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...
Figure 3. Photoswitching of RNA cleavage at the GU site by Dz7X. Visible light was applied at 0, 60, 120, and 180 min. UV light irradiation was carried out at 30, 90, and 150 min. The irradiation time for visible and UV light was 1 and 10 min, respectively. Figure 4. Effect of the topological constraint on RNA digestion by the 10-23 DNA enzyme (see sequences in Scheme 1 b).
We demonstrated the generality of a strategy for photoswitching the activity of functional oligonucleotides by modulating their topological structure. Our strategy was proved to be versatile because it can be used to photoregulate functional oligonucleotides, e.g., ribozymes and DNAzymes, which have two binding arms and a catalytic loop. Repeated reversible photoregulation of RNA cleavage by a ribozyme or a DNAzyme was achieved by attaching two photoresponsive strands, artificial oligomers involving azobenzene moieties and nucleobases capable of forming a duplex as the supraphotoswitch. Individual strands were attached to the 3' and 5' ends of a RNA-cleavage oligonucleotide. Thus, the topological structure of the ribozyme or DNAzyme was constrained, and RNA cleavage was greatly suppressed when the supraphotoswitch duplex formed (OFF state). In contrast, RNA cleavage resumed when the supraphotoswitch duplex dissociated (ON state). Light irradiation was used to repeatedly switch the supraphotoswitch between the ON and OFF states so that RNA cleavage activity could be efficiently photoregulated. Analysis of the regulatory mechanism showed that topological constraints suppressed the RNA cleavage by causing both structural changes at the catalytic site and lower binding affinity between the RNA substrates and the functional oligonucleotides.
Due to the high homology of the ATP sites of the JAK family, the development of selective inhibitors for a certain JAK isoform is extremely challenging. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Based on the clinical compound BMS-986165, through structure-activity relationship studies, a class of acyl compounds with excellent TYK2 inhibitory activity and selectivity to other subtypes of the JAK family was discovered.
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