Single-Molecule Spectroscopy and Fluorescence Correlation Spectroscopy of the Lateral Transport of the T3 Promoter Primer at a Chemical Interface

Abstract

Fluorescence correlation spectroscopy reveals that an oligonucleotide, the T3 promoter primer, undergoes only lateral diffusion when adsorbed to the interface of water and silica chemically modified with a hydrocarbon. The autocorrelation decay fits well to the model of simple diffusion, reporting a diffusion coefficient of 1.8 × 10⁻⁶ cm²/s. Single-molecule resolution of bursts for the T3 promoter primer reveals that rare, strong adsorption punctuates the lateral diffusion. Removal of the strong adsorption events from the data set, followed by autocorrelation, shows the actual diffusion coefficient to be 2.8 × 10⁻⁶ cm²/s, which is comparable to other oligonucleotides of the same size at the same interface. The single-molecule measurements show that average duration of strong adsorption is 0.2 s, and the average fraction of strongly adsorbed molecules is 10% of the molecules at the interface. While single-molecule spectroscopy reveals a process not evident in fluorescence correlation spectroscopy, the precision of the parameters describing strong adsorption is limited by the statistics of small numbers. Fluorescence correlation spectroscopy is suited to observing a much larger number of events, which is needed for high precision. The two methods are complementary: single-molecule spectroscopy gives estimates of the chemical parameters needed for design of the fluorescence correlation spectroscopy, achieving precise measurements with an accurate interpretation.