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bioRxiv · 10.1101/2023.11.28.569002

In vivo detection of DNA secondary structures using Permanganate/S1 Footprinting with Direct Adapter Ligation and Sequencing (PDAL-Seq)

Lahnsteiner, A.·Craig, S. J. C.·Kamali, K.·Weissensteiner, B.·McGrath, B.·Risch, A.·Makova, K.
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Abstract

DNA secondary structures are essential elements of the genomic landscape, playing a critical role in regulating various cellular processes. These structures refer to G-quadruplexes, cruciforms, Z-DNA or H-DNA structures, amongst others (collectively called non-B DN), which DNA molecules can adopt beyond the B conformation. DNA secondary structures have significant biological roles, and their landscape is dynamic and can rearrange due to various factors, including changes in cellular conditions, temperature, and DNA-binding proteins. Understanding this dynamic nature is crucial for unraveling their functions in cellular processes. Detecting DNA secondary structures remains a challenge. Conventional methods, such as gel electrophoresis and chemical probing, have limitations in terms of sensitivity and specificity. Emerging techniques, including next-generation sequencing and single-molecule approaches, offer promise but face challenges since these techniques are mostly limited to only one type of secondary structure. Here we describe an updated version of a technique permanganate/S1 nuclease footprinting, which uses potassium permanganate to trap single-stranded DNA regions as found in non-B structures, in combination with S1 nuclease digest and adapter ligation to detect genome-wide non-B formation. To overcome technical hurdles, we combined this method with direct adapter ligation and sequencing (PDAL-Seq). Furthermore, we established a user-friendly pipeline available on Galaxy to standardize PDAL-Seq data analysis. This optimized method allows the analysis of many types of DNA secondary structures that form in a living cell and will advance our knowledge of their roles in health and disease.

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Lahnsteiner, A., Craig, S. J. C., Kamali, K., Weissensteiner, B., McGrath, B., Risch, A., Makova, K.. 2023-11-28. In vivo detection of DNA secondary structures using Permanganate/S1 Footprinting with Direct Adapter Ligation and Sequencing (PDAL-Seq). https://doi.org/10.1101/2023.11.28.569002

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