Multiphoton characterization and live cell imaging using fluorescent adenine analogue 2CNqA
Artikel i vetenskaplig tidskrift, 2023

Fluorescent nucleobase analogues (FBAs) are established tools for studying oligonucleotide structure, dynamics and interactions, and have recently also emerged as an attractive option for labeling RNA-based therapeutics. A recognized drawback of FBAs, however, is that they typically require excitation in the UV region, which for imaging in biological samples may have disadvantages related to phototoxicity, tissue penetration, and out-of-focus photobleaching. Multiphoton excitation has the potential to alleviate these issues and therefore, in this work, we characterize the multiphoton absorption properties and detectability of the highly fluorescent quadracyclic adenine analogue 2CNqA as a ribonucleotide monomer as well as incorporated, at one or two positions, into a 16mer antisense oligonucleotide (ASO). We found that 2CNqA has a two-photon absorption cross section that, among FBAs, is exceptionally high, with values of & sigma;(2PA)(700 nm) = 5.8 GM, 6.8 GM, and 13 GM for the monomer, single-, and double-labelled oligonucleotide, respectively. Using fluorescence correlation spectroscopy, we show that the 2CNqA has a high 2P brightness as the monomer and when incorporated into the ASO, comparing favorably to other FBAs. We furthermore demonstrate the usefulness of the 2P imaging mode for improving detectability of 2CNqA-labelled ASOs in live cells.


Jesper Nilsson

Chalmers, Kemi och kemiteknik, Kemi och biokemi

Carlos Benitez-Martin

Chalmers, Kemi och kemiteknik, Kemi och biokemi

Henry G. G. Sansom

University of Glasgow

Pauline Pfeiffer

Chalmers, Kemi och kemiteknik, Kemi och biokemi

Tom Baladi

AstraZeneca AB

Hoang-Ngoan Le

AstraZeneca AB

Anders Dahlen

AstraZeneca AB

Steven W. W. Magennis

University of Glasgow

Marcus Wilhelmsson

Chalmers, Kemi och kemiteknik, Kemi och biokemi

Physical Chemistry Chemical Physics

1463-9076 (ISSN) 1463-9084 (eISSN)

Vol. 25 30 20218-20224

Funktionell leverans av nukleotid-baserade läkemedel

Stiftelsen för Strategisk forskning (SSF) (IRC15-0065), 2017-03-01 -- 2024-12-31.


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Biokemi och molekylärbiologi

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