Single-molecule detection of oligonucleotides using the fluorescent nucleobase analogue ABN

In this study, we investigate the properties of the fluorescent pyrimidine analogue ABN in duplex DNA oligonucleotides using ensemble measurements, time-resolved fluorescence, and single-molecule total internal reflection fluorescence (smTIRF) microscopy. As a free nucleoside, ABN exhibits ε442 nm = 20,000 M−1cm−1 and Φem = 0.39 in 1× PBS buffer at pH 7.4 and its high brightness is retained when base-paired and stacked in duplex DNA. The ABN nucleoside’s time-resolved fluorescence shows that it emits primarily from a thymine-like tautomeric form T1 that matches its ground state, but is photo-tautomerized to a cytosine-like form T2 that contributes ~ 20% to the emission intensity, with a shorter lifetime. In duplex DNA when base-paired with adenine, fluorescent emission is again dominated by T1, with ~ 20% of the intensity deriving from photo-induced double proton transfer to form a Watson–Crick-like base pair between T2 and the imino tautomer of adenine. When base-paired with guanine, ABN adopts predominantly the T2 tautomer form to enable Watson–Crick base pairing and this tautomer exhibits red-shifted absorption and blue-shifted emission; around 20% of ABN remains in the T1 tautomeric form and engages in a wobble base pair with G, contributing to the bulk fluorescent properties. The high brightness of ABN and its relatively long absorption and emission wavelengths position it uniquely among fluorescent nucleobase analogues (FBAs) for applications in single-molecule fluorescence. We immobilized ABN using strand hybridization and biotin pull-down on a PLL-PEG–streptavidin surface and imaged it using a 488 nm laser and an smTIRF microscope. The brightness and photostability of ABN in this context are enhanced by the use of the GO/CAT oxygen scavenging system and triplet depopulation using Trolox. Under these conditions, ABN-containing oligos are readily observed as bright spots with time traces of up to 9 seconds and single-step photobleaching. These results show that ABN is the first FBA to enable single-molecule fluorescence studies of oligonucleotides and requires only a conventional smTIRF configuration.