A Light‐Driven DNA Nanomachine for the Efficient Photoswitching of RNA Digestion

Abstract

Recently, DNA has gained attention as one of the most promising molecules for use in bottom-up nanotechnology. [1] In the last two decades, numerous DNA nanostructures with mechanical functions such as DNA tweezers, DNA walkers, and DNA gears have been constructed. [2,3] However, the practical use of DNA nanotechnology remains a great challenge. One of the problems limiting the application of DNA nanomachines is that oligo-DNAs or other small molecules have to be added as the "fuel" during each operation cycle, and "waste" molecules detrimentally accumulate in the system after several cycles.[3] Previously, we have tried to solve this problem by constructing a model photon-fueled nanomachine involving azobenzene moieties as photoswitches, based on the photoregulation of DNA hybridization. [4,5] No waste was produced because only light, the cleanest source of energy, was used to drive the nanomachine, and the operation could be repeated for many cycles without loss of efficiency.[4a] Efforts should be made to construct nanomachines that work on the single-molecule level, which is highly favorable for nanotechnology applications.[4c] In the present study, we constructed a machinelike photoresponsive DNAzyme that can work at the singlemolecule level (intramolecular nanomachine). The change of its topological conformation can be regulated simply by light irradiation. For the first time, complete ON-OFF photoswitching of RNA digestion has been realized by regulating the higher order structure of a DNAzyme-RNA complex.The 10-23 DNAzyme, captured from a DNA pool of random sequences by in vitro selection, was used here as the model system for constructing an RNA-cleaving nanomachine driven by photons.[6] As shown in Scheme 1 a, a photoresponsive machinelike DNAzyme (Dz7X) was constructed by attaching complementary azobenzene-modified sequences to both ends of the 10-23 DNAzyme. As in our previous design, the two azobenzene-modified sequences were able to form an interstrand-wedged duplex after hybridization. [4c,d] When visible light is applied, the azobenzene residues (X) take the trans form, and a very stable duplex structure involving seven azobenzene units and nine base pairs is formed.[4c] The modified DNAzyme Dz7X can be regarded as a DNA hairpin structure with a big loop (Scheme 1 a). In this case, the RNA-cleavage activity is expected to be suppressed because the topologically constrained higher order structure of the catalytic loop cannot form the correct conformation for cleavage, even when the RNA substrate hybridizes with both arms. On the other hand, when UV light is applied and azobenzene residues take the cis Scheme 1. Design of the machinelike photoresponsive DNAzyme (a) and sequences of DNAzymes and RNA targets used in this study (b). The azobenzene-modified DNAzymes show high activity only when azobenzenes (in red; its structure (X) is also shown) take the cis form after irradiation with UV light. RNA substrate (in green) was labeled with the fluorophore FITC at the 5' end. The sequence of catalytic lo...

Optochemical Control of Biological Processes in Cells and Animals

“…[136a,193,207] Photoswitchable oligonucleotides have enabled precise reversible control of nucleic acid based processes such as DNA crosslinking [208] and DNA–RNA hybridization. [209] More complex photoswitching tools have also been reported, such as azobenzene-modified DNAzymes and ribozymes [210] to reversibly control RNA cleavage. Diarylethene photoswitches incorporated at the C5 position of uracil were installed in the T7 promoter region of a double-stranded DNA template, thereby allowing photo-switching of transcription.…”

Functionally-interdependent shape-switching nanoparticles with controllable properties

“…Rationally designed DNA nanomachines can carry out a rotary motion by switching from B- to Z-DNA at high ionic strength (14), sense the pH (15,16) and respond to changes from visible to UV light (17). DNA ‘walkers’ are capable of directional movement based on strand displacement (18,19), enzymatic activity (20,21) or in accordance with the prescriptive DNA origami landscapes (22).…”

Pseudo-complementary PNA actuators as reversible switches in dynamic DNA nanotechnology

“…Hitherto existing applications have shown that architecturally simpler DNA nanostructures like molecular tweezers (21) and beacons (22), DNAzymes (23,24) or DNA tetrahedrons (25) are suited for light-induced switching, but this has not yet been implemented for the switching of more complex origami-based DNA structures. By using a double-stranded DNA (dsDNA) rotaxane architecture (26) that we reversibly switched between a mobile and stalled macrocycle, we have recently shown that light-induced switching operations based on dimethylazobenzene (DMAB)-functionalized DNA sequences result in a more robust switching behavior than oligodeoxynucleotides (ODNs) functionalized with unmodified azobenzene (27), and thus may find broader applications in DNA nanotechnology.…”