When R > 0.8 R0: fluorescence anisotropy, non-additive intensity, and cluster size

Zolmajd-Haghighi, Z. and Hanley, Q.S. ORCID: 0000-0002-8189-9550, 2016. When R > 0.8 R0: fluorescence anisotropy, non-additive intensity, and cluster size. Method and Applications in Fluorescence, 4, 024006. ISSN 2050-6120

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Assembly and clustering feature in many biological processes and homo-FRET and fluorescence anisotropy can assist in estimating the aggregation state of a system. The distance dependence of resonance energy transfer is well described and tested. Similarly, assessment of cluster size using steady state anisotropy is well described for non-oriented systems when R < 0.8 R0, however, these methods break down when R > 0.8 R0. Fused trimeric DNA clusters labelled with fluorescein were engineered to provide inter-fluorophore distances from 0.7 to 1.6 R/R0 and intensity and anisotropy were measured. These constructs cover a range where anisotropy effects depend on distance. Analytical expressions were derived for fully labelled and fractionally labelled clusters and the experimental results analysed. The experimental results showed that: 1) the system underwent distance dependent quenching; 2) when incompletely labelled both doubly and triply labelled forms could be assessed to obtain distance dependent intensity factors; 3) the anisotropy behaviour of a multiply labelled cluster of a particular size depends on the behaviour of the fluorophores and their distance in a cluster. This work establishes that when emission intensity data are available the analytically useful range for investigating clusters does not have to be restricted to R < 0.8 R0 and is applicable to cases where the anisotropy of a cluster of N fluorophores is not well approximated by r1/N.

Item Type: Journal article
Alternative Title: When R > 0.8 R0: the effects of non-additivity, distance, and homo-FRET on intensity and anisotropy
Publication Title: Method and Applications in Fluorescence
Creators: Zolmajd-Haghighi, Z. and Hanley, Q.S.
Publisher: IOP Publishing
Date: 15 June 2016
Volume: 4
ISSN: 2050-6120
Divisions: Schools > School of Science and Technology
Record created by: Linda Sullivan
Date Added: 23 May 2016 10:56
Last Modified: 16 Jun 2017 03:00
URI: https://irep.ntu.ac.uk/id/eprint/27858

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