We propose to develop and analyze water-soluble nanomaterials as reporters in FRET optical bioassay technologies to probe conformational dynamics for nucleo-protein interactions. The ability to interrogate bimolecular interactions in the context of binding and dissociation kinetics and thermodynamics is essential to obtain a molecular understanding of the interplay between structure and function. Using variations on existing organic dye based bioassays, we propose to develop the chemistry, probe the properties, and demonstrate the applicability of nanomaterials in the bio-nano conjugates, while providing a foundation for interfacing more complex and diverse protein-DNA-systems. The selected protein """"""""systems"""""""" include the well studied EcoRV endonuciease (R.ECORV), the EcoRi DNA methyltransferase (M.ECORI), and the Papl regulon that controls the expression of pill-related proteins in pathogenic E.coli bacteria. Each of the three systems involves dramatic conformation changes, ranging from base flipping (M.ECORi) and localized DNA bending (M.EcoRi and R.EcoRV) to large scale DNA looping (Papl regulon). The use of nanoscale materials in FRET based assays results in substantial enhanced photostabiiity in comparison to organic dyes (20x), and enhanced long range energy transfer (> 10 nm) for FRET applications. The nanomaterial bimolecular interface, which involves components of similar size scales, remains poorly understood and any application in bioassay technologies requires analysis of the biocompatibility of these materials. The development of long-range FRET assays based on nanomaterial bio-conjugates will allow a significant extension of the current technology into multiple protein binding assays for analysis of conformational changes in the nucleic acid structures. This information is needed for a basic understanding of protein ligand interactions, for the intelligent redesign of such systems, and for the development of modulators of such interactions (e.g., novel drugs). ? ?
Showing the most recent 10 out of 17 publications
