RNA-stabilized protein nanorings: high-precision adapters for biohybrid design

Abstract

The equipment of technical devices with biogenic adapter structures enables effective integration of biofunctional units into advanced sensor, filtration or catalytic layouts. Plant virus-derived self-organizing supramolecular complexes are among the most promising soft-matter adapters due to their multivalence on the nanometer scale, precise dimensions, high availability and compatibility with routine conjugation chemistry. Bioengineering can tailor both the templates’ shapes and coupling sites and has been applied here to develop nucleoprotein ‘nanorings’ made of tobacco mosaic virus (TMV) building blocks. Short RNA-scaffolded four-turn helices of ≈68 protein subunits and ≈9 nm length, with 18 nm outer and 4 nm inner diameters, were generated efficiently in vitro. A structure-directing single-stranded 204-nucleotide ribonucleic acid containing the TMV origin of assembly-yielded colloidal preparations that have structural integrity and are well dispersed in a pH range from about 7 to 9. Two selectively addressable protein types with either an amino or a thiol group accessible were combined in the ring-like objects, allowing dual covalent coupling of a fluorescent dye first and to an isothiocyanate-covered substrate thereafter. Such precisely shaped nanoconstructs with distinct functional groups exposed in high surface densities offer novel opportunities as versatile adapter elements for the fabrication of extended bio/synthetic hybrid materials.

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

“…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, ). The final length of the nanotube is thus almost exclusively defined by that of the RNA, with the overall duration of assembly depending on the position of the OAs in the RNA, the CP aggregation states in the preparation, and the reaction conditions including educt concentrations and temperature (Butler, ; Butler & Lomonossoff, ; Klug, ; Kraft et al, ).…”

“…Brackets indicate four‐turn helices. Reproduced with permission from Altintoprak et al (). (c) Schematics of TMV particles in various lengths.…”

“…It should be added here, however, that in parallel to the RNA‐stabilized nanorings with superior pH stability and dispersal, also a number of engineered TMV CP types have been developed that allow the fabrication of similarly robust disks devoid of RNA, due to enhanced CP‐CP interactions (Bruckman et al, ; Dedeo et al, ; Finbloom et al, ; J. Zhang, Wang, Zhou, Chen, & Wang, ; J. Zhang, Zhou, & Wang, ; Zhou, Li, Dai, Meng, & Wang, ). Although several of them show an increased propensity for aggregation under certain conditions, they reflect the high interest in such bionanostructures for various further purposes, including vaccination, delivery of biopharmaceuticals and intravital imaging and nuclear magnetic resonance contrast agents, plasmonics and energy transfer (see Altintoprak et al, for an overview and Eiben et al, and the above references for details on the applications).…”

TMV Particles: The Journey From Fundamental Studies to Bionanotechnology Applications

“…RNA-scaffolded four-turn helices constitute the lower size limit of TMV-derived assemblies. Such nanorings of 9.2 nm thickness, stabilized by a 204 nt OAS-RNA and containing blends of distinct CP types, were not only used for attaching functional molecules to technical substrates (Altintoprak et al, 2017; Fig. 3) but recently have been employed as "pore-in-pore" inlays to implant their central 4 nm nanopores into solid-state membrane templates (Farajollahi et al, 2018).…”

Electrochemically-Driven Insertion of Biological Nanodiscs into Solid State Membrane Pores as a Basis for “Pore-In-Pore” Membranes

“…In this work, we describe a combination of biological functional units with an inorganic template according to [ 31 ] by applying concentration gradient-based and electrophoresis-driven diffusion: the insertion of biologically bottom-up self-assembled TMV-based nucleoprotein nanodisc structures [ 32 ] into the hexagonally-arranged conical pores of an artificial solid state membrane (SSM, Figure 1 ). The SSM is prepared using a top-down micelle technique [ 33 ].…”