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Browsing by Subject "dna barcoding"

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    Adaptation of HUH endonucleases for protein-DNA conjugation
    (2018-10) Lovendahl, Klaus
    The conjugation of proteins to DNA is a useful technique for basic research and medicine. Current methods for conjugation are limited in both specificity and efficiency, as they generally depend on nonspecific exogenous chemical crosslinkers or reversible DNA-binding proteins. In this work we have adapted a natural protein domain, known as the HUH domain, which targets DNA and forms a covalent phosphotyrosine adduct with the DNA backbone. We show that HUH domains react efficiently with specific sequences of unmodified single-stranded DNA, and we have identified five proteins which react orthogonally with distinct DNA sequences. We demonstrate a number of applications for this technology. HUH fusion proteins can be use to label proteins both in vitro and in cultured mammalian cells with excellent specificity. Fusing an HUH domain to the gene-editing Cas9 protein enhances the integration of a single-stranded donor DNA template in mammalian cells. Surface-adhered HUH protein can be used to tether DNA molecules for sensitive measurements in a single- molecule magnetic tweezer. Additionally, we show that an HUH domain protein can be rationally mutated to increase its stability and reactivity. This work presents a novel technology with wide applicability and presents several avenues for future work.