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Light-regulated DNA catalysis and a photoaffinity labelling exploration of nucleic acid structures.

机译：光调节的DNA催化和核酸结构的光亲和标记探索。

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摘要

The 8-17 deoxyribozyme is an efficient RNA-cleaving DNA molecule, holding considerable promise for applications. By covalently tethering photoisomerizable azobenzene (Az) into different positions within the 8-17, light has been successfully used as a versatile "effector" to regulate the enzyme's activity via trans-cis conversion of Az. Trans-Az can stack comfortably within a DNA double helix, stabilizing it, while cis-Az has a helix-destabilizing effect. I created two classes of Az-modified 8-17 constructs, in which two Az are substituted for nucleotides---either in the substrate binding arm (SBAs); or within the catalytic core. Kinetics measurement for RNA cleavage under single-turnover conditions revealed that the SBA constructs El 1 and E13 had 5-6-fold higher catalytic rates when the reaction was carried out under visible light (favouring trans-Az) as compared to UV light (promoting cis-Az). Surprisingly, the reverse result was observed with the catalytic core construct E17, where UV irradiation resulted in 5-6-fold faster catalytic activity relative to visible light irradiation. The development of such light-responsive nucleic acid enzymes may enable the use of light as a regulator in the control of gene expression within cells and organisms.;Although the 8-17 deoxyribozyme has been subjected to intensive structural and mechanistic studies, high-resolution insight on its 3-dimensional structure remains unknown to date. Thus, a systematic substitution of structurally and catalytically important nucleotides within the enzyme and its substrate with photoactivated thio-substituted and halogenated nucleotide analogues was carried out to enable the formation of contact crosslinks within the folded enzyme-substrate complex. Key nucleotides immediately flanking the scissile phosphodiester within the substrate showed strong crosslinking patterns with both a catalytically conserved internal bulge loop and a terminal loop within the folded 8-17 and vice versa. Based on the significant set of distance constraints obtained from photocrosslinking experiments, a model for the tertiary structure of 8-17 deoxyribozyme active site has been proposed.;In order to improve on the 5-6-fold discrimination caused by trans-cis isomerization of Az, efforts were made to select for photo-allosteric ribozymes and photo-aptamers using SELEX, and the results are reported here.
机译：8-17脱氧核酶是一种有效的RNA切割DNA分子，具有广阔的应用前景。通过将光可异构化的偶氮苯（Az）共价束缚在8-17的不同位置，光已成功用作通用“效应子”，可通过反式顺式转化Az来调节酶的活性。反式-Az可以舒适地堆叠在DNA双螺旋中，使其稳定，而顺式-Az具有螺旋去稳定作用。我创建了两类经过Az修饰的8-17结构，其中两个Az取代了核苷酸-在底物结合臂（SBA）中；或在催化核内。在单周转条件下进行RNA裂解的动力学测量结果表明，与可见光（促进反式Az）相比，在可见光（有利于反式Az）下进行反应时，SBA构建体El 1和E13的催化速率高5-6倍。顺式-Az）。出乎意料的是，用催化核心构建体E17观察到相反的结果，其中UV辐射导致的催化活性比可见光辐射快5-6倍。这种光响应性核酸酶的发展可能使光可以用作调节细胞和生物体内基因表达的调节剂。尽管8-17脱氧核酶已经进行了深入的结构和机制研究，但高分辨率迄今为止，对其3维结构的见解仍然未知。因此，用光活化的硫代取代的和卤代的核苷酸类似物对酶及其底物中的结构上和催化上重要的核苷酸进行了系统的取代，以使折叠的酶-底物复合物中形成接触交联。紧接在底物内易裂磷酸二酯侧翼的关键核苷酸显示出强的交联模式，具有催化保守的内部凸起环和折叠的8-17内的末端环，反之亦然。基于光交联实验获得的重要距离限制，建立了8-17个脱氧核酶活性位点三级结构模型。;为了改善反式-顺式异构化引起的5-6倍分辨力Az，使用SELEX努力选择了光变构核酶和光适体，结果报告在这里。

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  • 作者

    Liu, Yong.;

  • 作者单位

    Simon Fraser University (Canada).;

  • 授予单位 Simon Fraser University (Canada).;
  • 学科 Biology Molecular.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2008
  • 页码 195 p.
  • 总页数 195
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 能源与动力工程;
  • 关键词

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