Peptide Assembly Directed and Quantified Using Megadalton DNA Nanostructures

Abstract

In nature, co-assembly of polypeptides, nucleic acids, and polysaccharides is used to create functional supramolecular structures. Here, we show that DNA nanostructures can be used to template interactions between peptides and to enable the quantification of multivalent interactions that would otherwise not be observable. Our functional building blocks are peptide–oligonucleotide conjugates comprising de novo designed dimeric coiled-coil peptides covalently linked to oligonucleotide tags. These conjugates are incorporated in megadalton DNA origami nanostructures and direct nanostructure association through peptide–peptide interactions. Free and bound nanostructures can be counted directly from electron micrographs, allowing estimation of the dissociation constants of the peptides linking them. Results for a single peptide–peptide interaction are consistent with the measured solution-phase free energy; DNA nanostructures displaying multiple peptides allow the effects of polyvalency to be probed. This use of DNA nanostructures as identifiers allows the binding strengths of homo- and heterodimeric peptide combinations to be measured in a single experiment and gives access to dissociation constants that are too low to be quantified by conventional techniques. The work also demonstrates that hybrid biomolecules can be programmed to achieve spatial organization of complex synthetic biomolecular assemblies.

Self‐Assembly of DNA–Peptide Supermolecules: Coiled‐Coil Peptide Structures Templated by d‐DNA and l‐DNA Triplexes Exhibit Chirality‐Independent but Orientation‐Dependent Stabilizing Cooperativity

“…The programmed self‐assembly of oligonucleotides (ONs) through hydrogen bond and π‐stacking interactions has inspired the burgeoning development of elaborate two‐ and three‐dimensional DNA nanostructures during the last decades . With sizes ranging from nano‐ to micrometers, the user‐defined geometry and the facile chemical functionalization of DNA nanostructures offer ideal nanoscale molecular construction tools for peptide and protein engineering with unparalleled spatial accuracy down to the ångström‐scale level with various chemical and biological applications as long term goals . However, only a few examples have demonstrated bottom‐up construction of artificial protein mimics using the programmability and adaptability of nucleic acid secondary structures, including DNA triplexes, G‐quadruplexes and multiple DNA junctions .…”

Suprakolloidale Selbstorganisation von bivalenten Janus‐3D‐DNA‐Origami über programmierbare, multivalente Wirt/Gast‐Wechselwirkungen

“…Die K a zwischen zwei Origami‐Interaktionsstellen haben wir mithilfe der von Turberfield et al. eingeführten Bildanalyse ermittelt . Tatsächlich skaliert die K a ‐Konstante nichtlinear als Funktion der Wirt/Gast‐Partnermenge und liegt um mehrere Größenordnungen über den auf dem Origami vorhandenen monovalenten Wirt/Gast‐Wechselwirkungen auf molekularer Ebene (ca.…”

Supracolloidal Self‐Assembly of Divalent Janus 3D DNA Origami via Programmable Multivalent Host/Guest Interactions

“…The host/guest interactions are located on both ends of the same duplexes running through the origami lattice, which allows for a perfect alignment of the interactions once two origami patches approach each other. We designed four cuboids with different numbers of docking strands (9,18,27,36; Figure 1 b). This can be achieved by adding the appropriate docking strands during the folding, to assure that indeed only a certain valency is generated.…”

“…[16] In contrast, typical 3D DNA origami nanoparticle concentrations are only in the nm to mm regime and we employ a concentration of 20 nm. Depending on the valency of the cuboid (9,18,27,36), the total concentration of Adm and bCD units is still only at a maximum of around 1 mm, hence a factor of 10 3 lower than needed for a greater than 80 % binding. We however hypothesized that the multi- valency, arising from placing the interacting motifs into close proximity at a patch may facilitate chelate cooperativity, greatly supporting robust supracolloidal fibril formation.…”

“…In the absence of Adm-ssDNA and bCD-ssDNA most of the docking-strand-modified cuboids are in monomeric form (Figure 2 a). Statistical analysis of TEM images gives an average degree of polymerization (X n ) of 1.1-1.2 for the four different cuboids (9,18,27,36), corresponding to a unimer fraction of greater than 80 %. Addition of the host and guest ssDNA strands triggers the supramolecular polymerization and the X n increases with the number of connector positions, hence the multivalency available on the cuboids (Figure 2 b).…”