Fluorescence Fluctuation Analysis for the Study of Interactions between Oligonucleotides and Polycationic Polymers
-
Elsa Van Rompaey
Abstract
The interactions between a cationic polymer, poly(2- dimethylamino)ethyl methacrylate (pDMAEMA), and negatively charged rhodaminelabeled 25-mer phosphodiester oligonucleotides (RhONs) were studied by fluorescence fluctuation spectroscopy and other techniques. The composition of the pDMAEMA/Rh ON complexes was investigated as a function of the charge ratio (+/) by increasing the pDMAEMA concentration and keeping the RhON concentration constant. We applied two different methods for analyzing the fluorescence fluctuation profiles of the pDMAEMA/RhON 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 RhONs 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 (e), as revealed from PCH analysis, was greater than e for the free Rh ONs, indicating that the RhONs were attached to pDMAEMA chains.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
Articles in the same Issue
- Highlight: Fluorescence Correlation Spectroscopy
- Heuristic Statistical Analysis of Fluorescence Fluctuation Data with Bright Spikes: Application to Ligand Binding to the Human Serotonin Receptor Expressed in Escherichia coli Cells
- Fluorescence Correlation Spectroscopy as a Tool to Investigate Single Molecule Probe Dynamics in Thin Polymer Films
- Ligand-Receptor Interactions in the Membrane of Cultured Cells Monitored by Fluorescence Correlation Spectroscopy
- Fluorescence Fluctuation Analysis for the Study of Interactions between Oligonucleotides and Polycationic Polymers
- Determination of the Net Exchange Rate of Tubulin Dimer in Steady-State Microtubules by Fluorescence Correlation Spectroscopy
- UV-Fluorescence Correlation Spectroscopy of 2-Aminopurine
- Isolation of Two cDNAs Encoding Functional Human Cytoplasmic Cysteinyl-tRNA Synthetase
- Involvement of Intracellular Ca2+in the Regulation of Bovine Leukemia Virus Expression
- Nucleotide-Binding Sites in the Functional Unit of Sarcoplasmic Reticulum Ca2+-ATPase as Studied by Photoaffinity Spin-Labeled 2-N3-SL-ATP
- Interaction between Lipopolysaccharide (LPS), LPS-Binding Protein (LBP), and Planar Membranes
- Differential Binding of Urokinase and Peptide Antagonists to the Urokinase Receptor: Evidence from Characterization of the Receptor in Four Primate Species
- Effects of Cysteine to Serine Substitutions in the Two Inter-Chain Disulfide Bonds of Insulin
- Tricorn Protease in Bacteria: Characterization of the Enzyme from Streptomyces coelicolor
- Structures of Tryparedoxins Revealing Interaction with Trypanothione
- Quantitative Two-Color Fluorescence Cross-Correlation Spectroscopy in the Analysis of Polymerase Chain Reaction
- Fluorescence Correlation Spectroscopy as a New Method for the Investigation of Aptamer/Target Interactions
- Localization of Lysobisphosphatidic Acid-Rich Membrane Domains in Late Endosomes
- Fluorescence Correlation Spectroscopy for the Characterisation of Drug Delivery Systems
- Accessing Molecular Dynamics in Cells by Fluorescence Correlation Spectroscopy
- Tailor-Made Dyes for Fluorescence Correlation Spectroscopy (FCS)
- Synergistic Inhibition of the Glucocorticoid Receptor by Radicicol and Benzoquinone Ansamycins
Articles in the same Issue
- Highlight: Fluorescence Correlation Spectroscopy
- Heuristic Statistical Analysis of Fluorescence Fluctuation Data with Bright Spikes: Application to Ligand Binding to the Human Serotonin Receptor Expressed in Escherichia coli Cells
- Fluorescence Correlation Spectroscopy as a Tool to Investigate Single Molecule Probe Dynamics in Thin Polymer Films
- Ligand-Receptor Interactions in the Membrane of Cultured Cells Monitored by Fluorescence Correlation Spectroscopy
- Fluorescence Fluctuation Analysis for the Study of Interactions between Oligonucleotides and Polycationic Polymers
- Determination of the Net Exchange Rate of Tubulin Dimer in Steady-State Microtubules by Fluorescence Correlation Spectroscopy
- UV-Fluorescence Correlation Spectroscopy of 2-Aminopurine
- Isolation of Two cDNAs Encoding Functional Human Cytoplasmic Cysteinyl-tRNA Synthetase
- Involvement of Intracellular Ca2+in the Regulation of Bovine Leukemia Virus Expression
- Nucleotide-Binding Sites in the Functional Unit of Sarcoplasmic Reticulum Ca2+-ATPase as Studied by Photoaffinity Spin-Labeled 2-N3-SL-ATP
- Interaction between Lipopolysaccharide (LPS), LPS-Binding Protein (LBP), and Planar Membranes
- Differential Binding of Urokinase and Peptide Antagonists to the Urokinase Receptor: Evidence from Characterization of the Receptor in Four Primate Species
- Effects of Cysteine to Serine Substitutions in the Two Inter-Chain Disulfide Bonds of Insulin
- Tricorn Protease in Bacteria: Characterization of the Enzyme from Streptomyces coelicolor
- Structures of Tryparedoxins Revealing Interaction with Trypanothione
- Quantitative Two-Color Fluorescence Cross-Correlation Spectroscopy in the Analysis of Polymerase Chain Reaction
- Fluorescence Correlation Spectroscopy as a New Method for the Investigation of Aptamer/Target Interactions
- Localization of Lysobisphosphatidic Acid-Rich Membrane Domains in Late Endosomes
- Fluorescence Correlation Spectroscopy for the Characterisation of Drug Delivery Systems
- Accessing Molecular Dynamics in Cells by Fluorescence Correlation Spectroscopy
- Tailor-Made Dyes for Fluorescence Correlation Spectroscopy (FCS)
- Synergistic Inhibition of the Glucocorticoid Receptor by Radicicol and Benzoquinone Ansamycins
