2'-Deoxy-5-(hydroxymethyl)cytidine: estimation in human cancer cells with a simple chemosensor

Abstract

A pyrrole-based rhodamine conjugate (CS-1) has been developed and characterized for the selective detection and quantification of 2'-deoxy-5-(hydroxymethyl)cytidine (5hmC) in human cancer cells with a simple chemosensing method.

Scheme 1. 5hmC-induced FRET OFF–ON mechanism of the chemosensor CS-1.

Fig. 1. ORTEP diagram of CS-1 (ellipsoids are drawn at 40% probability level).

Fig. 2. (a) UV-vis absorption spectra of CS-1 (1 μM) upon gradual addition of 5hmC up to 1.2 equiv. in H₂O–CH₃CN (15 : 1, v/v) at neutral pH. (b) Fluorescence emission spectra of CS-1 (1 μM) upon addition of 1.2 equiv. of 5hmC in H₂O–CH₃CN (15 : 1, v/v) at neutral pH (λ_ex = 325 nm).

Fig. 3. Visible color (top) and fluorescence changes (bottom) of CS-1 (1 μM) in aqueous medium upon addition of 1.2 equiv. of various pyrimidine/purine derivatives (λ_ex = 325 nm) in H₂O–CH₃CN (15 : 1, v/v) at neutral pH.

Fig. 4. Energy diagram showing the energy differences between CS-1 and CS-1–5hmC complex.

Fig. 5. Confocal microscopic images of A549 cells treated with CS-1 and 5hmC. (a) Cells treated with only CS-1 at 1 μM concentration. (b) Bright field image of (a). (c) Cells treated with CS-1 and 5hmC at concentration 10 μM. (d) Bright field image of (c). All images were acquired with a 60× objective lens with the applied wavelengths: For (a) and (b), E_ex = 341 nm, E_em = 414 nm, filter used: DIDS; for (c) and (d) E_ex = 550 nm, E_em = 571 nm, filter used: Rhod-2.

Fig. 6. (a) Calibration curve obtained for the estimation of 5hmC. (b) Estimation of the concentration of 5hmC (red point) from the calibration curve.