Single-Molecule FRET Studies of RNA Folding: A Diels鈥揂lderase Ribozyme with Photolabile Nucleotide Modifications

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• 作者：Andrei Yu. Kobitski ; Stefan Sch盲fer ; Alexander Nierth ; Marco Singer ; Andres J盲schke ; G. Ulrich Nienhaus
• 刊名：Journal of Physical Chemistry B
• 出版年：2013
• 出版时间：October 24, 2013
• 年：2013
• 卷：117
• 期：42
• 页码：12800-12806
• 全文大小：459K
• 年卷期：v.117,no.42(October 24, 2013)
• ISSN：1520-5207

文摘

Enzymology at the single-molecule level by using fluorescence resonance energy transfer (smFRET) offers unprecedented insight into mechanistic aspects of catalytic reactions. Implementing spatiotemporal control of the reaction by using an external trigger is highly valuable in these challenging experiments. Here, we have incorporated a light-cleavable caging moiety into specific nucleotides of the Diels鈥揂lderase (DAse) ribozyme. In this way, the folding energy landscape was significantly perturbed, and the catalytic activity was essentially suppressed. A careful smFRET efficiency histogram analysis at various Mg²⁺ ion concentrations revealed an additional intermediate state that is not observed for the unmodified DAse ribozyme. We also observed that only a fraction of DAse molecules returns to the native state upon cleavage of the caged group by UV light. These constructs are attractive model RNA systems for further real-time single-molecule observation of the coupling between conformational changes and catalytic activity.