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, tC^O, 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.

Figure 1

(a) Two fluorescent base analogues from the tricyclic cytosine family (tC and tC^O) and two adenine analogues (2CNqA and pA) were used in this study; R = deoxyribose. (b) Sequence and chemistry of the MALAT1-targeting gapmer (U) explored in this study. The three flanking bases on each side (wings, bold font) have constrained ethyl (cEt) sugars, all other sugars are deoxyribose. The nucleotides in the gapmer are linked by phosphorothioate (PS) groups.

Figure 2

Circular dichroism (CD) spectra of tC (green), tC^O (red), 2CNqA (blue), pA (dark gray), Cy3 (violet), and unmodified U (black dotted line) gapmers hybridized to the RNA target T. The duplex concentration was 4 µM and the spectra were collected at 10–15 °C to ensure full hybridization. Insets: A zoom-out indicating the region (black rectangle) of the main figure. (a) Single-labeled gapmers. (b) Double-labeled gapmers. To illustrate the similarity to the A-form CD signature, an RNA:RNA duplex (red dashed line, normalized, see Material and Methods section for sequence) is included.

Figure 3

Normalized UV–vis absorption spectra (solid lines) and emission spectra (dashed lines) of the tC-2 (green), tC^O-2 (red), 2CNqA-2 (blue), pA-2 (black), and Cy3 (violet) gapmers.

Figure 4

Fluorescence quantum yield (Φ_F, red bars) and lifetime (τ_F, green bars) of the single-stranded (plain bars) and RNA-bound (striped bars) gapmers. Presented values are mean ± standard deviation from two independent replicates. The corresponding numeric data are provided in Supplementary Table S1.

Figure 5

Confocal microscopy images of live HEK 293 T cells continuously exposed to unformulated gapmers (3 µM) for 24 h. Samples were excited at 405 nm (emission: 407–700 nm) for FBA detection and at 561 nm (emission: 563–700 nm) for Cy3 detection. Scale bars are 20 µm.

Figure 6

Quantification of gapmer uptake in HEK 293 T cells after 24 h continuous exposure, evaluated using flow cytometry. All data were collected using the same excitation power (excitation at 405 nm) and detector settings. To enable comparisons of the FBA-labeled gapmer concentrations inside cells, a relative uptake was calculated based on the observed mean fluorescence intensity (MFI, see Supplementary Fig. S24 for the untreated data). Briefly, this was achieved by (1) subtracting the corresponding MFI from the negative control (unmodified gapmer U at equal concentration), (2) dividing by the brightness of the single-stranded gapmer (B) and (3) dividing by a spectral correction factor (SCF), see supplementary Sect. 3.5 for details. (a) Dose-dependent relative uptake. (b) Relative uptake at the top concentration in (a). Error bars are propagated standard deviations (N = 6) based on the cytometry- and brightness data (Supplementary Fig. S18). *Probability for equal mean from a paired sample t-test.