The fluorescence of a nucleoside base is highly dependent on the environment of the base and the measurement of its fluorescence is a powerful and sensitive tool for the analysis of DNA and RNA structure. It is especially useful for analyzing the interaction of DNA and RNA with their corresponding binding proteins. Fluorescence measurements allow real-time probing of these structural interactions. Unfortunately, the intrinsic fluorescence of the regular bases is extremely low to non-existent, so fluorescent analogues have been sought for a long time.
For probing DNA structure, the ideal fluorescent nucleoside:
Should have bright fluorescence, which is sensitive to its environment, and a large Stokes shift;
Should be amenable to phosphoramidite preparation for incorporation into oligo-nucleotides by solid-phase synthesis;
Should not disrupt duplex formation and should mimic one of the regular nucleosides;
Should behave as a regular nucleoside in its interaction with proteins and enzymes; and
Should be capable of being converted to the triphosphate and be incorporated into DNA with high efficiency by current commercial polymerases.