The 5'UTR of HCoV-OC43 adopts a topologically constrained structure to intrinsically repress translation.

The emergence of SARS-CoV-2, which is responsible for the COVID-19 pandemic, has highlighted the need for rapid characterization of viral mechanisms associated with cellular pathogenesis. Viral UTRs represent conserved genomic elements that contribute to such mechanisms. Structural details of most CoV UTRs are not available, however. Experimental approaches are needed to allow for the facile generation of high-quality viral RNA tertiary structural models, which can facilitate comparative mechanistic efforts. By integrating experimental and computational techniques, we herein report the efficient characterization of conserved RNA structures within the 5'UTR of the HCoV-OC43 genome, a lab-tractable model coronavirus. We provide evidence that the 5'UTR folds into a structure with well-defined stem-loops (SLs) as determined by chemical probing and direct detection of hydrogen bonds by NMR. We combine experimental base-pair restraints with global structural information from SAXS to generate a 3D model that reveals that SL1-4 adopts a topologically constrained structure wherein SLs 3 and 4 coaxially stack. Coaxial stacking is mediated by short linker nucleotides and allows SLs 1 to 2 to sample different cojoint orientations by pivoting about the SL3,4 helical axis. To evaluate the functional relevance of the SL3,4 coaxial helix, we engineered luciferase reporter constructs harboring the HCoV-OC43 5'UTR with mutations designed to abrogate coaxial stacking. Our results reveal that the SL3,4 helix intrinsically represses translation efficiency since the destabilizing mutations correlate with increased luciferase expression relative to wildtype without affecting reporter mRNA levels, thus highlighting how the 5'UTR structure contributes to the viral mechanism.

Characterization of tautomeric forms of anti-cancer drug gemcitabine and their interconversion upon mechano-chemical treatment, using ATR-FTIR spectroscopy and complementary methods.

Gemcitabine is a widely used anti-cancer drug of pyrimidine structure, which can exist as a free base molecular form in crystals. Tautomers are structural isomers of molecules, which interconvert via proton transfer. Mechano-chemistry studies reactions of solids under mechanical impact. We investigated gemcitabine free base for the presence of specific molecular tautomers, using ATR-FTIR spectroscopic analysis, powder XRD, optical microscopy and HPLC. The amino-keto tautomer has the characteristic infrared (IR) peak of the amino group at 3390 cm-1. For the first time, the imino-keto tautomer of gemcitabine free base was detected. The imino-keto tautomer has the characteristic IR peak of the =N-H group, and its peak due to the CO group in pyrimidine ring is shifted vs. that of the amino-keto tautomer. This serves as the unique spectroscopic "fingerprints" of these tautomers. The ATR-FTIR spectroscopic analysis shows that gemcitabine free base can be enriched with the amino-keto or the imino-keto tautomer. Further, we studied the transformation of gemcitabine free base in crystals between its tautomers under conditions of liquid-assisted grinding (LAG). The imino-keto tautomer undergoes tautomerization to the amino-keto tautomer, while the amino-keto tautomer remains stable. No destruction of molecules of gemcitabine free base, when present as either tautomer, occurs during LAG as was verified by the HPLC-UV analysis. LAG is a new, straightforward, facile and fast method to interconvert tautomers in crystals, and ATR-FTIR spectroscopy is a method of choice to study tautomerization reactions of pharmaceuticals. The presented approach is promising for analysis of crystals of drugs containing one or more than one tautomer, and the knowledge-driven design of composite materials, which contain specific tautomeric molecular forms of pyrimidines, purines and other biologically active heterocyclic compounds.

The Repurposing of Cellular Proteins during Enterovirus A71 Infection.

Viruses pose a great threat to people's lives. Enterovirus A71 (EV-A71) infects children and infants all over the world with no FDA-approved treatment to date. Understanding the basic mechanisms of viral processes aids in selecting more efficient drug targets and designing more effective antivirals to thwart this virus. The 5'-untranslated region (5'-UTR) of the viral RNA genome is composed of a cloverleaf structure and an internal ribosome entry site (IRES). Cellular proteins that bind to the cloverleaf structure regulate viral RNA synthesis, while those that bind to the IRES also known as IRES trans-acting factors (ITAFs) regulate viral translation. In this review, we survey the cellular proteins currently known to bind the 5'-UTR and influence viral gene expression with emphasis on comparing proteins' functions and localizations pre- and post-(EV-A71) infection. A comprehensive understanding of how the host cell's machinery is hijacked and reprogrammed by the virus to facilitate its replication is crucial for developing effective antivirals.

Hygroscopic metal-organic framework MIL-160(Al): In-situ time-dependent ATR-FTIR and gravimetric study of mechanism and kinetics of water vapor sorption.

Metal-organic frameworks (MOFs) are advanced highly porous coordination polymers of high interest to separations, environmental remediation, catalysis, and biomedicine. While many MOFs are unstable in water and aqueous solutions, aluminum MOFs (Al-MOFs) offer an unprecedented stability. First, we synthesize unusual highly hygroscopic Al-MOF MIL-160(Al), purify it and assign FTIR peaks to specific groups as potential water binding sites. Further, we introduce a novel method of in-situ time-dependent ATR-FTIR spectroscopy to detect specific binding sites in MIL-160(Al) and investigate the progress of reaction. Specifically, we combine in-situ time-dependent ATR-FTIR spectroscopy with using water as "spectroscopic probe" to determine binding sites in MIL-160(Al) and their evolution during the reaction. The in-situ time-dependent ATR-FTIR spectra provide evidence of water bonding to: the µ-OH group, the carboxylate anion COO- in 2,5-FDCA2- linker, oxygen atom in the furan ring of the linker, and the C-C and C-H bonds of the furan ring of the linker. Then, we conduct mechanistic and kinetic study of sorption of water vapor on MIL-160(Al) in air using the combination of two complementary in-situ time-dependent methods: the ATR-FTIR spectroscopy and gravimetric analysis. Water vapor sorption on MIL-160(Al) results in the solid-state adsorption complex with up to four water molecules per unit of MIL-160(Al). Chemical kinetics of water sorption on MIL-160(Al) follows a pseudo-first order rate law and it is consistent with dynamics and timescale revealed by in-situ time-dependent ATR-FTIR. The combination of two in-situ time-dependent methods, the ATR-FTIR spectroscopy and gravimetry, forms a new powerful experimental approach to facilely study mechanisms, stoichiometry and chemical kinetics of various solid-gas reactions in the ambient and controlled environments.