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