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Highly Sensitive FRET-FCS Detects Amyloid beta-Peptide Oligomers in Solution at Physiological Concentrations
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-1850-5440
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.ORCID iD: 0000-0003-3200-0374
2015 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 23, 11700-11705 p.Article in journal (Refereed) Published
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Text
Abstract [en]

Oligomers formed by the amyloid beta-peptide (A beta) are pathogens in Alzheimers disease. Increased knowledge on the oligomerization process is crucial for understanding the disease and for finding treatments. Ideally, A beta oligomerization should be studied in solution and at physiologically relevant concentrations, but most popular techniques of today are not capable of such analyses. We demonstrate here that the combination of FOrster Resonance Energy Transfer and Fluorescence Correlation Spectroscopy (FRET-FCS) has a unique ability to detect small subpopulations of FRET-active molecules and oligomers. FRET-FCS could readily detect a FRET-active oligonucleotide present at levels as low as 0.5% compared to FRET-inactive dye molecules. In contrast, three established fluorescence fluctuation techniques (FCS, FCCS, and PCH) required fractions between 7 and 11%. When applied to the analysis of A beta, FRET-FCS detected oligomers consisting of less than 10 A beta molecules, which coexisted with the monomers at fractions as low as 2 +/- 2%. Thus, we demonstrate for the first time direct detection of small fractions of A beta oligomers in solution at physiological concentrations. This ability of FRET-FCS could be an indispensable tool for studying biological oligomerization processes, in general, and for finding therapeutically useful oligomerization inhibitors.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2015. Vol. 87, no 23, 11700-11705 p.
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-180226DOI: 10.1021/acs.analchem.5b02630ISI: 000365931100016PubMedID: 26489794Scopus ID: 2-s2.0-84948448366OAI: oai:DiVA.org:kth-180226DiVA: diva2:895437
Note

QC 20160119

Available from: 2016-01-19 Created: 2016-01-08 Last updated: 2017-11-30Bibliographically approved
In thesis
1. Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells
Open this publication in new window or tab >>Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescence spectroscopy and imaging have a very broad spectrum of applicationswithin the life sciences, in particular for detection and characterization ofbiomolecular dynamics and interactions in different environments. This thesis comprisesprojects that strive to further expand the information content extracted fromthe detected fluorescence, leading to sensitive readout parameters for studies ofbiomolecular dynamics and interactions. Two major strategies are presented toachieve this aim. The first strategy is based on the expansion of the availablereadout parameters beyond the "traditional" fluorescence parameters: intensity,wavelength, polarization and fluorescence lifetime. The additional parameters arebased on blinking properties of fluorescent labels. In particular on transitions betweensinglet and triplet states, and transitions between the trans- and cis-isomersof fluorophores. Two publications in the thesis are based on this strategy (paperI and IV). The second strategy is based on the utilization of fluorescence intensityfluctuations in order to detect the oligomerization mechanisms of fluorescentlylabeled peptides and proteins. This strategy combines the intensity fluctuationanalysis and the readout of distance dependent energy transfer between fluorescentmolecules together with the correlation analysis of fluorescence from two labeledproteins emitting at different wavelengths. Another two publications presented inthe thesis are based on the second comprehensive strategy (papers II and III).The work presented in this thesis shows that the blinking kinetics of fluorescentlabels contain significant information that can be exploited by a combination of fluctuationsanalysis with distance dependent excitation energy transfer between thefluorescent molecules, or by analysis of fluorescence covariance between moleculesthat emit at different wavelengths. These fluorescence-based methods have a significantpotential for molecular interaction studies in the biomedical field.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. x, 59 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2016:22
Keyword
FCS, FCCS, Isomerization, TRAST, NMR, FRET, biomombrane, fluidity
National Category
Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-187708 (URN)978-91-7729-026-1 (ISBN)
Public defence
2016-06-13, FB52, KTH, AlbaNova University Center, Roslagsvägen 30 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20160527

Available from: 2016-05-27 Created: 2016-05-26 Last updated: 2016-05-31Bibliographically approved

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Wennmalm, Stefan

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