NMR instrument time for a 27 nt model RNA. The method was further extended to assigning a 6 nt bulge from a 61 nt viral RNA element justifying its use for a wide range RNA chemical shift resonance assignment problems.

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.

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.

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).