Oligonucleotide Encapsulated Ag Nanoclusters as Single Molecule Labels

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DESCRIPTION (provided by applicant): The long term goal of this research is the development of ultra-bright, single molecule labels for biological systems through the integration of nanomaterials and biology. Unlike ensemble measurements, single molecule spectroscopy allows for the resolution of mechanistic dynamics of complex biological activity. Despite recent success in the application of single molecule spectroscopy to biological systems, studies are limited by the brightness and photostability of available fluorophores. The creation of bright and extremely stable fluorophores will facilitate advances in understanding biological function on both much faster and longer lasting timescales. Through the three Specific Aims we will integrate nanomaterials, in the form of highly fluorescent Ag nanoclusters, with biocompatible oligonucleotide scaffolds, to create a new class of biological labels. In Specific Aim I, the in vitro photophysical parameters of oligonucleotide encapsulated Ag nanoclusters will be studied. Characterization of blinking dynamics, emission rates, and photostability of individual molecules in solution will allow for the identification of fluorescent Ag nanocluster species that show the greatest promise as single molecule labels. In Specific Aim II, the encapsulation of the nanomaterials identified in the previous aim will be targeted through the use of DMA and RNA microarrays. By varying oligonucleotide strand length and base sequence, the optimum scaffold for the Ag nanocluster of interest will be determined. In Specific Aim III, the optimized oligonucleotide-Ag nanocluster combination will be evaluated for biocompatibility. Cellular uptake mechanisms will be determined and enhanced through backbone modification and conjugation with cell penetrating peptides. The proposed development of a new class of single molecule fluorophores should help lead to advancements in unraveling the dynamics of complex biological systems. Relevance to Public Health Brighter and more robust biological labels would allow for advanced single molecule studies leading to a more complete understanding of the function of biomolecules within living systems. The ability to resolve these functions and determine the dynamics of complex interactions between disease causing biomolecules and cellular components is paramount to understanding infection mechanisms.