Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics
Artikel i vetenskaplig tidskrift, 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.


Jesper Nilsson

Chalmers, Kemi och kemiteknik, Kemi och biokemi

Tom Baladi

Chalmers, Kemi och kemiteknik, Kemi och biokemi

AstraZeneca AB

Audrey Gallud

Chalmers, Biologi och bioteknik, Kemisk biologi

AstraZeneca AB

Dženita Baždarević

AstraZeneca AB

Malin Lemurell

AstraZeneca AB

Elin Esbjörner Winters

Chalmers, Biologi och bioteknik, Kemisk biologi

Marcus Wilhelmsson

Chalmers, Kemi och kemiteknik, Kemi och biokemi

Anders Dahlén

AstraZeneca AB

Scientific Reports

2045-2322 (ISSN) 20452322 (eISSN)

Vol. 11 1 11365


Nanovetenskap och nanoteknik

Hälsa och teknik


Biokemi och molekylärbiologi

Bioinformatik (beräkningsbiologi)

Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)





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