Nucleotide-Type Chemical Shift Assignment of the Encapsulated 40 kbp dsDNA in Intact Bacteriophage T7 by MAS Solid-State NMR

Gili Abramov, Chemistry, Tel Aviv University, Tel Aviv, Israel
Amir Goldbourt, Chemistry, Tel Aviv University, Tel Aviv, Israel

The icosahedral bacteriophage T7 is a 50 MDa virus that infects rough F^- Escherichia coli strains. The genome of T7 is a 40 kbp dsDNA molecule, which constitutes slightly over half of the total viral mass and arranged in a highly condensed form within the capsid shell. While the physical and morphological properties of T7 were extensively studied, mostly by Raman spectroscopy and cryo-EM, structural information on its capsid and particularly on its dsDNA, is scarce.

Here, we apply the magic-angle spinning (MAS) solid-state NMR technique to study the secondary structure of the 40 kbp dsDNA in a uniformly ¹³C and ¹⁵N labeled intact T7 phage. The isotopically enriched wild-type phage sample was prepared under fully hydrated conditions, and we present the complete ¹³C and the near-complete ¹⁵N nucleotide-type specific assignment of the sugar and base moieties, by using two-dimensional ¹³C–¹³C and ¹⁵N–¹³C correlation experiments. The obtained chemical shifts are interpreted as reporters of a B-form conformation of the encapsulated dsDNA. While MAS solid-state NMR 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 shows the feasibility of characterizing large and complex nucleic acid systems by solid-state NMR.