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

Jesper R. Nilsson,Malin Lemurell,Audrey Gallud,Dženita Baždarević,L. Marcus Wilhelmsson,Anders Dahlén,Elin K. Esbjörner,Tom Baladi

doi:10.1038/s41598-021-90629-1

Jesper R. Nilsson, Malin Lemurell + Show 6 more

Open Access

https://doi.org/10.1038/s41598-021-90629-1

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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.

Highlights

To expand the antisense oligonucleotide (ASO) fluorescence labeling toolbox beyond covalent conjugation of external dyes (e.g. ATTO, Alexa Fluor, or cyanine dyes), we explore fluorescent base analogues (FBAs) as a novel approach to endow fluorescent properties to antisense oligonucleotides (ASOs)
It has previously been shown that the effect on T m of substituting canonical bases for FBAs typically depends on the neighboring bases in the oligonucleotide; for example, tC substitution in 10mer DNA renders a ΔTm of 5 °C for TT neighbors, but − 1 °C for GA n eighbours[42], which are the positions for the cytosine analogues in this work
We report that incorporation of one or two FBAs in the gap region of the gapmer does not lead to substantial changes in ribonucleic acids (RNAs) affinity for any of the investigated FBAs

Summary

Introduction

To expand the antisense oligonucleotide (ASO) fluorescence labeling toolbox beyond covalent conjugation of external dyes (e.g. ATTO-, Alexa Fluor-, or cyanine dyes), we explore fluorescent base analogues (FBAs) as a novel approach to endow fluorescent properties to ASOs. The currently favored approach for introducing fluorescent labels to ASOs is by covalent conjugation of fluorophores, such as ATTO-, Alexa Fluor-, or cyanine (Cy) dyes, to the end of the oligonucleotide This external end-labeling scheme typically renders the gapmer highly emissive, the significant size, amphiphilicity, and/or additional charges associated with the fluorophores can be problematic as they may affect properties that are critical to the gapmer’s therapeutic performance, including the affinity to the target RNA and/or interactions with RNase H1, and may affect the affinity to lipid structures. We perform a comprehensive photophysical characterization, which is essential for accurate interpretation of fluorescence-based data (e.g. from live cell microscopy studies), and study how different FBAs, and positions in the gapmer sequence, affect their RNA target affinities, secondary structures, knockdown activities, and uptake characteristics. The study includes the tricyclic cytosine analogues tC32 and tCO33, as well as the more recently developed quadracyclic adenine analogue 2 CNqA34, and pentacyclic adenine analogue p A35 (Fig. 1a), all of which were incorporated into a 16 nt gapmer sequence (Fig. 1b) targeting the long non-coding RNA Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1)[36]

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