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[寡脱氧核苷酸的高效互补寻址激光修饰(切割)]

[Highly effective complementary addressed laser modification (cleavage) of oligodeoxynucleotides].

作者信息

Benimetskaia L Z, Bulychev N V, Kozionov A L, Koshkin A A, Lebedev A V

出版信息

Bioorg Khim. 1988 Jan;14(1):48-57.

PMID:3382432
Abstract

Complementary addressed nonlinear photomodification of oligodeoxynucleotide dAGAGTATTGACTTA ("target") has been carried out by means of fluorescent derivatives of oligonucleotide dpAATACTCT ("addressed chromophore"). Three different ethidium derivatives were used as a chromophore. The photomodification was induced by nitrogen laser radiation (337 nm, 15 MW/cm2), which led to the target cleavage in the addressation region with the yield of the main fragment (8 bases long) about 10%, formation of specific covalent adduct target-addressed chromophore with the yield 20-70%, "hidden" (not revealed by gel electrophoresis) target damages with 7-27% yield (for different chromophores). The total yield of specific (i. e. localized in the vicinity of the addressation site) modification was 50-80%. The target cleavage and hidden damage generation are optically nonlinear processes. Piperidine treatment of the irradiated samples caused addressed cleavage of the target with up to 40% yield. All kinds of observed modification are not effected by high concentrations of free radical scavengers, 1,3 M t-BuOH or 10 mM cystamine. The bulk of the data is in agreement with the mechanism of nonlinear photomodification (the cleavage and hidden damage generation) based on the transfer of two-photon excitation energy from the chromophore to the target.

摘要

利用寡核苷酸dpAATACTCT(“寻址发色团”)的荧光衍生物对寡脱氧核苷酸dAGAGTATTGACTTA(“靶标”)进行了互补寻址非线性光修饰。使用了三种不同的溴化乙锭衍生物作为发色团。光修饰由氮激光辐射(337nm,15MW/cm²)诱导,这导致靶标在寻址区域发生切割,主要片段(8个碱基长)的产率约为10%,形成特定的共价加合物靶标-寻址发色团,产率为20-70%,“隐藏”(凝胶电泳未显示)的靶标损伤产率为7-27%(针对不同发色团)。特异性(即位于寻址位点附近)修饰的总产率为50-80%。靶标切割和隐藏损伤的产生是光学非线性过程。用哌啶处理辐照后的样品会导致靶标发生寻址切割,产率高达40%。各种观察到的修饰不受高浓度自由基清除剂、1,3M叔丁醇或10mM胱胺的影响。大部分数据与基于从发色团到靶标的双光子激发能量转移的非线性光修饰机制(切割和隐藏损伤的产生)一致。

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