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

Abstract

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.

From stars to stripes: RNA‐directed shaping of plant viral protein templates—structural synthetic virology for smart biohybrid nanostructures

“…This may be of major importance for presenting cell targeting, adhesion and differentiation‐mediating ligands in biomedical applications and 3D matrices used for tissue culture (L. A. Lee et al, ; X. Liu et al, ; Luckanagul et al, ; Maturavongsadit et al, ; Sitasuwan, Lee, Li, Nguyen, & Wang, ; Zan, Sitasuwan, Powell, Dreher, & Wang, ; Zhao, Lin, & Wang, ), but is also advantageous for applications relying for example, on enzyme and antibody display (Koch et al, ). Even smaller distances of coupling sites on TLPs have been generated for double‐mutant TMV CPs displaying both an additional lysine and an additional cysteine residue on the outer tube surface (Wege & Geiger, ). In addition, the zeta potential of the viral nanorod surface may be adjusted through incorporating a fraction of CP molecules with altered charge, in order to control the particles' deposition on surfaces and interactions with inorganic matter (Atanasova et al, ; Atanasova, Kim, Chen, Eiben, & Bill, ).…”

“…However, simple and efficient one-step conjugation to reactive amino acid side chains is typically achieved for the ε-NH 2groups of lysines by way of N-hydroxy succinimide (NHS) esters (Maedler, Bich, Touboul, & Zenobi, 2009), and for the thiol groups of cysteines often with maleimide-based reagents (Gunnoo & Madder, 2016). To make use of the large choice of commercial NHS ester-or maleimide-modified linkers and functional molecules such as dyes, enzymes or magnetic beads, TMV has been genetically engineered in a number of labs to display the preferred amino acids on the outer virion surface, with either a single new residue per CP (Demir & Stowell, 2002;Geiger et al, 2013;Smith et al, 2006;Yi et al, 2005), or both a lysine and a cysteine introduced into accessible CP regions (Wege & Geiger, 2018). These regions are either CP domains at or close to the protein termini, or a surface-exposed loop between aa 53 and 72 that could even be used to accommodate a 12 aa malarial epitope inserted downstream its proline (Pro) 63 in a pioneering study (Turpen et al, 1995).…”

Surface Functionalization of Rod-Shaped Viral Particles for Biomedical Applications

“…Steric hindrance of functional ligands on neighboring subunits (∼3 nm apart) can slow down or even prevent reactions from taking place at every exposed reaction site. , Furthermore, not all chemical reactions are equally efficient. Most standard residues and their associated chemistry suffer from poor kinetics and can require a large excess of reagent to achieve high labeling densities . This often makes downstream purification necessary, adding more production steps relative to direct genetic fusion.…”

“…These nucleophiles are so competent that VLP mutants presenting either residue on their outer surface are routinely used . Both groups can react with reactive electrophiles including acids, acrylates, maleimides, and N -hydroxy succinimide (NHS) esters . For example, a TMV-cysteine mutant was recently conjugated to the anticancer, topoisomerase II inhibitor doxorubicin with a maleimide group and an acid-cleavable linker, which facilitated drug release in cells .…”

Rip It, Stitch It, Click It: A Chemist’s Guide to VLP Manipulation

“…(Lee et al, 2020) high relaxivity MRI contrast agents(Bruckman et al, 2013) E97, E106 Texas Red maleimide,(Yi et al, 2005) RBITC,(Liu et al, 2016) FITC, Cy5,(Dharmarwardana et al, 2018) Mitoxantrone,(Lin and Steinmetz, 2018) Phenanthriplatin(Lin and Steinmetz, 2018;Liu et al, 2016;Shukla et al, 2021;Yi et al, 2005) T153K Prostate specific antigen,(Shukla et al, 2021) thrombolytic therapy(Geiger et al, 2013;Shukla et al, 2021) S123CMaleimide PEG conjugation(Koch et al, 2015;Zhou et al, 2013) K53, K68 Doxorubicin(Finbloom et al, 2016) E50Q, D77N Disassembly of TMV(Lu et al, 1996) T103C Au nanowires(Zhou et al, 2013) MBP TMV Gold nanoparticles(Love et al, 2015) S3C + T153K Dual functionalization(Wege and Geiger, 2018) …”