On the Feasibility of Single-Molecule Detection of the Guanosine-Analogue 3-MI

We present UV fluorescence fluctuation correlation spectroscopy (FCS) measurements on the guanosineanalogue 3-MI [3-methyl-8-(2-deoxy-‚-D-ribofuranosyl)isoxanthopterin], a pteridine widely used in studies of DNA binding and dynamics. We measure the photon count rate and signal-to-background ratio per molecule, for both monomeric 3-MI and a 3-MI-containing oligonucleotide. For the monomer, we find a maximum photon count rate per molecule above 4 kHz and a maximum signal-to-background ratio of 5. For incorporated 3-MI, both parameters are a factor of 4 smaller. We discuss the triplet and photobleaching behavior of 3-MI and the possibilities of using this analogue in single-molecule studies of DNA dynamics. Comparisons are made to the behavior of stilbene 3, a brilliant laser dye with a similar fluorescence spectrum. Nucleic acid analogues offer an attractive alternative to the use of linker-attached dyes in the study of DNA binding and dynamics. For bulk assays, analogues are widely exploited as a way to avoid artifacts associated with linker and dye structure and dynamics (for a review, see ref 1). For single-molecule studies, the use of analogues has been thwarted by their smaller absorption cross sections, absorption maxima that tend to be in the UV, and a propensity to photobleach. Here, we investigate the potential of 3-MI [3-methyl-8-(2-deoxy-‚-D-ribofuranosyl)isoxanthopterin], a guanosine analogue, 2 in the study of single DNA molecules. We show that single-molecule detection of 3-MI monomer is possible and suggest that single-molecule spectroscopic techniques can successfully be applied to DNA incorporated with 3-MI. Nucleoside analogues offer some advantages over labeling