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Trans-cis isomerization kinetics of cyanine dyes reports on the folding states of RNA G-quadruplexes in live cells
KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.ORCID iD: 0000-0002-6191-9921
Hokkaido University, Sapporo.
Hokkaido University, Sapporo.
KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.ORCID iD: 0000-0003-3200-0374
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Guanine (G)-rich sequences in nucleic acids are prone to assemble into four-stranded structures, called G-quadruplexes. Abnormal GGGGCC repeat elongations have been associated with Amyotrophic lateral sclerosis and fronto-temporal dementia. In particular, the folding states of such elongations are believed to play a central role in the development of these diseases. Since most studies of G-quadruplex structures are made in vitro, it is highly relevant to clarify what the structures of elongated GGGGCC repeats look like in vivo. However, due to methodological constraints, evidence of specific structures of such GGGGCC repeats in vivo is sparse.

In this work, we devised a readout strategy, exploiting the sensitivity of trans-cis isomerization of cyanine dyes to local viscosity and sterical constraints. We show that the folding states of cyanine-labeled RNA molecules, and in particular of G-quadruplexes, can be identified in a sensitive manner by this strategy. The isomerization kinetics, monitored via the fluorescence blinking generated upon transitions between a fluorescent trans isomer and a non-fluorescent cis isomer, was first characterized for RNA molecules with GGGGCC repeats in aqueous solution using Fluorescence correlation spectroscopy (FCS) and transient state (TRAST) monitoring. With TRAST, monitoring the isomerization kinetics from how the average fluorescence intensity varies with modulation characteristics of a laser excitation source, we could then also detect the folding states of RNA molecules in living cells. This approach is robust, applicable on a broad range of biological samples and can also be extended to study folding or misfolding of proteins and biomolecules in general.

National Category
Other Physics Topics
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-246018OAI: oai:DiVA.org:kth-246018DiVA, id: diva2:1295296
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationSwedish Foundation for Strategic Research
Note

QC 20190312

Available from: 2019-03-11 Created: 2019-03-11 Last updated: 2019-03-12Bibliographically approved
In thesis
1. Fluorescence-based Transient State Monitoring for biomolecular, cellular and label-free studies
Open this publication in new window or tab >>Fluorescence-based Transient State Monitoring for biomolecular, cellular and label-free studies
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorophore blinking dynamics are highly sensitive to the local environment and can be used as an additional readout parameter to increase the information gained from existing fluorescence techniques.The origin of these blinking patterns are photophysical transitions to and from a manifold of non-luminescent states. The long lifetime of these dark transient states, typically 103 to 106 times longer than the fluorescent state, gives them correspondingly more time to sense their environment. For this reason, fluorophore blinking dynamics are particularly sensitive to low frequency events, such as diffusion-mediated interactions between the fluorophore and dilute species.

Transient State (TRAST) monitoring has been developed to quantify fluorophore blinking dynamics in a simple and widely applicable manner. TRAST does not need to resolve individual blinking events, but instead monitors the average fluorescence intensity in response to a modulated excitation. By systematically varying the modulation parameters, the transient state kinetics of the sample are mapped out. Without the need for time-resolved detection, a regular camera can be used to image blinking dynamics with high spatial resolution.

This thesis presents TRAST characterizations of common autofluorescent compounds and demonstrates their ability to sense relevant biological parameters such as oxygen concentration and redox potential. In Papers I and II, the autofluorescent co-enzymes flavin and NAD(P)H were studied, and label-free imaging of local redox variations within cells was demonstrated. Perturbing the cells, through dilute additions of mitochondrial uncouplers, revealed a strong andlocalized response in the TRAST images. In Paper III we studied tryptophan autofluorescence and used it to detect conformational changes in an unlabeled spider silk protein.

Labeling with external fluorophores can add further specificity to the TRAST measurements. In Paper IV, TRAST was used to monitor diffusion-mediated interactions between lipids and receptors in a cell membrane, including the influence of receptor activation. In Paper V we tracked folding of RNA into G-quadruplexes in live cells, monitored via the isomerization properties of an attached cyanine dye.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. p. i-vi; 116
Series
TRITA-SCI-FOU ; 2019:13
National Category
Other Physics Topics
Research subject
Biological Physics; Physics
Identifiers
urn:nbn:se:kth:diva-246020 (URN)978-91-7873-142-8 (ISBN)
Public defence
2019-04-05, FB53, KTH, Roslagstullsbacken 21, Stockholm, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilSwedish Cancer SocietySwedish Foundation for Strategic Research Knut and Alice Wallenberg Foundation
Note

QC 20190312

Available from: 2019-03-12 Created: 2019-03-11 Last updated: 2019-03-13Bibliographically approved

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Tornmalm, JohanWidengren, Jerker

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