Fluorescence fluctuation analysis for the study of interactions between oligonucleotides and polycationic polymers

E. Van Rompaey, Y. Chen, J. D. Müller, E. Gratton, E. Van Craenenbroeck, Y. Engelborghs, S. De Smedt, J. Demeester

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The interactions between a cationic polymer, poly(2-dimethylamino) ethyl methacrylate (pDMAEMA), and negatively charged rhodamine-labeled 25-mer phosphodiester oligonucleotides (Rh-ONs) were studied by fluorescence fluctuation spectroscopy and other techniques. The composition of the pDMAEMA/RhON complexes was investigated as a function of the charge ratio (+/-) by increasing the pDMAEMA concentration and keeping the Rh-ON concentration constant. We applied two different methods for analyzing the fluorescence fluctuation profiles of the pDMAEMA/Rh-ON complexes, which depended on their composition. First, we analyzed the data with the photon counting histogram (PCH) technique, which determines the molecular brightness and the concentration of fluorophores (Chen et al., 1999). A particular challenge for the data analysis is the occurrence of sudden fluorescence bursts in the fluorescence fluctuation profiles, which are linked to the appearance of multimolecular complexes (i. e. when several Rh-ONs were present in one complex). A quantitative interpretation of the analysis for the complexes remains challenging and is connected to the rarity of the fluorescent bursts, which do not provide sufficient data statistics. To specifically address the problem of the fluorescent bursts we employed a method described by Van Craenenbroeck et al. (1999). This method, applicable only when data were integrated over much longer time bins, allowed us to estimate the number of fluorescence bursts which could be considered as a relative measure of the amount of multimolecular complexes present. When monomolecular complexes were formed, i.e. at high values of the charge ratio, highly intense fluorescence peaks were not present and the interpretation of the PCH analysis was more straightforward. The molecular brightness of the species (ε), as revealed from PCH analysis, was greater than ε for the free RhONs, indicating that the Rh-ONs were attached to pDMAEMA chains.

Original languageEnglish (US)
Pages (from-to)379-386
Number of pages8
JournalBiological Chemistry
Issue number3
StatePublished - 2001
Externally publishedYes

Bibliographical note

Funding Information:
Elke Van Craenenbroeck is a research assistant of FWO-Vlaan-deren. The installation of FCS at the University of Leuven was financially supported by grant 9.0320.97 from FWO-Vlaanderen, which is acknowledged with gratitude. The University of Ghent (UG-BOF) supported this project through instrumentation credits. We thank the group of Professor W. Hennink (University of Utrecht, The Netherlands) for synthesizing pDMAEMA. The work performed at the Laboratory for Fluorescence Dynamics was supported by the National Institute of Health (RR03155).


  • Fluorescence correlation spectroscopy
  • Gene therapy
  • Interpolyelectrolyte complexes
  • Photon counting histograms
  • Polyplexes
  • Self-assembling systems

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