The promutagenic process known as translesion DNA synthesis reflects the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template. To study the underlying mechanism of nucleotide selection during this process, we quantified the incorporation of various non-natural nucleotide analogs opposite an abasic site, a non-templating DNA lesion. Our kinetic studies using the bacteriophage T4 DNA polymerase reveal that the π-electron surface area of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site. A remaining question is whether the selective insertion of these non-hydrogen-bonding analogs can be achieved through optimization of shape and π-electron density. In this report, we describe the synthesis and kinetic characterization of four novel nucleotide analogs, 5-cyanoindolyl-2′-deoxyriboside 5′-triphosphate (5-CyITP), 5-ethyleneindolyl-2′-deoxyriboside 5′-triphosphate (5-EyITP), 5-methylindolyl-2′-deoxyriboside 5′-triphosphate (5-MeITP), and 5-ethylindolyl-2′-deoxyriboside 5′-triphosphate (5-EtITP). Kinetic analyses indicate that the overall catalytic efficiencies of all four nucleotides are related to their base-stacking properties. In fact, the catalytic efficiency for nucleotide incorporation opposite an abasic site displays a parabolic trend in the overall π-electron surface area of the non-natural nucleotide. In addition, each non-natural nucleotide is incorporated opposite templating DNA ∼100-fold worse than opposite an abasic site. These data indicate that selectivity for incorporation opposite damaged DNA can be achieved through optimization of the base-stacking properties of the incoming nucleotide.

Measles, hepatitis C, and COVID-19 are significant human diseases caused by RNA viruses. While vaccines exist to prevent infections, there are a small number of currently available therapeutic agents that can effectively treat these diseases after infection occurs. This study explores a new therapeutic strategy using a small molecule designated polyprenyl immunostimulant (PI) to increase innate immune responses and combat viral infections. Using a multi-disciplinary approach, this study quantifies the effects of PI in mice and THP-1 cells using flow cytometry to identify immune phenotypic markers and mass spectroscopy to monitor the metabolomic profiles of immune cells perturbed by PI treatment. The metabolomic studies identified that sphinganine and ceramide, which are precursors of sphingosine-1-phosphate (S1P), were the common metabolites upregulated in THP-1 and mice blood. Sphingosine-1-phosphate can mediate the trafficking of T cells, whereas ceramide can signal the activation and proliferation of T cells, thereby modulating the mammalian host’s immunity. To demonstrate proof-of-principle, a case study was conducted to examine the benefit of administering PI to improve the outcomes of a feline co-infected with two distinct RNA viruses—feline leukemia virus and feline infectious peritonitis virus. Both viruses produce deadly symptoms that closely resemble RNA viruses that infect humans. The results identify quantifiable cellular and metabolic markers arising from PI treatment that can be used to establish a platform measuring the efficacy of PI in modulating the innate immune system.

As an ATP-dependent protease, the quality control functions of Lon have been extensively studied and reviewed in the literature. By contrast, very little research has been conducted to investigate Lon’s physiological functions and its mechanism as a regulatory protease. In this manuscript, we provided a survey of literature and data to convey that the lambda N (λN) protein is a suitable Escherichia coli Lon (ELon) substrate for studying the role played by Lon in regulating an RNA transcription process. For proof of principle, we demonstrated that the minimal component of the RNA transcription complex containing RNA polymerase (RNAP) and the σ factor can inhibit λN degradation by ELon through SDS-PAGE, and the carboxyl-terminal of λN is important for Lon competing with RNAP interaction. Using negative stain electron microscopy, we obtained structural evidence to show that λN lacking the carboxyl-terminal flanked by residues 99-107 interacted with ELon differently than full-length λN. Taken together, the activity and EM data provide a starting point for performing a physiological enzymology study on the contribution of ELon toward RNA transcription.