Modified Antisense Oligonucleotides Light Controlled Gene Expression

Azobenzene is widely used as a photo-induced switch due to its high efficient photoisomerization and structural differences between cis and trans isomers.On the other hand,azobenzene derivatives have similar base sizes and similar polarity distributions.Through ingenious molecular design,it can regulate the structure of nucleic acids,RNA silencing,gene expression,aptamer recognition,enzyme activity and so on.Thus,the reversible regulation of functional nucleic acids and their application in the biological field have become a hot topic in the field of nucleic acid chemistry.We based on the most studied azobenzene 4,4'-dimethylolazobenzene(compound 0),introduced three different substituents in the N=N double bond ortho,designed and synthesized four kinds of photosensitizing azobenzene derivatives(compounds 0-3)to achieve visible light regulation and stability of azobenzene compounds.The results of the photo-isomerization of azobenzene derivatives revealed that the azobenzene containing substituents achieved photoconversion under visible light.Among them,the compound 0 underwent trans-cis isomerization only under UV light,the chlorine-substituted azobenzene(compound 1),the methyl-substituted azobenzene(compound 2)and the fluoro-substituted azobenzene(compound 3)underwent trans-cis isomerization under irradiation with yellow light,yellow light and green light,respectively.Next,we studied the isomerization time of azobenzene derivatives under optimal light illumination.The experimental results showed that the cis-trans isomerization time of the compound 1,the compound 2 and the compound 3 were 13 min,6 min and 50 minr,respectively;Cis-trans isomerization time were 20 s,2 min,and 12 min,respectively.The photoisomerization efficiency of azobenzene derivatives was investigated by the change of the methylene chemical shift of azobenzene derivatives before and after light irradiation.The experimental results showed that the compound 1 converted from cis to cis under yellow light illumination with a degree of 69%,while the degree of isomerization of cis-trans under blue light irradiation was 84%.The trans-cis isomerization degree of the compound 2 and the compound 3 were 64% and 78%,respectively,and the degree of isomerization from cis-trans were 84% and 71%,respectively.The thermal stability experimental results of the cis-structure of azobenzene derivatives showed that introduced an electron-donating group into the azobenzene N=N double-stranded ortho-position can improve the cis-structure thermal stability.The compound 0 underwent a complete thermal conversion at 49 h.The compound 1 extended the cis-structure thermal stability to 69 h.In particular,the compound 3 had the best stability,only some of the heat conversion occurred after 73 h.The thermal stability of the compound 2 was only 36 h.The glutathione reducibility test results of azobenzene derivatives showed that the stability of the four azobenzene derivatives was the compound 1> the compound 3> the compound 0> the compound 2.Similarly,introducd an ortho-electron –donating group into the N=N double bond can enhance the reduction resistance of the azobenzene derivatives.The results of light modulation experiments on the melting and hybridization of the nucleic acid hairpin structure showed that the compound 0 and the compound 3 had the best light control effect,and the misalignment of the ortho positions of the azobenzenes favored the occurrence of isomerization.The compound 1,although it underwent photoisomerization after it was introduced into the oligonucleotide,had no effect on Tm value.While the compound 2 neither underwent photoisomerization nor affected the Tm value.The binding experiments of the dumbbell-type photosensitivity oligonucleotides GFP10 and GFP20 before and after irradiation with the three target molecules showed that the binding of the dumbbell-type photosensitizing oligonucleotides to the target molecules were mainly affected by the UV illumination and the number of pairs of bases in the stem of the target molecule.UV illumination promoted the combination of the two and when there was eight pairs of matched bases in the stem of the target molecule,the difference in binding before and after light irradiation was most obvious.