Dual Functionalization of Rod-Shaped Viruses on Single Coat Protein Subunits

Plant viruses are emerging as versatile tools for nanotechnology applications since it is possible to modify their multivalent protein surfaces and thereby introduce and display new functionalities. In this chapter, we describe a tobacco mosaic virus (TMV) variant that exposes two selectively addressable amino acid moieties on each of its 2130 coat protein (CP) subunits. A lysine as well as a cysteine introduced at accessible sites of every CP can be modified with amino- and/or thiol-reactive chemistry such as N-hydroxysuccinimide esters (NHS ester) and maleimide containing reagents alone or simultaneously. This enables the pairwise immobilization of distinct molecules in close vicinity to each other on the TMV surface by simple standard conjugation protocols. We describe the generation of the mutations, the virus propagation and isolation as well as the dual functionalization of the TMV variant with two fluorescent dyes. The labeling is evaluated by SDS-PAGE and spectrophotometry and the degree of labeling (DOL) calculated.