New Far-red and Near-infrared Fluorescent Probes with Large Stokes Shifts for Dual Covalent Labeling of DNA

摘要

Imaging biological structures by means of so-called farred, near infrared (NIR) fluorophores is becoming more and more important for their high sensitivity, excellent temporal and spatial resolution, and their potential for multichannel imaging. Being less or not at all interfered by biological background luminescence, these fluorophores are particularly suitable for biological labeling both in vitro and in vivo. Very recently, we have reported on the synthesis of a series of "clickable" dyes that emit in the far-red/NIR regime having very large Stokes shifts. We have demonstrated that these dyes are suitable for bioorthogonal labeling protocols by labeling metabolically modified cell-cultures. The copper catalyzed 1,3-dipolar cycloaddition between azides and terminal alkynes, the most so-called "click" ligation, ensures covalent and postsynthetic attachment of these probes to oligonucleotides of interest in a fast, high yielding, biocompatible reaction between functional groups that do not interfere with other naturally occurring reactive sites. We recently reported the single labeling of oligonucleotides with one representative coumarin dye using "click" chemistry. The dyes were ligated to a propargyl group that was attached to the 2'-OH function of the ribofuranoside of uridine. Herein we want to extend this chemistry to three different dyes of this fluorophore class. The large Stokes shifts make these dyes ideal candidates for Forster resonance energy transfer (FRET) applications. It is an often encountered problem in FRET assays composed of fluorophores with ordinary Stokes shifts that direct excitation of the acceptor occurs by the light used to excite the donor. This problem largely reduces the dynamic range of assays where most of the acceptor is unbound. Another problem that has to be taken into account in such cases is the overlap between the emission bands of the donor and acceptor dyes. The large Stokes shifts of the fluorophores applied herein are likely to reduce these difficulties as the excitation and emission spectra are well resolved. In the present study we demonstrate the applicability and power of our clickable megaStokes dyes in FRET systems by dual modification of DNA sequences.