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

Zolmajd-Haghighi, Z and Hanley, QS ORCID: https://orcid.org/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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Abstract

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
Identifiers:	Number Type 10.1088/2050-6120/4/2/024006 DOI
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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