Fluorescence is a powerful tool for biological analysis, including high sensitivity detection and in vivo imaging. The proposed studies consider a new class of biocompatible and biofunctional fluorescent labels that are comprised of small (< 10 atoms) silver clusters encapsulated by oligonucleotides. These molecular metals have discrete electronic energy levels from which strong fluorescence is observed. The DNA stabilizes the clusters in a base and sequence specific manner. Via the following experiments, our goal is to vary the types of clusters and hence their fluorescence color by varying the sequence of the DNA. I. DNA arrays will be used to systematically evaluate the sequence dependence of cluster formation. Interesting sequences will be identified based on high fluorescence intensities and distinctive fluorescence spectra. Two types of binding motifs - single-stranded and hairpin oligonucleotides - will be used as templates for synthesis of the silver clusters. This approach will also be used to consider how reaction conditions such as pH influence cluster formation. II. On the basis of the rapid screening using DNA arrays, the photophysical characteristics of specific complexes will be evaluated. Limited photostability is characteristic of other chromophores. For example, reaction with oxygen results in photodestruction of the chromophore. In addition, long residence times in spin-forbidden excited states result in intermittent fluorescence that reduces the net fluorescence yield and inhibits single molecule detection. Preliminary studies indicate that the silver clusters are much less susceptible to these problems, and our studies will focus on the sequence dependence of the photostability using the sequences identified using the arrays. III. The array studies provide the basis for assessing how the clusters are formed, so the silver cation complexes with these DNA sequences will be studied. Isothermal titration calorimetry will characterize the thermodynamic parameters of the cation-DNA conjugates. The interaction of Ag+ with the binding sites of the individual bases will be studied using the variations of the thermodynamic parameters with the pH and ionic strength of the solution. In addition, the effect of Ag+ on the conformation of the single-stranded oligonucleotides will be studied by measuring the cation-induced changes in the solvent exposure of the bases. Optical spectroscopy will be used to determine the structure and stabilities of the complexes.
Fluorescence is a powerful tool in medicine with applications such as molecular diagnostics and in vivo imaging. We are developing a new class of biocompatible and biofunctional fluorescence probes based on DNA encapsulated silver clusters. Their favorable photostability, bright fluorescence, and distinctive spectra indicate the promise of this new class of fluorescence labels. ? ? ?
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