Improved hydration-sensitive dual-fluorescence labels for monitoring peptide-nucleic acid interactions.

Environmentally sensitive labels constitute a new, attractive tool for monitoring biomolecular interactions. 3-Hydroxychromone derivatives are of particular interest because they undergo excited-state intramolecular proton transfer (ESIPT) showing dual emission highly sensitive to environmental hydration. To overcome the drawbacks of the previously developed label for sensing protein-DNA interactions based on 2-furanyl-3-hydroxychromone (FC), a series of hydration-sensitive labels based on 3-hydroxy-4'-methoxyflavone have been synthesized. As compared to FC, the new labels display higher sensitivity of the ratio of their two emission bands (N*/T*) to solvent polarity and H-bond donor ability, as well as higher fluorescence quantum yields in water. Moreover, they show higher pK(a) values of their 3-hydroxyl group, allowing their application at neutral pH without interference of anionic forms. To illustrate the applications of these labels, we covalently coupled them to the N-terminus of the Tat(44-61) peptide that corresponds to the basic domain of the HIV-1 Tat protein. This coupling did not modify the nucleic acid chaperone properties of the peptide. Binding of oligonucleotides of varying length, sequence, and strandedness to the labeled peptides induced dramatic change in the N*/T* ratio of their two emission bands. This change indicated that the level of probe hydration in the peptide/oligonucleotide complexes decreases in the following order: short ssDNAs ≫ long ssDNAs > DNA hairpins > dsDNAs. The level of probe hydration was related to the ability of the probe to stack with the DNA bases or base pairs in the various complexes. The changes in the N*/T* ratio upon interaction of the labeled Tat peptides with DNA were about 3-fold larger with the new probes as compared to the parent FC label, in line with the higher sensitivity of the new probes to the environment. One of these labels, presenting the most compact geometry, showed the highest sensitivity, probably due to its optimal stacking with the DNA bases. Thus, the new hydration-sensitive labels appear as improved highly sensitive tools to site-selectively monitor the binding of peptides to oligonucleotides and nucleic acids.