New fluorescent nucleosides for real-time exploration of nucleic acids

Abstract

Nucleic acids experience a variety of perturbations. These may include strand cleavage and ligation, local conformational changes, base flipping, as well as structural and environmental perturbations that are induced upon protein and low MW ligand binding. Since the native nucleobases are practically non-emissive, synthetic fluorescent nucleoside analogs that are sensitive to their local environment have become powerful tools for investigating nucleic acids structure, dynamics, recognition and damage. Our criteria for ideal fluorescent nucleoside analogs include: (a) high structural similarity to the native nucleobases to faithfully mimic their size and shape, as well as hybridization and recognition properties; (b) red shifted absorption (> 290 nm) to minimize overlap with the absorption of the natural bases; (c) red shifted emission (preferably in the visible range); (d) reasonable emission quantum efficiency; and, importantly, (e) sensitivity/responsiveness of one or all photophysical parameters (λem and/or ΦF, τ) to changes in the probe's microenvironment. The design and synthesis of new fluorescent isosteric nucleobase analogs and their utilization for the fabrication of real-time fluorescence-based discovery and detection assays are outlined.