Building expanded structures from tetrahedral DNA branching elements, RNA and TMV protein

Abstract

By combining both chemical and enzymatic ligation with procedures guiding the self-assembly of nanotubular tobacco mosaic virus (TMV)-like particles (TLPs), novel nucleoprotein structures based on DNA-terminated branching elements, RNA scaffolds and TMV coat protein (CP) are made accessible. Tetrahedral tetrakis(hydroxybiphenyl)adamantane cores with four 5'-phosphorylated dinucleotide arms were coupled to DNA linkers by chemical ligation. The resulting three-dimensional (3D) branching elements were enzymatically ligated to the 3' termini of RNA scaffolds either prior to or after the RNAs' incorporation into TLPs. Thus, architectures with interconnected nanotube domains in two different length classes were generated, each containing 70 CP subunits per 10 nm length. Short TMV origin-of-assembly-containing RNA scaffolds ligated to the DNA allowed the growth of protein-coated 34 nm tubes on the terminal RNA strands in situ. Alternatively, 290 nm pre-fabricated tubes with accessible RNA 3' termini, attained by DNA blocking elements hybridized to the RNAs, were ligated with the branched cores. Both approaches resulted in four-armed nanoobjects, demonstrating a so far unique combination of organic synthesis of branching elements, enzymatic modifications, nucleic acid-based scaffolding and RNA-guided and DNA-controlled assembly of tubular RNA-encapsidating protein domains, yielding a novel class of 3D nucleoprotein architectures with polyvalent protein elements. In the long term, the production route might give rise to supramolecular systems with complex functionalities, installed via the orthogonal coupling of effector molecules to TLP domains.

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

“…Particles were visualized using small‐angle Pt shadowing. Reproduced from Wenz et al () with permission from The Royal Society of Chemistry. CP, coat protein; OAs, origin of assembly; TEM, transmission electron microscopy; TMV, tobacco mosaic virus…”

“…To exploit the rigidity of interconnected TLPs at the best, for example, for constructing functionalized TLP lattices and 3D meshworks, it is tempting to develop methods that may install them in fixed angular arrangements. Since TMV components have proven their compatibility with heterologous compounds in many studies, a multistep protocol integrating chemical and enzymatic ligation has been developed to interconnect TLPs in tetrahedral orientations by organic adamantane-based cores (Wenz et al, 2018). To achieve this challenging goal, four-armed organic branching elements (tetrakis(hydroxybiphenyl) adamantane (TBA) cores with phosphorylated dinucleotide (5 0 -pCC) arms [(5 0 -pCC) 4 TBA hybrids]) were elongated by DNA linkers through phosphoramidate ligation, purified via gel electrophoresis, phosphorylated enzymatically and ligated to the 3 0 -termini of OAs-containing RNA scaffolds in the presence of DNA splints by T4 RNA ligase 2.…”

DNA Functional Materials Assembled from Branched DNA: Design, Synthesis, and Applications

“…165 This adamantane-core branched DNA could be assembled with eight arms simultaneously, almost reaching the limit of arm number, and could derivate into DNAprotein hybrids under the manipulation of ligase enzymes. 161,166 In addition, the geometry and flexibility of organic-molecule cores and the length of spacer units both affected the assembly of branched DNA in well-defined supramolecular conformations, which were demonstrated by synergistic experimental study and molecular dynamics simulations. 167,168 The highly flexible cores (e.g., tris(oxypropyloxymethyl)methyl) tended to form cage dimers, while relatively rigid organic cores (e.g., tetraphenylmethane) greatly biases the assembly of polymeric nanoparticles.…”

“…The assembled duplex had higher thermostability than linear counterparts judged from melting temperature. , More than that, the branched DNA hybrid with adamantane as an organic core even formed a new material when reaching 95 °C in case of only two palindrome complementary bases . This adamantane-core branched DNA could be assembled with eight arms simultaneously, almost reaching the limit of arm number, and could derivate into DNA-protein hybrids under the manipulation of ligase enzymes. , In addition, the geometry and flexibility of organic-molecule cores and the length of spacer units both affected the assembly of branched DNA in well-defined supramolecular conformations, which were demonstrated by synergistic experimental study and molecular dynamics simulations. , The highly flexible cores (e.g., tris­(oxypropyl­oxymethyl)­methyl) tended to form cage dimers, while relatively rigid organic cores (e.g., tetraphenyl­methane) greatly biases the assembly of polymeric nanoparticles . The nanoparticle assemblies had the narrowly dispersed size and size-tunability by controlled-adjusting assembly conditions, including monomers concentrations, time, and salt concentrations, as well as good cellular internalization and DNase resistance.…”

Synthesis and applications of anisotropic nanoparticles with precisely defined dimensions

“…We believe that templated assembly has great untapped potential to address this problem. The redirection of viral coat protein assembly using non-natural nanostructure templates is a rapidly developing field, with recent examples using branched RNA, 302,303 DNA, 304,305 polymers, 306 metal nanorods 307 and supramolecular amphiphile structures. 308 This idea was recently extended to the assembly of viral coat protein mimics.…”