Flipping the script: Understanding riboswitches from an alternative perspective.

Riboswitches are broadly distributed regulatory elements most frequently found in the 5'-leader sequence of bacterial mRNAs that regulate gene expression in response to the binding of a small molecule effector. The occupancy status of the ligand-binding aptamer domain manipulates downstream information in the message that instructs the expression machinery. Currently, there are over 55 validated riboswitch classes, where each class is defined based on the identity of the ligand it binds and/or sequence and structure conservation patterns within the aptamer domain. This classification reflects an "aptamer-centric" perspective that dominates our understanding of riboswitches. In this review, we propose a conceptual framework that groups riboswitches based on the mechanism by which RNA manipulates information directly instructing the expression machinery. This scheme does not replace the established aptamer domain-based classification of riboswitches but rather serves to facilitate hypothesis-driven investigation of riboswitch regulatory mechanisms. Based on current bioinformatic, structural, and biochemical studies of a broad spectrum of riboswitches, we propose three major mechanistic groups: (1) "direct occlusion", (2) "interdomain docking", and (3) "strand exchange". We discuss the defining features of each group, present representative examples of riboswitches from each group, and illustrate how these RNAs couple small molecule binding to gene regulation. While mechanistic studies of the occlusion and docking groups have yielded compelling models for how these riboswitches function, much less is known about strand exchange processes. To conclude, we outline the limitations of our mechanism-based conceptual framework and discuss how critical information within riboswitch expression platforms can inform gene regulation.

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.

Virtual Screening of Hepatitis B Virus Pre-Genomic RNA as a Novel Therapeutic Target.

The global burden imposed by hepatitis B virus (HBV) infection necessitates the discovery and design of novel antiviral drugs to complement existing treatments. One attractive and underexploited therapeutic target is ε, an ~85-nucleotide (nt) cis-acting regulatory stem-loop RNA located at the 3'- and 5'-ends of the pre-genomic RNA (pgRNA). Binding of the 5'-end ε to the viral polymerase protein (P) triggers two early events in HBV replication: pgRNA and P packaging and reverse transcription. Our recent solution nuclear magnetic resonance spectroscopy structure of ε permits structure-informed drug discovery efforts that are currently lacking for P. Here, we employ a virtual screen against ε using a Food and Drug Administration (FDA)-approved compound library, followed by in vitro binding assays. This approach revealed that the anti-hepatitis C virus drug Daclatasvir is a selective ε-targeting ligand. Additional molecular dynamics simulations demonstrated that Daclatasvir targets ε at its flexible 6-nt priming loop (PL) bulge and modulates its dynamics. Given the functional importance of the PL, our work supports the notion that targeting ε dynamics may be an effective anti-HBV therapeutic strategy.

Synthesis of an Atom-Specifically 2 H/13 C-Labeled Uridine Ribonucleoside Phosphoramidite.

A combined enzymatic and chemical synthesis of a 2'-O-cyanoethoxymethyl (CEM) protected [1',6-13 C2 , 5-2 H]-uridine phosphoramidite is described herein. This is the first report of an atom-specific nucleobase and ribose labeled 2'-O-CEM protected ribonucleoside phosphoramidite. Importantly, the CEM 2'-OH protecting group permits the efficient solid-phase synthesis of large (>60 nucleotides) RNAs with good yield and purity. The new isotope-labeled phosphoramidite can therefore be applied to nuclear magnetic resonance (NMR) spectroscopy studies. Specifically, the [1',6-13 C2 , 5-2 H]-uridine phosphoramidite can be used to make position-specifically labeled RNAs for NMR analysis without complications from resonance overlap and scalar and dipolar couplings. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of the ribonucleoside 6 Basic Protocol 2: Synthesis of the ribonucleoside phosphoramidite 11.

Conformational Dynamics of the Hepatitis B Virus Pre-genomic RNA on Multiple Time Scales: Implications for Viral Replication.

Human hepatitis B virus (HBV) replication is initiated by the binding of the viral polymerase (P) to epsilon (ε), an ≈85-nucleotide (nt) cis-acting regulatory stem-loop RNA located at the 5'-end of the pre-genomic RNA (pgRNA). This interaction triggers P and pgRNA packaging and protein-primed reverse transcription and is therefore an attractive therapeutic target. Our recent nuclear magnetic resonance (NMR) structure of ε provides a useful starting point toward a detailed understanding of HBV replication, and hints at the functional importance of ε dynamics. Here, we present a detailed description of ε motions on the ps to ns and µs to ms time scales by NMR spin relaxation and relaxation dispersion, respectively. We also carried out molecular dynamics simulations to provide additional insight into ε conformational dynamics. These data outline a series of complex motions on multiple time scales within ε. Moreover, these motions occur in mostly conserved nucleotides from structural regions (i.e., priming loop, pseudo-triloop, and U43 bulge) that biochemical and mutational studies have shown to be essential for P binding, P-pgRNA packaging, protein-priming, and DNA synthesis. Taken together, our work implicates RNA dynamics as an integral feature that governs HBV replication.

Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs.

RNA is central to the proper function of cellular processes important for life on earth and implicated in various medical dysfunctions. Yet, RNA structural biology lags significantly behind that of proteins, limiting mechanistic understanding of RNA chemical biology. Fortunately, solution NMR spectroscopy can probe the structural dynamics of RNA in solution at atomic resolution, opening the door to their functional understanding. However, NMR analysis of RNA, with only four unique ribonucleotide building blocks, suffers from spectral crowding and broad linewidths, especially as RNAs grow in size. One effective strategy to overcome these challenges is to introduce NMR-active stable isotopes into RNA. However, traditional uniform labeling methods introduce scalar and dipolar couplings that complicate the implementation and analysis of NMR measurements. This challenge can be circumvented with selective isotope labeling. In this review, we outline the development of labeling technologies and their application to study biologically relevant RNAs and their complexes ranging in size from 5 to 300 kDa by NMR spectroscopy.

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.

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.

Quantifying the effects of long-range 13C-13C dipolar coupling on measured relaxation rates in RNA.

Selective stable isotope labeling has transformed structural and dynamics analysis of RNA by NMR spectroscopy. These methods can remove 13C-13C dipolar couplings that complicate 13C relaxation analyses. While these phenomena are well documented for sites with adjacent 13C nuclei (e.g. ribose C1'), less is known about so-called isolated sites (e.g. adenosine C2). To investigate and quantify the effects of long-range (> 2 Å) 13C-13C dipolar interactions on RNA dynamics, we simulated adenosine C2 relaxation rates in uniformly [U-13C/15N]-ATP or selectively [2-13C]-ATP labeled RNAs. Our simulations predict non-negligible 13C-13C dipolar contributions from adenosine C4, C5, and C6 to C2 longitudinal (R1) relaxation rates in [U-13C/15N]-ATP labeled RNAs. Moreover, these contributions increase at higher magnetic fields and molecular weights to introduce discrepancies that exceed 50%. This will become increasingly important at GHz fields. Experimental R1 measurements in the 61 nucleotide human hepatitis B virus encapsidation signal ε RNA labeled with [U-13C/15N]-ATP or [2-13C]-ATP corroborate these simulations. Thus, in the absence of selectively labeled samples, long-range 13C-13C dipolar contributions must be explicitly taken into account when interpreting adenosine C2 R1 rates in terms of motional models for large RNAs.

Solid-Phase Chemical Synthesis of Stable Isotope-Labeled RNA to Aid Structure and Dynamics Studies by NMR Spectroscopy.

RNA structure and dynamic studies by NMR spectroscopy suffer from chemical shift overlap and line broadening, both of which become worse as RNA size increases. Incorporation of stable isotope labels into RNA has provided several solutions to these limitations. Nevertheless, the only method to circumvent the problem of spectral overlap completely is the solid-phase chemical synthesis of RNA with labeled RNA phosphoramidites. In this review, we summarize the practical aspects of this methodology for NMR spectroscopy studies of RNA. These types of investigations lie at the intersection of chemistry and biophysics and highlight the need for collaborative efforts to tackle the integrative structural biology problems that exist in the RNA world. Finally, examples of RNA structure and dynamic studies using labeled phosphoramidites are highlighted.

Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine.

The vibrational reporter unnatural amino acid (UAA) 4-cyano-l-phenylalanine (pCNF) was genetically incorporated individually at three sites (5, 36, and 78) in the heme protein Caldanaerobacter subterraneus H-NOX to probe local hydration environments. The UAA pCNF was incorporated site-specifically using an engineered, orthogonal tRNA synthetase in E. coli. The ability of all of the pCNF-containing H-NOX proteins to form the ferrous CO, NO, or O2 ligated and unligated states was confirmed with UV-Vis spectroscopy. The solvation state at each site of the three sites of pCNF incorporation was assessed using temperature-dependent infrared spectroscopy. Specifically, the frequency-temperature line slope (FTLS) method was utilized to show that the nitrile group at site 36 was fully solvated and the nitrile group at site 78 was de-solvated (buried) in the heme pocket. The nitrile group at site 5 was found to be partially solvated suggesting that the nitrile group was involved in moderate strength hydrogen bonds. These results were confirmed by the determination of the X-ray crystal structure of the H-NOX protein construct containing pCNF at site 5.