Molecular simulations suggest that the stability of a folded macromolecule increases in a confined space due to entropic effects. However, due to the interactions between the confined molecular structure and the walls of the container, clear-cut experimental evidence for this prediction is lacking. Here, using DNA origami nanocages, we show the pure effect of confined space on the property of individual human telomeric DNA G-quadruplexes. We induce targeted mechanical unfolding of the G-quadruplex while leaving the nanocage unperturbed. We find that the mechanical and thermodynamic stabilities of the G-quadruplex inside the nanocage increase with decreasing cage size. Compared to the case of diluted or molecularly crowded buffer solutions, the G-quadruplex inside the nanocage is significantly more stable, showing a 100 times faster folding rate. Our findings suggest the possibility of co-replicational or co-transcriptional folding of G-quadruplex inside the polymerase machinery in cells.

While single-molecule sensing offers the ultimate detection limit, its throughput is often restricted as sensing events are carried out one at a time in most cases. 2D and 3D DNA origami nanostructures are used as expanded single-molecule platforms in a new mechanochemical sensing strategy. As a proof of concept, six sensing probes are incorporated in a 7-tile DNA origami nanoassembly, wherein binding of a target molecule to any of these probes leads to mechanochemical rearrangement of the origami nanostructure, which is monitored in real time by optical tweezers. Using these platforms, 10 pM platelet-derived growth factor (PDGF) are detected within 10 minutes, while demonstrating multiplex sensing of the PDGF and a target DNA in the same solution. By tapping into the rapid development of versatile DNA origami nanostructures, this mechanochemical platform is anticipated to offer a long sought solution for single-molecule sensing with improved throughput.

A photofunctionalized square bipyramidal DNA nanocapsule (NC) was designed and prepared for the creation of a nanomaterial carrier. Photocontrollable open/close system and toehold system were introduced into the NC for the inclusion and release of a gold nanoparticle (AuNP) by photoirradiation and strand displacement. The reversible open and closed states were examined by gel electrophoresis and atomic force microscopy (AFM), and the open behavior was directly observed by high-speed AFM. The encapsulation of the DNA-modified AuNP within the NC was carried out by hybridization of a specific DNA strand (capture strand), and the release of the AuNP was examined by addition of toehold-containing complementary DNA strand (release strand). The release of the AuNP from the NC was achieved by the opening of the NC and subsequent strand displacement.

Due to the small size of a nanoconfinement, the property of water contained inside is rather challenging to probe. Herein, we measured the amount of water molecules released during the folding of individual G-quadruplex and i-motif structures, from which water activities are estimated in the DNA nanocages prepared by 5 × 5 to 7 × 7 helix bundles (cross-sections, 9 × 9 to 15 × 15 nm). We found water activities decrease with reducing cage size. In the 9 × 9-nm cage, water activity was reduced beyond the reach of regular cosolutes such as polyethylene glycol (PEG). With this set of nanocages, we were able to retrieve the change in water molecules throughout the folding trajectory of G-quadruplex or i-motif. We found that water molecules absorbed from the unfolded to the transition states are much fewer than those lost from the transition to the folded states. The overall loss of water therefore drives the folding of G-quadruplex or i-motif in nanocages with reduced water activities.

Two-dimensional self-assembly of DNA origami structures was carried out using a connector that has connection sites at all four edges. By utilizing this four-way connector, five and eight origami monomers were assembled to form a cruciate and a hollow square structure, respectively.

No abstract

We demonstrate direct observation of the dynamic opening and closing behavior of photocontrollable DNA origami nanoscissors using high-speed atomic force microscopy (HS-AFM). First the conformational change between the open and closed state controlled by adjustment of surrounding salt concentration could be directly observed during AFM scanning. Then light-responsive moieties were incorporated into the nanoscissors to control these structural changes by photoirradiation. Using photoswitchable DNA strands, we created a photoresponsive nanoscissors variant and were able to distinguish between the open and closed conformations after respective irradiation with ultraviolet (UV) and visible (Vis) light by gel electrophoresis and AFM imaging. Additionally, these reversible changes in shape during photoirradiation were directly visualized using HS-AFM. Moreover, four photoswitchable nanoscissors were assembled into a scissor-actuator-like higher-order object, the configuration of which could be controlled by the open and closed switching induced by irradiation with UV and Vis light.

Regnase-1-mediated mRNA decay (RMD), in which inflammatory mRNAs harboring specific stem–loop structures are degraded, is a critical part of proper immune homeostasis. Prior to initial translation, Regnase-1 associates with target stem–loops but does not carry out endoribonucleolytic cleavage. Single molecule imaging revealed that UPF1 is required to first unwind the stem–loops, thus licensing Regnase-1 to proceed with RNA degradation. Following translation, Regnase-1 physically associates with UPF1 using two distinct points of interaction: The Regnase-1 RNase domain binds to SMG1-phosphorylated residue T28 in UPF1; in addition, an intrinsically disordered segment in Regnase-1 binds to the UPF1 RecA domain, enhancing the helicase activity of UPF1. The SMG1-UPF1–Regnase-1 axis targets pioneer rounds of translation and is critical for rapid resolution of inflammation through restriction of the number of proteins translated by a given mRNA. Furthermore, small-molecule inhibition of SMG1 prevents RNA unwinding in dendritic cells, allowing post-transcriptional control of innate immune responses.