Single-Molecule Detection of a Fluorescent Nucleobase Analogue via Multiphoton Excitation

David Nobis,Anita C Jones,Rachel S Fisher,Patrycja A Hopkins,Yitzhak Tor,Steven W Magennis,Mats Simmermacher

doi:10.1021/acs.jpclett.9b02108

Abstract

The ability to routinely detect fluorescent nucleobase analogues at the single-molecule level would create a wealth of opportunities to study nucleic acids. We report the multiphoton-induced fluorescence and single-molecule detection of a dimethylamine-substituted extended-6-aza-uridine (DMAthaU). We show that DMAthaU can exist in a highly fluorescent form, emitting strongly in the visible region (470-560 nm). Using pulse-shaped broadband Ti:sapphire laser excitation, DMAthaU undergoes two-photon (2P) absorption at low excitation powers, switching to three-photon (3P) absorption at high incident intensity. The assignment of a 3P process is supported by cubic response calculations. Under both 2P and 3P excitation, the single-molecule brightness was over an order of magnitude higher than reported previously for any fluorescent base analogue, which facilitated the first single-molecule detection of an emissive nucleoside with multiphoton excitation.

Highlights

Supporting Information Placeholder magnitude higher than reported previously for any fluorescent base analog, which facilitated the first singlemolecule detection of an emissive nucleoside with multiphoton excitation
We show that DMAthaU can exist in a highly fluorescent form, emitting strongly in the visible region (470–560 nm)
The assignment of a 3P process is supported by cubic response calculations. Under both 2P and 3P excitation, the single-molecule brightness was over an order of Fluorescence-based methods are powerful tools for studying the structure, dynamics and interactions of nucleic acids in vitro and in vivo, and for technologies such as DNA sequencing and high-throughput screening

Summary

Introduction

Supporting Information Placeholder magnitude higher than reported previously for any fluorescent base analog, which facilitated the first singlemolecule detection of an emissive nucleoside with multiphoton excitation. We report the multiphoton-induced fluorescence and single-molecule detection of a dimethylaminesubstituted extended-6-aza-uridine (DMAthaU).

Results

Conclusion