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Nucleotide-type chemical shift assignment of the encapsulated 40 kbp dsDNA in intact bacteriophage T7 by MAS solid-state NMR

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Abstract

The icosahedral bacteriophage T7 is a 50 MDa double-stranded DNA (dsDNA) virus that infects Escherichia coli. Although there is substantial information on the physical and morphological properties of T7, structural information, based mostly on Raman spectroscopy and cryo-electron microscopy, is limited. Here, we apply the magic-angle spinning (MAS) solid-state NMR (SSNMR) technique to study a uniformly 13C and 15N labeled wild-type T7 phage. We describe the details of the large-scale preparation and purification of an isotopically enriched phage sample under fully hydrated conditions, and show a complete 13C and a near-complete 15N nucleotide-type specific assignment of the sugar and base moieties in the 40 kbp dsDNA of T7 using two-dimensional 13C–13C and 15N–13C correlation experiments. The chemical shifts are interpreted as reporters of a B-form conformation of the encapsulated dsDNA. While MAS SSNMR was found to be extremely useful in determining the structures of proteins in native-like environments, its application to nucleic acids has lagged behind, leaving a missing 13C and 15N chemical shift database. This work therefore expands the 13C and 15N database of real B-form DNA systems, and opens routes to characterize more complex nucleic acid systems by SSNMR.

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Acknowledgments

We would like to thank Dr. Udi Qimron from the Department of Human Microbiology, Faculty of Medicine in Tel Aviv University, for providing initial stocks of wild-type T7 phage and its host. We thank Anat Haimovich for the TEDOR simulations. Financial support was provided by the Israel Science Foundation. Partial support for the spectrometer was given by the center for Nanoscience and Nanotechnology of Tel Aviv University.

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  1. School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, 69978, Ramat Aviv, Tel Aviv, Israel

    Gili Abramov & Amir Goldbourt

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  1. Gili Abramov
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  2. Amir Goldbourt
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Correspondence to Amir Goldbourt.

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Abramov, G., Goldbourt, A. Nucleotide-type chemical shift assignment of the encapsulated 40 kbp dsDNA in intact bacteriophage T7 by MAS solid-state NMR. J Biomol NMR 59, 219–230 (2014). https://doi.org/10.1007/s10858-014-9840-4

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