Multiphoton characterization and live cell imaging using fluorescent adenine analogue 2CNqA

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Fluorescent nucleobase analogues (FBAs) are crucial for studying oligonucleotide behavior and labeling RNA therapeutics.
• Conventional FBAs often require UV excitation, posing challenges like phototoxicity and limited tissue penetration in biological imaging.
• Multiphoton excitation offers a promising alternative to overcome these limitations.

Purpose of the Study:

• To characterize the multiphoton absorption properties and detectability of the fluorescent nucleobase analogue 2CNqA.
• To evaluate 2CNqA as a monomer and when incorporated into antisense oligonucleotides (ASOs).
• To assess the utility of two-photon (2P) imaging for enhanced detection of 2CNqA-labeled ASOs in live cells.

Main Methods:

• Characterization of two-photon absorption cross-section (σ2PA) for 2CNqA monomer and ASOs.
• Utilizing fluorescence correlation spectroscopy (FCS) to determine 2P brightness.
• Demonstrating 2P imaging capabilities in live cellular environments.

Main Results:

• 2CNqA exhibits exceptionally high two-photon absorption cross-section values, increasing with oligonucleotide labeling.
• 2CNqA demonstrates high 2P brightness, comparable or superior to other FBAs, in both monomeric and oligonucleotide-incorporated forms.
• Two-photon imaging significantly enhances the detectability of 2CNqA-labeled ASOs within live cells.

Conclusions:

• 2CNqA is a highly fluorescent nucleobase analogue with superior two-photon absorption properties.
• Its incorporation into ASOs maintains or enhances brightness, making it suitable for advanced imaging applications.
• Two-photon excitation microscopy provides a powerful tool for visualizing 2CNqA-labeled ASOs in biological systems, advancing RNA therapeutics research.