Complex RNA Folding Kinetics Revealed by Single-Molecule FRET and Hidden Markov Models

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• 作者：Bettina G. Keller ; Andrei Kobitski ; Andres J盲schke ; G. Ulrich Nienhaus ; Frank No茅
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：March 26, 2014
• 年：2014
• 卷：136
• 期：12
• 页码：4534-4543
• 全文大小：617K
• 年卷期：v.136,no.12(March 26, 2014)
• ISSN：1520-5126

文摘

We have developed a hidden Markov model and optimization procedure for photon-based single-molecule FRET data, which takes into account the trace-dependent background intensities. This analysis technique reveals an unprecedented amount of detail in the folding kinetics of the Diels鈥揂lderase ribozyme. We find a multitude of extended (low-FRET) and compact (high-FRET) states. Five states were consistently and independently identified in two FRET constructs and at three Mg²⁺ concentrations. Structures generally tend to become more compact upon addition of Mg²⁺. Some compact structures are observed to significantly depend on Mg²⁺ concentration, suggesting a tertiary fold stabilized by Mg²⁺ ions. One compact structure was observed to be Mg²⁺-independent, consistent with stabilization by tertiary Watson鈥揅rick base pairing found in the folded Diels鈥揂lderase structure. A hierarchy of time scales was discovered, including dynamics of 10 ms or faster, likely due to tertiary structure fluctuations, and slow dynamics on the seconds time scale, presumably associated with significant changes in secondary structure. The folding pathways proceed through a series of intermediate secondary structures. There exist both compact pathways and more complex ones, which display tertiary unfolding, then secondary refolding, and, subsequently, again tertiary refolding.