Single-molecule fluorescence detection of a tricyclic nucleoside analogue.

George N Samaan,Lisa-Maria Needham,Susan Andersen,Julian M Cizmic,Katrina Ngo,Steven W Magennis,Byron W Purse,David Nobis,Mckenzie K Wyllie,Steven F Lee

doi:10.1039/d0sc03903a

Abstract

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 ε442 = 20 000 M−1 cm−1 and Φem,540 = 0.39 in water, increasing to Φem = 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.

Highlights

Single-molecule uorescence studies of biological molecules have a unique capacity to provide mechanistic insights into the relationships between structural dynamics and function, which are lost to averaging in ensemble measurements.[1,2,3] Most of these studies have used extrinsic uorophores, which can potentially interfere with the native biomolecular behavior and obscure local structural details.[4,5,6,7] An ideal approach in this regard would be the use of intrinsically uorescent biomolecules, prepared by the synthetic introduction of only minimal changes.[8]
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
The resulting nucleoside analogue, whose synthesis and characterization we report here, includes 8-(diethylamino)benzo[b] [1,8]naphthyridin-2(1H)-one as a uorescent nucleobase surrogate, and we name this new compound ABN

Summary

Introduction

Single-molecule uorescence studies of biological molecules have a unique capacity to provide mechanistic insights into the relationships between structural dynamics and function, which are lost to averaging in ensemble measurements.[1,2,3] Most of these studies have used extrinsic uorophores, which can potentially interfere with the native biomolecular behavior and obscure local structural details.[4,5,6,7] An ideal approach in this regard would be the use of intrinsically uorescent biomolecules, prepared by the synthetic introduction of only minimal changes.[8]. 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.

Results

Conclusion