QDS · New strategies for bioconjugation to quantum dots. Study of protein-nucleic acids and protein-protein interactions using fluorescence resonance energy transfer through quantum-dot-protein conjugates

Quantum Dots (QDs) are a relative new semiconductor nanoparticles made from Cd/Se or Cd/Te with a shell of ZnS that have very excellent spectroscopic properties: broad absorption spectra, low photobleaching levels, narrow and symmetric emission bands, high quantum yields and large stoke shifts, which make them very attractive for fluorescence applications and, principally, for research studies in the biomedical field. The use of nanoparticles in vitro and in vivo has come in parallel with the development of water soluble QDs. However, together with the improvement in the solubility properties, there is a nascent necessity of developing efficient chemoselective methods of bioconjugation. This purpose is where is addressed this proposal to. We are going to try to use chemoselective ligations to attach peptides and proteins to QDs. In principle, we are interested in oxime, hydrazone reactions and [3+2] azide-alkyne cycloadditions, because it´s known that are very selective and happen with efficient rates at low µM concentrations (~10 µM) forming thermodynamically and kinetically stable products. Moreover, these ligation reactions have been used with success in peptide-peptide and peptide-dye conjugations. Once we have developed the conjugation methodology, we have in mind to apply it to the study of protein-nucleic acids and protein-protein interactions through the well-known FRET technique (Fluorescence Resonance Energy Transfer). We think that attaching a low number of proteins per quantum dot (around 5-10 protein molecules/QD) we can get high amplifications in the FRET signal, which will allow us to measure thermodynamic parameters like binding constants. To evaluate the method, we´ll study the system formed by GCN4 (a natural Transcription Factor) and its CRE recognition site (5´-…ATGACGTCAT…-3´), principally because there is a high literature data that help us to validate the approach.