Single-molecule study of an adsorbed oligonucleotide undergoing both lateral diffusion and strong adsorption

Single-molecule fluorescence spectroscopy was used to probe the lateral tra nsport of an adsorbed oligonucleotide, the SP6 promoter primer, which is a 24-mer labeled at the 5' end with tetramethylrhodamine. The oligonucleotide was adsorbed to the interface of an aqueous solution of 0.01 M KC1 and sil ica chemically modified by chlorodimethyloctadecylsilane. Confocal fluoresc ence microscopy achieved single-molecule resolution, with a molecule in the beam 7% of the time. Autocorrelation of the data fit well to a model havin g two species, one diffusing and the other one undergoing transient strong adsorption. A small number of bursts, 0.3%, had unusually long durations, a ccounting for the slow component in the autocorrelation. When these long bu rsts were excised from the data, the autocorrelation fit well to simple dif fusion, with D = 4 x 10(-6) cm(2)/s. Autocorrelation of the long bursts alo ne gave a rate constant for desorption of 3 s(-1). The strongly adsorbed mo lecules were found to comprise 10% of the total population of adsorbates. I t is concluded that the lateral transport of the SP6 promoter primer is des cribed by fast lateral diffusion interrupted by rare, reversible, strong ad sorption to defect sites. During strong adsorption, the tetramethylrhodamin e label undergoes hindered motion, suggesting it is not the adsorbing moiet y.