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When R  >  0.8R 0: fluorescence anisotropy, non-additive intensity, and cluster size.

Z Zolmajd-Haghighi1, Q S Hanley

  • 1School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK. Current address: Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.

Methods and Applications in Fluorescence
|August 16, 2017
PubMed
Summary
This summary is machine-generated.

This study extends the use of fluorescence anisotropy to measure DNA cluster sizes beyond previously established limits. New analytical methods allow for more accurate distance-dependent intensity factor assessments in biological systems.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Biochemistry

Background:

  • Assembly and clustering are crucial in biological processes.
  • Homo-Förster Resonance Energy Transfer (FRET) and fluorescence anisotropy are used to estimate aggregation states.
  • Current methods for cluster size assessment using anisotropy are limited to distances R < 0.8R 0.

Purpose of the Study:

  • To extend the application range of fluorescence anisotropy for cluster size estimation.
  • To develop analytical expressions for analyzing labeled DNA clusters.
  • To investigate distance-dependent quenching and anisotropy in engineered DNA clusters.

Main Methods:

  • Engineered fused trimeric DNA clusters labeled with fluorescein.
  • Measured fluorescence intensity and anisotropy at varying inter-fluorophore distances (0.7 to 1.6 R/R 0).
  • Derived analytical expressions for fully and fractionally labeled clusters.

Main Results:

  • Observed distance-dependent quenching in the DNA clusters.
  • Successfully assessed doubly and triply labeled forms to obtain distance-dependent intensity factors.
  • Demonstrated that multiply labeled cluster anisotropy depends on fluorophore behavior and distance.

Conclusions:

  • Fluorescence anisotropy can be reliably used for cluster investigation beyond R < 0.8R 0 when emission intensity data are available.
  • The derived analytical expressions enable more accurate assessment of distance-dependent intensity factors.
  • This work expands the utility of fluorescence anisotropy in studying biological aggregation states.