Fluorescent probes for the detection of DNA
- Marie-Paule Teulade-Fichou
- Florence Mahuteau-Betzer
- Delphine Martin
During the past decade, there has been a great interest in the conception of fluorescent probes for the detection of nucleic acids. The requested characteristics for a good fluorescent DNA probe are a better affinity for DNA than for other cell components, a large increase in quantum yield upon binging with DNA, a low mutagenicity and emission inside the physiological optical window (700-1300 nm) where biological tissues absorb less. In this context, we have developed conjugated systems with high charge transfer based on the triphenylamine core (TP). The first generation of vinyl triphenylamines was based on the introduction of pyridinium acceptors tethered to the central triphenylamine donor via vinyl arms which resulted in the classical quadrupolar and octupolar (TP-2Py, TP-3Py) arrangements.
These dyes are light-up fluorescent DNA probes with a long wavelength emission, good DNA affinity and good 2PA properties. In order to optimize the photophysical properties, different analogues were prepared. This optimization led to the identification of the TP-Bzim series bearing two or three vinyl branches terminated by a N-methyl benzimidazolium moiety. Compared to their pyridinium models, the TP-Bzim dyes exhibit a remarkable improvement of both their DNA affinity and fluorescence quantum yield, especially for the two-branch derivative (TP-2Bzim: ΦF = 0.54, Ka = 107 M−1), resulting in a large fluorescence emission turn-on ratio of up to 140. Concomitantly, the two-photon absorption cross-section of TP-2Bzim is dramatically enhanced upon DNA binding (δ = 1080 vs 110 GM for the free form). This effect of the DNA matrix on the nonlinear absorption is uncovered for the first time.
These properties enable to image nuclear DNA in fixed cells at submicromolar concentration ([TP-2Bzim] = 100 nM) and to visualize ultrabright foci of centromeric AT-rich chromatin. Finally TP-2Bzim exhibits a high photostability, is live-cell permeant, and does not require RNase treatment. This outstanding combination of optical and biological properties makes TP-2Bzim a bioprobe surpassing the best DNA stainers and paves the way for studying further nonlinear optical processes in DNA.
By contrast, two-branch triphenylamines accumulate mostly in the mitochondria of living cells and efficiently trigger cell apoptosis upon prolonged irradiation. This process occurs concomitantly with their subcellular re-localization to the nucleus, enabling fluorescence imaging of apoptosis.
Finally, as the resulting TP-dyes represent a new family of switchable biolabels that display essential criteria for single and two-photon excitation fluorescence imaging in cells, we were interested in developing protein probes. We first studied the affinity for a model protein ((human serum albumin) using complementary absorption and fluorescence spectroscopy characterization tools. By replacing the cationic acceptors by anionic ones, we switched from DNA probes to protein probes (anionic TP-rhodanine series) without affecting the optical properties.
