Dynamic DNA-controlled “stop-and-go” assembly of well-defined protein domains on RNA-scaffolded TMV-like nanotubes

Abstract

A DNA-based approach allows external control over the self-assembly process of tobacco mosaic virus (TMV)-like ribonucleoprotein nanotubes: their growth from viral coat protein (CP) subunits on five distinct RNA scaffolds containing the TMV origin of assembly (OAs) could be temporarily blocked by a stopper DNA oligomer hybridized downstream (3') of the OAs. At two upstream (5') sites tested, simple hybridization was not sufficient for stable stalling, which correlates with previous findings on a non-symmetric assembly of TMV. The growth of DNA-arrested particles could be restarted efficiently by displacement of the stopper via its toehold by using a release DNA oligomer, even after storage for twelve days. This novel strategy for growing proteinaceous tubes under tight kinetic and spatial control combines RNA guidance and its site-specific but reversible interruption by DNA blocking elements. As three of the RNA scaffolds contained long heterologous non-TMV sequence portions that included the stopping sites, this method is applicable to all RNAs amenable to TMV CP encapsidation, albeit with variable efficiency most likely depending on the scaffolds' secondary structures. The use of two distinct, selectively addressable CP variants during the serial assembly stages finally enabled an externally configured fabrication of nanotubes with highly defined subdomains. The "stop-and-go" strategy thus might pave the way towards production routines of TMV-like particles with variable aspect ratios from a single RNA scaffold, and of nanotubes with two or even more adjacent protein domains of tightly pre-defined lengths.

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

“…In contrast, the RNA 3′‐portion undergoes an about five to three times slower, noncooperative encapsidation by small CP oligomers such as A‐protein and monomers (Butler & Lomonossoff, ; Fukuda, Ohno, Okada, Otsuki, & Takebe, ; Otsuki, Takebe, Ohno, Fukuda, & Okada, ; Turner, McGuigan, & Butler, ). Although there is some bias towards G residues in the third positions along the wildtype TMV RNA, thought to stabilize the assembled complex (Steckert & Schuster, ; Wilson & McNicol, ), numerous heterologous OAs‐containing RNA sequences have been packaged efficiently with TMV CP in vitro, and endogenous plant transcripts can be found in natural virus particles (the original references are briefly reviewed in Schneider et al, ). Hence, except for the OAs nucleotide stretch folding into its major stem‐loop A (position 5,447–5,515 of the TMV vulgare sequence, National Center for Biotechnology Information NCBI reference NC_001367.1 (Butler, ; Goelet et al, ; Zimmern, ), no further sequence prerequisites seem essential for the scaffolding function of the integral TMV RNA (see also Altintoprak et al, ).…”

“…Extensive RNA secondary structures (Gaddipati & Siegel, ; Sleat, Turner, Finch, Butler, & Wilson, ) and also hybridized, cross‐linked DNA strands (Fairall, Finch, Hui, Cantor, & Butler, ) are known to stall the assembly of the TMV ribonucleoprotein helix in a site‐specific manner. Hence, a transient blockage of TLP growth at preselected sequences by DNA “stopper elements”, and their externally induced removal through strand displacement as applied in dynamic DNA nanotechnology (Y. Zhang, Pan, et al, ; D. Y. Zhang & Seelig, ), appeared a reasonable strategy for the step‐by‐step fabrication of TMV‐like rods from distinct CP types (Figure c, d; Schneider et al, ). After interrupting the assembly of a TLP segment with a first CP species by a site‐specifically hybridized DNA oligonucleotide, and subsequent exchange of the first to a second CP type, removal of the DNA stopper should restart elongation of the nucleoprotein tube from the second CP source.…”

Tobacco Mosaic Virus as a Versatile Platform for Molecular Assembly and Device Fabrication

“…can be organized in discrete sections along the length of the particle by sequential introduction of the different CP variants to the RNA scaffold in vitro. This is due to the assembly process which progressively adds CP to the 5’ end of the RNA OAS, a process which can be more tightly controlled by the use of DNA oligonucleotides that can hybridize with or release from specific sequences of the exposed RNA to halt or allow continued assembly . Because the TMV rod length is primarily constrained by the length of its RNA, custom length RNA containing the OAS can be used to control particle length in vitro or in planta .…”

Covalent incorporation of tobacco mosaic virus increases the stiffness of poly(ethylene glycol) diacrylate hydrogels

“…29 In addition, we have recently achieved to produce VLPs with highly dened longitudinal domains. 30 With regard to hydrogels, TMV by itself and in combination with alginate hydrogels has been shown to have a positive effect on osteogenesis without provoking adverse immune reactions in vivo. [31][32][33] For these alginate gels, the impact of TMV particles on their mechanical properties has been characterised, demonstrating that TMV rods induced nonlinear responses to compression at high strains.…”