RNA dimerization monitored by fluorescence correlation spectroscopy.

Area of Science:

• Biophysics
• Biochemistry
• Molecular Biology

Background:

• Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying molecular interactions.
• A key limitation of FCS is the requirement for a significant difference in molecular weights between interacting species.
• Previous methods needed an 8-fold molecular weight difference to distinguish species.

Purpose of the Study:

• To investigate the hybridization of equally sized RNA single strands using FCS.
• To determine if FCS can resolve diffusion differences between molecules of similar size.
• To expand the applicability of FCS for molecular interaction analysis.

Main Methods:

• Utilized the photostable fluorophore Atto647N for high signal-to-noise ratio.
• Applied multicomponent model analysis of single autocorrelation functions.
• Employed global fitting of autocorrelation functions with a priori knowledge.

Main Results:

• Accurately resolved 1.3- to 1.4-fold differences in diffusion coefficients for RNA strands of varying lengths (26, 41, 54 nucleotides).
• Achieved highly accurate quantification of diffusion species fractions and mobilities through global fitting.
• Demonstrated robust representation of heterogeneous data using median fitting at high signal-to-noise ratios.

Conclusions:

• FCS can effectively study molecular interactions of equally sized molecules by analyzing diffusional behavior.
• The presented method significantly broadens the application spectrum of FCS in molecular sciences.
• This work enables the investigation of interactions previously inaccessible to FCS.