Single-molecule fluorescence detection of a tricyclic nucleoside analogue

Fluorescent nucleobase surrogates capable of Watson-Crick hydrogen bonding are essential probes of nucleic acid structure and dynamics, but their limited brightness and short absorption and emission wavelengths have rendered them unsuitable for single-molecule detection. Aiming to improve on these properties, we designed a new tricyclic pyrimidine nucleoside analogue with a push-pull conjugated system and synthesized it in seven sequential steps. The resulting C-linked 8-(diethylamino)benzo[b][1,8]naphthyridin-2(1H)-one nucleoside, which we name ABN, exhibits epsilon(442) = 20 000 M-1 cm(-1) and phi(em,540) = 0.39 in water, increasing to phi(em) = 0.50-0.53 when base paired with adenine in duplex DNA oligonucleotides. Single-molecule fluorescence measurements of ABN using both one-photon and two-photon excitation demonstrate its excellent photostability and indicate that the nucleoside is present to > 95% in a bright state with count rates of at least 15 kHz per molecule. This new fluorescent nucleobase analogue, which, in duplex DNA, is the brightest and most red-shifted known, is the first to offer robust and accessible single-molecule fluorescence detection capabilities.

Automated summary

A new tricyclic pyrimidine nucleoside analogue named ABN was designed and synthesized, which exhibited improved brightness and fluorescence properties in water, becoming the brightest and most red-shifted nucleobase analogue known in duplex DNA. Single-molecule fluorescence measurements of ABN showed excellent photostability with high count rates, offering robust and accessible single-molecule fluorescence detection capabilities.