Measuring, in solution, multiple-fluorophore labeling by combining fluorescence correlation spectroscopy and...

Area of Science:

• Biophysics
• Molecular Biology
• Spectroscopy

Background:

• Accurate quantification of fluorescent labels on biomolecules is essential for single-molecule studies and understanding molecular interactions.
• Characterizing labeling processes and molecular complexes requires reliable measurement techniques.

Purpose of the Study:

• To develop and validate a method for determining the number of fluorescent labels per molecule.
• To measure the effective number of molecules and their brightness using fluorescence correlation spectroscopy (FCS) and photobleaching.

Main Methods:

• Combined fluorescence correlation spectroscopy (FCS) with photobleaching experiments on cDNA labeled with Alexa Fluor 647.
• Used photobleaching as a control parameter to vary experimental outputs.
• Analyzed photon count distribution using the cumulant method.

Main Results:

• Successfully determined the mean number of fluorescent labels per cDNA strand to be approximately two, assuming a Poissonian distribution.
• Showed that cDNA brightness varies between molecules due to statistical label distribution and altered fluorophore properties.
• Observed a 30% decrease in Alexa Fluor 647 photobleaching decay rate and a 25% decrease in individual label brightness upon incorporation into cDNA.

Conclusions:

• FCS combined with photobleaching provides a robust method for quantifying fluorescent labels on nucleic acids.
• The study highlights the importance of considering label distribution and photophysical changes for accurate molecular characterization.
• This approach is vital for advancing single-molecule biophysics and molecular interaction studies.