Photo-regulated Hybridization And Light-driven Strand Displacement Of Azobenzene Tethered DNA

Azobenzene was first inserted into the DNA backbone via a three-carbon-atom linker at the end of 20 century. It was reported that trans-azobenzene (under visible light irradiation) stabilized the duplex by stacking interactions whereas cis-azobenzene (under UV light irradiation) greatly destabilized it by steric hindrance. Thus the photo-reversible duplex association and dissociation of azobenzene-modified DNA can be simply regulated by light irradiation. This photoresponsive DNA material promises a bright prospect in photo-controlled DNA technology.In this report, azobenzene and 2’,6’-dimethylazobenzene (DMazo) was introduced to DNA by a two-carbon-atom glycerol linker via an ether bond (azo-g-DNA or DMazo-g-DNA), which minimized the asymmetry and distortion of duplex formed when a native DNA is used as the complementary strand, and suppressed the instability when more azobenzene inserted in a long sequence. The trans-to-cis photoisomerization increased due to the flexible ether bond connecting azobenzene. As a result, the photo-regulated efficiency of duplex association and dissociation was greatly improved. At last these novel photoresponsive DNA were applied to strand displacement successfully. My work is divided into three sections as follow.1) Synthesis and property of the novel photoresponsive DNA materialThe photoresponsive azobenzene series were synthesized by diazo-coupling reaction at first. Then glycidol was hydroxy-protected, epoxide ring opened and phosphoramidite-protected orderly to give the azobenzene (DMazo) phosphoramidite. At last the phosphoramidite was tethered into DNA by commercial DNA synthesizer. The molar extinction coefficients, the content of photoisomerization and the thermo stability at cis-form of azobenzene tethered DNA were measured.The results show that the phosphoramidite monomers was synthesized successfully and inserted into DNA as designed. When irradiated by visible light at room temperature,94% of the tethered azobenzene and DMazo take trans form, while irradiated by UV the content of cis-azobenzene reached 80%. The half-life time of cis-form DMazo-g-DNA lasts for 24 h at 60℃.2) Photo-regulated association and dissociation of the photoresponsive DNA duplexIndicated by melting curve, the trans azo-g-DNA (or DMazo-g-DNA) duplex demonstrated a higher hybridization stability than that of the native duplex, in contrast the melting temperature (Tm) of cis form duplex decreased when tethered more azobenzene. The Tm of trans-form duplex (12 nucleotide consisting of 5 azobenzene) was 7.6 ℃ higher than that of the native duplex, and it was 38 ℃ lower for cis-form. When the ratio of azobenzene to nucleotide is 1:2 in the modified strand of 20 or 35 nucleotide, the difference of Tm of the same duplex at trans- and cis-forms reached to about 40 ℃, which represents a wide potential of photoregulation ability.The hybridization percentages were quantified by fluorescence spectroscopy so far as two stands were tagged with fluorescent molecules and quencher separately. For NX9/N’(20 nucleotide consisting of 9 azobenzene),95% of the duplex formed under visual light irradiation at room temperature, while 81% of the duplex dissociated under UV light irradiation. It was agreed with the UV/vis absorbance result. For D16/cb (35 nucleotide consisting of 16 DMazo), the duplex hybridization were photo-regulated with high efficiency (the difference of association percentage under UV and visible light irradiation) of 76% at 60～70 ℃. Repetitive irradiation by visible and UV light realized reversible photoregulation with small oscillations.The photoregulation of association and dissociation of azo-g-DNA was further confirmed by non-denaturing polyacrylamide gel electrophoresis.3) Light-driven DNA strand-displacement (DSD) of the photoresponsive DNAWhen azo-g-DNA (or DMazo-g-DNA), native complementary sequence and the native competing sequence were present in a DSD system, under visible light modified DNA will displace the native competing strand, for the trans-form modified DNA duplex demonstrate higher Tm than that of native DNA duplex. Under UV light, native competing DNA is preferred to hybridize with the complementary strand. Thus the light-driven DSD was implemented for the first time.From fluorescence spectroscopy, the DSD efficiency is quantified. Added with N14 (the native competing sequence of 14 nucleotide), the light-driven DSD of NX9/N14/N’ could be operated at 25～37 ℃. Under UV light N14 displaced NX9 from NX9/N’, while under visible light, more than 70% of NX9 bound back to N’. As the toehold became shorter, the DSD speed declined.For pre-hybridized D16D/Fbc, though the added cb20 (the native competing sequence of 20 nucleotide) would only displaced 57% of D16D under UV light at 37 ℃, it is much larger than photo-regulated hybridization scale of 7%. At 50 ℃, D16D displaced more than 85% of cb20 under visible light, while under UV light 90% of cb20 bound back to Fbc. When the toehold was shorter than 9 bases, the DSD speed declined and the final displacement efficiency (the difference of displacement degree under UV and visible light irradiation) got lower. Additionally confirmed by repetitive irradiation by visible/UV light and non-denaturing polyacrylamide gel electrophoresis, the "green" light-driven DSD was realized without any byproduct and recycle efficiency decay, and it is not essential to add any fuel strand.