Patel, Trushar

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https://opus.uleth.ca/handle/10133/5244

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Browsing Patel, Trushar by Author "D'souza, Simmone"

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    Current approaches for RNA-labelling to identify RNA-binding proteins
    (Canadian Science Publishing, 2020) Gemmill, Darren L.; D'souza, Simmone; Meier-Stephenson, Vanessa; Patel, Trushar R.
    RNA is involved in all domains of life, playing critical roles in a host of gene expression processes, host-defense mechanisms, cell proliferation, and diseases. A critical component in many of these events is the ability for RNA to interact with proteins. Over the past few decades, our understanding of such RNA–protein interactions and their importance has driven the search and development of new techniques for the identification of RNA-binding proteins. In determining which proteins bind to the RNA of interest, it is often useful to use the approach where the RNA molecule is the “bait” and allow it to capture proteins from a lysate or other relevant solution. Here, we review a collection of methods for modifying RNA to capture RNA-binding proteins. These include small-molecule modification, the addition of aptamers, DNA-anchoring, and nucleotide substitution. With each, we provide examples of their application, as well as highlight their advantages and potential challenges.
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    Development of a single-domain antibody to target a G-quadruplex located on the hepatitis B virus covalently closed circular DNA genome
    (American Society for Microbiology, 2024) Figueroa, Gerardo B.; D'souza, Simmone; Pereira, Higor S.; Vasudeva, Gunjan; Figueroa, Sara B.; Robinson, Zachary E.; Badmalia, Maulik D.; Meier-Stephenson, Vanessa; Corcoran, Jennifer A.; van Marle, Guido; Ni, Yi; Urban, Stephan; Coffin, Carla S.; Patel, Trushar R.
    To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine-quadruplexes (G4s) are a secondary structure that can be adopted by DNA and play a significant role in regulating viral replication, transcription, and translation. Antibody-based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein, we describe the development of a humanized single-domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small-Angle X-ray Scattering. Lentiviral transduction of S10 into HepG2-NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next-generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10.