Light-up properties of complexes between thiazole orange-small molecule conjugates and aptamers

Abstract

The full understanding of dynamics of cellular processes hinges on the development of efficient and non-invasive labels for intracellular RNA species. Light-up aptamers binding fluorogenic ligands show promise as specific labels for RNA species containing those aptamers. Herein, we took advantage of existing, non-light-up aptamers against small molecules and demonstrated a new class of light-up probes in vitro. We synthesized two conjugates of thiazole orange dye to small molecules (GMP and AMP) and characterized in vitro their interactions with corresponding RNA aptamers. The conjugates preserved specific binding to aptamers while showing several 100-fold increase in fluorescence of the dye (the 'light-up' property). In the presence of free small molecules, conjugates can be displaced from aptamers serving also as fluorescent sensors. Our in vitro results provide the proof-of-concept that the small-molecule conjugates with light-up properties can serve as a general approach to label RNA sequences containing aptamers.

Scheme 1.

Principle behind light-up probes for aptamers and their displacemnt with unconjugated small molecules: An RNA aptamer against a molecule (circle) binds a conjugate between molecule and TO dye, resulting in a fluorescent complex. Presence of free molecule in solution results in displacement of conjugate from the complex and reduction in fluorescence.

Figure 1.

(a) Fluorescence emission spectra of TO-GMP (65 nM) in the presence of varying concentrations of GTP Class I (0, 0.0195, 0.039, 0.078, 0.1563, 0.625 and 2.5 µM, frssom bottom to top). The insert shows for maximum fluorescence versus aptamer concentration curve fitting (K_d = 60 nM). (b) Dependence of the fluorescence on the concentration of GTP Class I aptamer for: 1; TO; 2 and TO-C₁₀ (all measurements at 50 nM of dyes).

Scheme 2.

Synthesis of GMP and AMP-TO conjugate (conjugates were made through an identical procedure and with similar yields).

Scheme 3.

Aptamers tested with their conjugates. Black elipsoid is NMP part of the conjugate, while green fluorescent rectangle is TO-C₁₀ part. In both cases the place of the insertion of TO dyes is shown aribitrarily, although in the case of AMP derivative we have indications based on aptamer modifications (Supplementary Data).

Figure 2.

Fluorescence response of TO-GMP (300 nM) containing GTP Class I (600 nM) in the presence of varying concentrations of GTP (0, 0.39, 0.78, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 µM). Small insert shows displacement (% remaining versus concentration) in the presence of GTP, and no response to other NTPs.