Pseudointrinsic probes for generating spectrally enhanced proteins and nucleic acids

The convergence of methods and techniques in biological fluorescence spectroscopy and molecular biotechnology have resulted in improved strategies for labelling specific sites in proteins and nucleic acids. Extrinsic probes, such as dansyl or fluorescein, are commonly used for labelling of proteins and nucleic acids. Introduction of extrinsic probes by covalent modification, however, is always accompanied by the potential risk of altering structure and/or function of these macromolecules. As an alternative to the use of extrinsic probes, there has been a developing interest in the use of tryptophan or nucleic acid base analogs as pseudo intrinsic probes in proteins and nucleic acids. The objective is to generate spectrally enhanced proteins or nucleic acids that are labelled at specific sites and that retain most or all of the functional features of the non-enhanced parent macromolecule. Base analogs with desirable spectroscopic properties can be introduced by direct synthesis. Tryptophan analogs with desirable spectroscopic properties can be introduced into proteins by expression in vivo or in vitro, or by direct chemical synthesis.