Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics

Abstract 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 cytosin...

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Autores principales:	Jesper R. Nilsson, Tom Baladi, Audrey Gallud, Dženita Baždarević, Malin Lemurell, Elin K. Esbjörner, L. Marcus Wilhelmsson, Anders Dahlén
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8abf403a3bcb4d9fa1479c960effbb9d
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spelling	oai:doaj.org-article:8abf403a3bcb4d9fa1479c960effbb9d2021-12-02T15:56:57ZFluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics10.1038/s41598-021-90629-12045-2322https://doaj.org/article/8abf403a3bcb4d9fa1479c960effbb9d2021-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-90629-1https://doaj.org/toc/2045-2322Abstract 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 R. NilssonTom BaladiAudrey GalludDženita BaždarevićMalin LemurellElin K. EsbjörnerL. Marcus WilhelmssonAnders DahlénNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jesper R. Nilsson Tom Baladi Audrey Gallud Dženita Baždarević Malin Lemurell Elin K. Esbjörner L. Marcus Wilhelmsson Anders Dahlén Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics
description	Abstract 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.
format	article
author	Jesper R. Nilsson Tom Baladi Audrey Gallud Dženita Baždarević Malin Lemurell Elin K. Esbjörner L. Marcus Wilhelmsson Anders Dahlén
author_facet	Jesper R. Nilsson Tom Baladi Audrey Gallud Dženita Baždarević Malin Lemurell Elin K. Esbjörner L. Marcus Wilhelmsson Anders Dahlén
author_sort	Jesper R. Nilsson
title	Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics
title_short	Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics
title_full	Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics
title_fullStr	Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics
title_full_unstemmed	Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics
title_sort	fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/8abf403a3bcb4d9fa1479c960effbb9d
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Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics

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