Single-molecule spectroscopy and fluorescence correlation spectroscopy of the lateral transport of the T3 promoter primer at a chemical interface

Fluorescence correlation spectroscopy reveals that an oligonucleotide, the T3 promoter primer, undergoes only lateral diffusion when adsorbed to the i nterface of water and silica chemically modified with a hydrocarbon. The au tocorrelation decay fits well to the model of simple diffusion, reporting a diffusion coefficient of 1.8 X 10(-6) cm(2)/s. Single-molecule resolution of bursts for the T3 promoter primer reveals that rare, strong adsorption p unctuates the lateral diffusion. Removal of the strong adsorption events fr om the data set, followed by autocorrelation, shows the actual diffusion co efficient to be 2.8 X 10-6 cm(2)/s, which is comparable to other oligonucle otides of the same size at the same interface. The single-molecule measurem ents show that average duration of strong adsorption is 0.2 s, and the aver age 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.