About 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS coronavirus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule–based interventions. All coronaviruses utilize a molecular mechanism called programmed −1 ribosomal frameshift (−1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high −1 PRF rates and that it also harbors a −1 PRF attenuation element. Altering −1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar −1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate −1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit −1 PRF was similarly effective against −1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.

Despite the threat to human health posed by some single stranded RNA viruses, little is understood about their assembly. The goal of this work is to introduce a new tool for watching an RNA genome direct its own packaging and encapsidation by proteins. Contrast Variation Small Angle X-Ray Scattering (CV-SAXS) is a powerful tool with the potential to monitor the changing structure of a viral RNA through this assembly process. The proteins, through present, do not contribute to the measured signal. As a first step in assessing the feasibility of viral genome studies, the structure of encapsidated MS2 RNA was exclusively detected with CV-SAXS and compared with a structure derived from asymmetric cryoEM reconstructions. Additional comparisons with free RNA highlight the significant structural rearrangements induced by capsid proteins, and invite the application of time-resolved CV-SAXS to reveal interactions that result in efficient viral assembly.

Surface-sensitive X-ray scattering and spectroscopy techniques reveal significant adsorption of iron ions and iron-hydroxide (Fe(III)) complexes to a charge-neutral zwitterionic template of phosphatidylcholine (PC). The PC template is formed by a Langmuir monolayer of dipalmitoyl-PC (DPPC) that is spread on the surface of 2 to 40 μM FeCl3 solutions at physiological levels of KCl (100 mM). At 40 μM of Fe(III) as many as ∼3 iron atoms are associated with each PC group. Grazing incidence X-ray diffraction measurements indicate a significant disruption in the in-plane ordering of DPPC molecules upon iron adsorption. The binding of iron-hydroxide complexes to a neutral PC surface is yet another example of nonelectrostatic, presumably covalent bonding to a charge-neutral organic template. The strong binding and the disruption of in-plane lipid structure has biological implications on the integrity of PC-derived lipid membranes, including those based on sphingomyelin.

Time-resolved mixing experiments, in which a microfluidic device is used to rapidly mix two species together to initiate a reaction, are a powerful tool to collect snapshots of the time-progression of macromolecular interactions. These mixing experiments are compatible with a variety of experimental techniques as structural probes, but Small Angle X-ray Scattering (SAXS) is particularly well suited to these studies as it can capture changes in the overall shape, size, and level of compactness of biological macromolecules with unconstrained motions in solution. Flowfocused diffusive mixers have been used successfully for time-resolved SAXS experiments, but they require that one of the reactants is small and highly soluble to achieve the rapid diffusion required to uniformly initiate a reaction. Although this requirement can be easily met for a broad range of biological macromolecule-ligand systems, many drug targets tend to be partially hydrophobic, not highly soluble, and not easily available in large quantities. Additionally, diffusive mixers preclude the study of the interaction of two large biological macromolecules, such as two proteins or a protein-nucleic acid system, as the diffusion times for these larger molecules can be longer than the reaction times of interest.We present a novel coupling of the Kenics-style chaotic advection mixer with SAXS to study diverse classes of macromolecular interactions, including reactions between two large biological macromolecules or one large biological macromolecule and a ligand. The mixer is comprised of a series of eight helical elements with alternating left-and right-handedness. Rapid mixing is achieved by creating ultra-thin layers of each species via baker's transformations (stretching, splitting, and layering of the liquids), so even large proteins can be mixed in as fast as a few milliseconds. The mixer itself was fabricated with a NanoScribe 3D Printer and our sample cell design presents a sufficiently large observation region which permits a good signal to noise ratio. Timepoints from 10-2000 ms can be reached by changing flowrates or the position of the X-ray beam relative to end of the Kenics mixer. We used this mixer to study a variety of different biological questions, such as RNA folding, protein conformational changes, protein-protein associations, and protein-nucleic acid complex formation. With this mixer, we captured transient reaction states that evade observation by typical equilibrium measurements and visualization of these short-lived states can elucidate the mechanism of these reactions or reveal the initial stages of comple