Chemistry

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https://hdl.handle.net/10323/11885

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Browsing Chemistry by Author "Avery, Adam"

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    Assessment of Frequency, Degradation, Normalization, Inhibition, and Potential Surrogates of the Sars-Cov-2 Gene Targets
    (2023-01-01) Hunawill, Emily; Szlag, David; Wu, Colin; Avery, Adam; Wendell, Doug; Westrick, Randal
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Our study focuses on identifying and quantifying the presence of SARS-CoV-2 in the sewage based on identifying three gene targets which target the nucleocapsid gene (N1 and N2) and the envelope gene (E). The first set of experiments looked at the degradation of these targets at 4C, 25C, and 35C. As temperature increased so did the rate constants. Based on the half-life data, N1 degraded faster than N2 at 4C, all targets degraded at the same rate at 25C, and E degraded faster than N1 and N2 at 35C. Frequency of the gene targets was then assessed. E occurred less frequently than N1 and N2 adding 2.3 more SARS-CoV-2 detections leading to its removal from testing. The N1 and N2 gene targets were both necessary with removal of one resulting in minimally 9.6 loss of SARS-CoV-2 detections. We investigated normalization of the N1 and N2 gene targets with four different fecal indicators (pepper mottled mild virus, HF183, lachno3, and crAssphage) to improve the correlation between the sewage signal and clinical cases. Normalizing the data did not result in an increased correlation between the clinical and sewage data. Bovine coronavirus (BCoV) and Phi6 viruses were evaluated as surrogates to estimate SARS-CoV-2 inhibition in a single duplex reaction. Duplexing these targets was successful without significant signal loss, and these targets were used to estimate SARS-CoV-2 inhibition. Compared to BCoV, Phi6 suggested over 10 more fully inhibited samples. Several workflow modifications including bovine serum albumin (BSA), dilution, polyvinylpyrrolidone, and pasteurization were applied to the sewage samples in an attempt to reduce sample inhibition. BSA was able to reduce inhibition for both N1 and N2. Lastly, BCoV, Phi6, and murine hepatitis virus (MHV) retrieval were compared to the retrieval of the SARS-CoV-2 gene targets in sewage to determine if they accurately depicted the amount of inhibition the SARS-CoV-2 targets exhibited. BCoV and MHV were better surrogates to assess SARS-CoV-2 inhibition. SARS-CoV-2 RNA concentration in sewage were shown to correlate with COVID-19 cases.
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    Mechanisms of G-quadruplex Recognition and Unfolding by the FANCJ Helicase and REV1 Polymerase
    (2024-01-01) Lowran, Kaitlin Antha; Wu, Colin G; Avery, Adam; Villa-Diaz, Luis G
    G-quadruplexes (G4s) are secondary structures formed by guanine-rich nucleic acid sequences. Accumulated G4s in cells can disrupt DNA replication, RNA transcription, and other cellular processes. G4 DNA can adopt parallel, antiparallel, or hybrid conformations depending on solution conditions and base sequence. Moreover, reactive oxygen species can readily convert guanine to 8-oxoguanine (8oxoG) to form 8oxoG4s. However, the precise mechanisms by which repair proteins recognize and remove such lesions remain unclear. The FANCJ DNA helicase possesses an AKKQ motif that functions as a G4-targeting site that binds to G4s and 8oxoG4s. Additionally, FANCJ interacts with the REV1 translesion synthesis polymerase to facilitate replication across G4-forming sites. Although REV1 can also target and disrupt G4s, it is unknown whether the activities of FANCJ and REV1 are redundant or if they act on specific G4 substrates. We hypothesized that the presence of 8oxoG would impact the folding and stability of parallel, antiparallel, and hybrid G4s in specific positions within the G4. Furthermore, we anticipated that FANCJ and REV1 would have distinct 8oxoG4 binding preferences that can be correlated with the physical properties of the 8oxoG4s. We predicted that the collaborative action of FANCJ and REV1 would repair tightly-folded 8oxoG4 structures, whereas REV1 alone may suffice for disrupting the loosely folded 8oxoG4s. Our results revealed that introducing 8oxo1 significantly reduced the thermal stability of both parallel and antiparallel G4 structures. While 8oxo5 destabilized hybrid structures. FANCJ and REV1 both preferred parallel G4 and 8oxoG4s due to their greater stability and tighter folding compared to hybrid or antiparallel conformations. Furthermore, FANCJ had the lowest affinity for antiparallel 8oxo3 G4s, while REV1 had a lower affinity for hybrid 8oxo1 G4s. These findings indicate that the presence of 8oxoG modification in G4 structures may hinder the binding of these proteins. The study revealed that both FANCJ and REV1 have distinct substrate-specific preferences for unfolding G4s. FANCJ demonstrated lower unfolding rates when 8oxo5 was present within the G4 stack, indicating that the position of the lesion plays a critical role in the unfolding efficiency. REV1, on the other hand, showed a lower unfolding rate for more stable G4 structures like the GGGT G4, but faster rates for the oxidized constructs. This research provides significant insights into the specific roles and mechanisms of FANCJ and REV1 in unfolding 8oxoG-damaged G4s, contributing to our understanding of DNA repair mechanisms.