TMV nanorods with programmed longitudinal domains of differently addressable coat proteins

Abstract

The spacing of functional nanoscopic elements may play a fundamental role in nanotechnological and biomedical applications, but is so far rarely achieved on this scale. In this study we show that tobacco mosaic virus (TMV) and the RNA-guided self-assembly process of its coat protein (CP) can be used to establish new nanorod scaffolds that can be loaded not only with homogeneously distributed functionalities, but with distinct molecule species grouped and ordered along the longitudinal axis. The arrangement of the resulting domains and final carrier rod length both were governed by RNA-templated two-step in vitro assembly. Two selectively addressable TMV CP mutants carrying either thiol (TMVCys) or amino (TMVLys) groups on the exposed surface were engineered and shown to retain reactivity towards maleimides or NHS esters, respectively, after acetic acid-based purification and re-assembly to novel carrier rod types. Stepwise combination of CP(Cys) and CP(Lys) with RNA allowed fabrication of TMV-like nanorods with a controlled total length of 300 or 330 nm, respectively, consisting of adjacent longitudinal 100-to-200 nm domains of differently addressable CP species. This technology paves the way towards rod-shaped scaffolds with pre-defined, selectively reactive barcode patterns on the nanometer scale.

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“…Lincei (Schlick et al 2005). In our experiments, we studied even a lysine mutant, TMV Lys (Geiger et al 2013), which shows a lysine residue that substitutes threonine at the position 158 of amino acid by genetic replacement. TMV presents 2130 identical copies of its coat protein, which means that the entire TMV shows 4260 solvent-exposed glutamic acids on the interior side and 2130 addressable tyrosines on its exterior surface; the TMV Lys mutants offer an additional 2130 surface-exposed lysine chains on the exterior surface (see Fig.…”

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“…TMV-lys displays a lysine residue instead of a serine at amino acid position 158; thus, the amine-functional lysine group is solvent-exposed and located at the C-terminus of the CP. 17 TMV-lys was extracted in yields of 1 mg of pure virus per gram of infected leaves. To obtain vcMMAEloaded TMV, the antimitotic drug was bioconjugated using a three-step reaction (Figure 1).…”

“…tuning the aspect ratio of TMV, selfassembling star-shapes or other higher order hierarchical assembles, or even switching shapes from rod-to-sphere, are also possible iterations of the TMV platform technology. [16][17][18][22][23][24] In this work, we bioconjugated TMV with a valine-citrulline monomethyl auristatin E (vcMMAE) pro-drug. Once targeted to the endolyosomal compartment of cancer cells, the dipeptide valine-citrulline (vc) is cleaved to release MMAE, an antimitotic and cytotoxic drug that inhibits polymerization of tubulin and thus interferes with cell replication, halts cancer growth, and induces apoptosis.…”

“…One example of this is the lysine-added mutant 'TMV-lys' that displays reactive lysine groups on the exterior protein shell and is easily modifiable using well known N-hydroxysuccinimide (NHS) chemistry. 17 In addition to the chemical and genetic engineering capabilities, precision shape-engineering, e.g. tuning the aspect ratio of TMV, selfassembling star-shapes or other higher order hierarchical assembles, or even switching shapes from rod-to-sphere, are also possible iterations of the TMV platform technology.…”

“…TMV is a stable, monodisperse, and biocompatible protein-based scaffold that presents an unparalleled opportunity for engineering. [14][15][16][17][18] The TMV structure is known to atomic resolution, 19 which allows for the identification of specific reactive groups to be targeted for bioconjugation of medical cargo, such as drugs, contrast agents, and/or targeting ligands. 20 Specifically, TMV is a 300 nm by 18 nm rigid hollow nanotube with a 4 nm wide interior channel.…”