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Obstacles in quantifying A-to-I RNA editing by Sanger sequencing.
Methods Enzymol
January 2025
Faculty of Biology, Technion - Israel Institute of Technology, Technion City, Haifa, Israel. Electronic address:
Adenosine-to-Inosine (A-to-I) RNA editing is the most prevalent type of RNA editing, in which adenosine within a completely or largely double-stranded RNA (dsRNA) is converted to inosine by deamination. RNA editing was shown to be involved in many neurological diseases and cancer; therefore, detection of A-to-I RNA editing and quantitation of editing levels are necessary for both basic and clinical biomedical research. While high-throughput sequencing (HTS) is widely used for global detection of editing events, Sanger sequencing is the method of choice for precise characterization of editing site clusters (hyper-editing) and for comparing levels of editing at a particular site under different environmental conditions, developmental stages, genetic backgrounds, or disease states.
View Article and Find Full Text PDFSingle-molecule assay reveals the impact of composition, RNA duplex, and inhibitors on the binding dynamics of SARS-CoV-2 polymerase complex.
bioRxiv
January 2025
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, T6G 2E1, Canada.
The genome replication of SARS-CoV-2, the causative agent of COVID-19, involves a multi-subunit replication complex consisting of non-structural proteins (nsps) 12, 7 and 8. While the structure of this complex is known, the dynamic behavior of the subunits interacting with RNA is missing. Here we report a single-molecule protein-induced fluorescence enhancement (SM-PIFE) assay to monitor binding dynamics between the reconstituted or co-expressed replication complex and RNA.
View Article and Find Full Text PDFSequence-Dependent Slowdown of DNA Translocation Using Transmembrane RNA-DNA Interactions in MoS Nanopore.
J Phys Chem B
January 2025
Institute of Quantitative Biology, College of Life Sciences, and School of Physics, Zhejiang University, Hangzhou, Zhejiang 310058, China.
The emergence of nanopores in two-dimensional (2D) nanomaterials offers an attractive solid-state platform for high-throughput and low-cost DNA sequencing. However, several challenges remain to be addressed before their wide application, including the too-fast DNA translocation speed (compared to state-of-the-art single nucleoside detection techniques) and too large noise/signal ratios due to DNA fluctuations inside the nanopores. Here, we use molecular dynamics (MD) simulations to demonstrate the feasibility of utilizing RNA-DNA interactions in modulating DNA translocations in 2D MoS nanopores.
View Article and Find Full Text PDFRegioselective N1-ribosylation of hydantoin: synthesis and properties of the first contracted uridine analog.
Chem Commun (Camb)
January 2025
Department of Chemistry, Bar-Ilan University, Ramat-Gan 5290002, Israel.
Modified nucleosides are vital in mRNA vaccines. We developed a contracted uridine analog, N1-hydantoinyl-ribose, HR, using steric shields to invert the regioselectivity of the classic Vorbrüggen reaction. We report synthetic routes and explore HR features such as acidity, stability, base pairing/stacking, and crystal/solution conformation compared to uridine.
View Article and Find Full Text PDFOne-pot ligation of multiple mRNA fragments on dsDNA splint advancing regional modification and translation.
Nucleic Acids Res
January 2025
Key Laboratory of Bio-resource and Eco-environment of Ministry of Education, The College of Life Sciences, Sichuan University, 24 South Section 1, 1st Ring Road, Chengdu, Sichuan 610064, P.R. China.
Region-specific RNA modifications are crucial for advancing RNA research and therapeutics, including messenger RNA (mRNA)-based vaccines and immunotherapy. However, the predominant method, synthesizing regionally modified mRNAs with short single-stranded DNA (ssDNA) splints, encounters challenges in ligating long mRNA fragments due to the formation of RNA self-folded complex structures. To address this issue, we developed an efficient strategy using an easily obtained long double-stranded DNA (dsDNA) as a ligation splint after in situ denaturing, while parts of this dsDNA are the templates for transcribing mRNA fragments.
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