DNA-Silver Cluster Complexes for Fluorescent Diagnostics

描述（申请人提供）：荧光是一种强大的生物分析工具，包括高灵敏度检测和体内成像。提出的研究考虑了一类新的生物相容性和生物功能荧光标记，它由小的（< 10个原子）银簇组成，由寡核苷酸包裹。这些分子金属具有离散的电子能级，从中可以观察到强烈的荧光。DNA以一种碱基和序列特定的方式稳定集群。通过下面的实验，我们的目标是通过改变DNA的序列来改变簇的类型，从而改变它们的荧光颜色。DNA阵列将用于系统地评估集群形成的序列依赖性。基于高荧光强度和独特的荧光光谱，将识别出有趣的序列。两种类型的结合基序-单链和发夹寡核苷酸-将被用作合成银簇的模板。这种方法还将用于考虑反应条件（如pH值）如何影响簇的形成。2。在DNA阵列快速筛选的基础上，评估特定复合物的光物理特性。有限的光稳定性是其他发色团的特点。例如，与氧的反应导致发色团的光破坏。此外，长时间停留在自旋禁止激发态会导致间歇性荧光，从而降低净荧光产率并抑制单分子检测。初步研究表明，银团簇不易受到这些问题的影响，我们的研究将集中在利用阵列识别的序列和光稳定性的序列依赖上。3。阵列研究为评估簇的形成提供了基础，因此将对这些DNA序列的银阳离子配合物进行研究。等温滴定量热法将表征阳离子- dna共轭物的热力学参数。利用热力学参数随溶液pH值和离子强度的变化，研究Ag+与单个碱基结合位点的相互作用。此外，Ag+对单链寡核苷酸构象的影响将通过测量碱基在溶剂暴露中阳离子诱导的变化来研究。光谱学将用于确定配合物的结构和稳定性。公共卫生相关性：荧光在医学上是一种强大的工具，应用于分子诊断和体内成像。我们正在开发一种基于DNA包裹银簇的新型生物相容性和生物功能荧光探针。它们良好的光稳定性、明亮的荧光和独特的光谱表明了这种新型荧光标签的前景。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.