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).
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.
sequence-specific hybridization and forms a highly regular double-helical structure with suitable flexibility. [1][2][3][4] DNA is probably one of the most promising biomolecules for future applications in nanotechnology and materials science.[5] Many 2D and 3D nanostructures with determined shapes and geometries have been reported recently in which DNA is used as the building blocks and mortar. [3,6,7] More excitingly, several types of DNA nanomachines, fuelled with DNA oligonucleotides [8] or other molecules such as intercalators [9] and metal ions, [10] have been constructed. [4,5] During these 10 years of development, substantial progress has been made in the design of DNA-based devices such as tweezers, walkers, and gears, which can perform mechanical functions such as scission, directional motion, or rolling. [11][12][13] The prospects of this field are extraordinarily promising, and several valuable applications of DNA nanomachines as sensors, transporters, and drug-delivery systems have also been reported. [5] For most of the DNA nanomachines constructed so far, oligonucleotides have been generally used as the fuel. In many of these systems, the mechanical motion was usually carried out by hybridization of one DNA fuel molecule to target sequences followed by its removal with another DNA sequence that is completely or partially complementary to the first. [5] Yurke et al. demonstrated the first DNA machine that functioned as "tweezers" fuelled by two strands of DNA with tailored complementarity.[8a] As the energy for operating these DNA nanomachines is produced by a strand-exchange strategy, a DNA duplex is produced as a waste product in every working cycle. Thus, the operating efficiency decreases gradually with the accumulation of "wastes". A new strategy is therefore required to overcome this problem for the further development of DNA nanotechnology.Over the past decade, we have developed a series of photoresponsive DNAs by covalently tethering azobenzene moieties onto the DNA strand. [14][15][16][17][18][19] Hybridization of these photoresponsive DNAs to single-stranded DNA (to form duplexes), RNA (to form DNA-RNA hybrids), or double-stranded DNA (to form triplexes) can be efficiently switched "on" and "off" by simply irradiating with UV and visible light. This is based on the following mechanism: the planar trans-azobenzene intercalates between adjacent base pairs and stabilizes the duplex or triplex structure by stacking interactions, whereas the nonplanar cisazobenzene destabilizes it by steric hindrance.[19] The successful photoregulation of primer elongation, transcription, and RNase H activity have also been demonstrated with photoresponsive DNAs. [20][21][22] Photoregulation efficiency can be amplified by the introduction of multiple azobenzene residues onto the DNA.[23] For example, nine azobenzene groups were introduced onto a DNA strand 20 nucleotides (nt) in length, and the clearcut photoswitching of DNA duplex formation was observed without loss of sequence specificity. Photoregulation of ...
Depurination has attracted considerable attention since a long time for it is closely related to the damage and repair of nucleic acids. In the present study, depurination using a pool of 30-nt short DNA pieces with various sequences at diverse pH values was analyzed by High Performance Liquid Chromatography (HPLC). Kinetic analysis results showed that non-enzymatic depurination of oligodeoxynucleotides exhibited typical first-order kinetics, and its temperature dependence obeyed Arrhenius’ law very well. Our results also clearly showed that the linear relationship between the logarithms of rate constants and pH values had a salient point around pH 2.5. Interestingly and unexpectedly, depurination depended greatly on the DNA sequences. The depurination of poly (dA) was found to be extremely slow, and thymine rich sequences depurinated faster than other sequences. These results could be explained to some extent by the protonation of nucleotide bases. Moreover, two equations were obtained based on our data for predicting the rate of depurination under various conditions. These results provide basic data for gene mutagenesis and nucleic acids metabolism in acidic gastric juice and some acidic organelles, and may also help to rectify some misconceptions about depurination.
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