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 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.

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

“…The 1P spectra of 2CNqA-1 and 2CNqA-2 were reported in previous work 13 and the 2CNqATP exhibits similar spectral characteristics, albeit with a slight blue-shift in absorption ( ca. 5 nm, λ max = 352 nm) and red-shift in emission ( ca.…”

“…14 2CNqATP was obtained in a good yield, similar to that observed for the former cytosine analogue, thus highlighting the robustness of the triphosphorylation process. The 2CNqA-labelled oligonucleotides were synthesized using a solid-phase oligonucleotide protocol, as described by Nilsson et al 13…”

“…Since the therapeutic mechanism of ASOs inside cells involves the ASO binding to its complementary target RNA, 29 we decided to also characterize the photophysical properties of 2CNqA-1 and 2CNqA-2 as RNA-bound duplexes and found that the absorption cross sections and multiplicities were largely retained (ESI† Fig S8). This, along with the nearly unchanged fluorescent quantum yield for these ASOs upon RNA binding, 13 could make 2CNqA a useful probe for multiphoton techniques aimed at quantifying oligonucleotide concentration via fluorescence readout.…”

“…11,12 FBAs have also been used to label and track oligonucleotides with therapeutic potential, such as antisense oligonucleotides (ASOs) and mRNA, in live cells. 13,14…”

“…, intracellular fluorescence is likely to emanate from intact oligonucleotides rather than degradation products, and (3) it is nontoxic to cells up to at least 3 μM. 13…”