A phosphoramidite monomer bearing an azobenzene is synthesized from D-threoninol. Using this monomer, azobenzene moieties can be introduced into oligodeoxyribonucleotide (DNA) at any position on a conventional DNA synthesizer. With this azobenzene-tethered DNA, formation and dissociation of a DNA duplex can be reversibly photo-regulated by cis-trans isomerization of the azobenzene. When the azobenzene takes a trans-form, a stable duplex is formed. After isomerization of the trans-azobenzene to its cis-form by UV-light irradiation (300 nm < lambda < 400 nm), the duplex can be dissociated into two strands. The duplex is reformed on photo-induced cis-trans isomerization (lambda > 400 nm). The introduction of azobenzenes into the T7 promoter at specific positions also efficiently and reversibly photo-regulates transcription by T7-RNA polymerase. The reversible regulation can be repeated many times without causing damage to the DNA or the azobenzene moiety. These procedures take approximately 10 d to complete.

The duplex-forming activity of an oligonucleotide has been photoregulated by making use of the isomerization of an azobenzene moiety in the side chain. When the azobenzene moiety is isomerized from the trans form to the cis form upon photoirradiation, the melting temperature of the duplex between the oligonucleotide and its complementary counterpart is significantly lowered, and the duplex is largely dissociated into two single-stranded oligonucleotides (shown schematically).

CONSPECTUS: DNA is regarded as an excellent nanomaterial due to its supramolecular property of duplex formation through A-T and G-C complementary pairs. By simply designing sequences, we can create any desired 2D or 3D nanoarchitecture with DNA. Based on these nanoarchitectures, motional DNA-based nanomachines have also been developed. Most of the nanomachines require molecular fuels to drive them. Typically, a toehold exchange reaction is applied with a complementary DNA strand as a fuel. However, repetitive operation of the machines accumulates waste DNA duplexes in the solution that gradually deteriorate the motional efficiency. Hence, we are facing an "environmental problem" even in the nanoworld. One of the direct solutions to this problem is to use clean energy, such as light. Since light does not contaminate the reaction system, a DNA nanomachine run by a photon engine can overcome the drawback of waste that is a problem with molecular-fueled engines. There are several photoresponsive molecules that convert light energy to mechanical motion through the change of geometry of the molecules; these include spiropyran, diarylethene, stilbene, and azobenzene. Although each molecule has both advantages and drawbacks, azobenzene derivatives are widely used as "molecular photon engines". In this Account, we review light-driven DNA nanomachines mainly focusing on the photoresponsive DNAs that we have developed for the past decade. The basis of our method is installation of an azobenzene into a DNA sequence through a d-threoninol scaffold. Reversible hybridization of the DNA duplex, triggered by trans-cis isomerization of azobenzene in the DNA sequences by irradiation with light, induces mechanical motion of the DNA nanomachine. Moreover we have successfully developed azobenzene derivatives that improve its photoisomerizaition properties. Use of these derivatives and techniques have allowed us to design various DNA machines that demonstrate sophisticated motion in response to lights of different wavelengths without a drop in photoregulatory efficiency. In this Account, we emphasize the advantages of our methods including (1) ease of preparation, (2) comprehensive sequence design of azobenzene-tethered DNA, (3) efficient photoisomerization, and (4) reversible photocontrol of hybridization by irradiation with appropriate wavelengths of light. We believe that photon-fueled DNA nanomachines driven by azobenzene-derivative molecular photon-fueled engines will be soon science rather than "science fiction".

A supra-photoswitch is designed for complete ON/OFF switching of DNA hybridization by light irradiation for the purpose of using DNA as a material for building nanostructures. Azobenzenes, attached to D-threoninols that function as scaffolds, are introduced into each DNA strand after every two natural nucleotides (in the form (NNX)n where N and X represent the natural nucleotide and the azobenzene moiety, respectively). Hybridization of these two modified strands forms a supra-photoswitch consisting of alternating natural base pairs and azobenzene moieties. In this newly designed sequence, each base pair is sandwiched between two azobenzene moieties and all the azobenzene moieties are separated by base pairs. When the duplex is irradiated by visible light, the azobenzene moieties take the trans form and this duplex is surprisingly stable compared to the corresponding native duplex composed of only natural oligonucleotides. On the other hand, when the azobenzene moieties are isomerized to the cis form by UV light irradiation, the duplex is completely dissociated. Based on this design, a DNA hairpin structure is synthesized that should be closed by visible light irradiation and opened by UV light irradiation at the level of a single molecule. Indeed, perfect ON/OFF photoregulation is attained. This is a promising strategy for the design of supra-photoswitches such as photoresponsive sticky ends on DNA nanodevices and other nanostructures.