Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics
Journal article, 2021

To expand the antisense oligonucleotide (ASO) fluorescence labeling toolbox beyond covalent conjugation of external dyes (e.g. ATTO-, Alexa Fluor-, or cyanine dyes), we herein explore fluorescent base analogues (FBAs) as a novel approach to endow fluorescent properties to ASOs. Both cytosine and adenine analogues (tC, tCO, 2CNqA, and pA) were incorporated into a 16mer ASO sequence with a 3-10-3 cEt-DNA-cEt (cEt = constrained ethyl) gapmer design. In addition to a comprehensive photophysical characterization, we assess the label-induced effects on the gapmers’ RNA affinities, RNA-hybridized secondary structures, and knockdown efficiencies. Importantly, we find practically no perturbing effects for gapmers with single FBA incorporations in the biologically critical gap region and, except for pA, the FBAs do not affect the knockdown efficiencies. Incorporating two cytosine FBAs in the gap is equally well tolerated, while two adenine analogues give rise to slightly reduced knockdown efficiencies and what could be perturbed secondary structures. We furthermore show that the FBAs can be used to visualize gapmers inside live cells using fluorescence microscopy and flow cytometry, enabling comparative assessment of their uptake. This altogether shows that FBAs are functional ASO probes that provide a minimally perturbing in-sequence labeling option for this highly relevant drug modality.

Author

Jesper Nilsson

Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry

Tom Baladi

Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry

AstraZeneca AB

Audrey Gallud

Chalmers, Biology and Biological Engineering, Chemical Biology

AstraZeneca AB

Dženita Baždarević

AstraZeneca AB

Malin Lemurell

AstraZeneca AB

Elin Esbjörner Winters

Chalmers, Biology and Biological Engineering, Chemical Biology

Marcus Wilhelmsson

Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry

Anders Dahlén

AstraZeneca AB

Scientific Reports

2045-2322 (ISSN)

Vol. 11 1 11365

Areas of Advance

Nanoscience and Nanotechnology (SO 2010-2017, EI 2018-)

Health Engineering

Subject Categories

Biochemistry and Molecular Biology

Bioinformatics (Computational Biology)

Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

DOI

10.1038/s41598-021-90629-1

PubMed

34059711

More information

Latest update

7/7/2021 9