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

Resource type
Thesis type
(Thesis) Ph.D.
Date created
2008
Authors/Contributors
Author: Liu, Yong
Abstract
The 8-17 deoxyribozyme is an efficient RNA-cleaving DNA molecule, holding considerable promise for applications. By covalently tethering photo-isomerizable 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 E11 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. 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. 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.
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Scholarly level
Language
English
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