用于DNA功能可逆光调控的偶氮苯连接DNA的合成：杂交与转录
Synthesis of azobenzene-tethered DNA for reversible photo-regulation of DNA functions: hybridization and transcription.
作者信息
Asanuma Hiroyuki, Liang Xingguo, Nishioka Hidenori, Matsunaga Daijiro, Liu Mingzhe, Komiyama Makoto
机构信息
Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
出版信息
Nat Protoc. 2007;2(1):203-12. doi: 10.1038/nprot.2006.465.
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.
一种带有偶氮苯的亚磷酰胺单体由D - 苏糖醇合成。使用这种单体，可在传统DNA合成仪的任何位置将偶氮苯部分引入到寡脱氧核糖核苷酸（DNA）中。利用这种连接有偶氮苯的DNA，DNA双链的形成和解离可通过偶氮苯的顺反异构化进行可逆的光调控。当偶氮苯呈反式构型时，会形成稳定的双链。在通过紫外线照射（300 nm < λ < 400 nm）使反式偶氮苯异构化为顺式构型后，双链可解离成两条链。在光诱导的顺反异构化（λ > 400 nm）时双链会重新形成。在T7启动子的特定位置引入偶氮苯也能有效且可逆地通过T7 - RNA聚合酶对转录进行光调控。这种可逆调控可重复多次而不会对DNA或偶氮苯部分造成损伤。这些步骤大约需要10天完成。
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