DNA-Silver Cluster Complexes for Fluorescent Diagnostics

描述（由申请人提供）：荧光是生物分析的有力工具，包括高灵敏度检测和体内成像。所提出的研究考虑了一类新的生物相容性和生物功能性荧光标记物，其由寡核苷酸包封的小（< 10个原子）银簇组成。这些分子金属具有离散的电子能级，从中观察到强荧光。DNA以碱基和序列特异性方式稳定簇。通过以下实验，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。I. DNA阵列将被用来系统地评估集群形成的序列依赖性。将基于高荧光强度和独特的荧光光谱来鉴定感兴趣的序列。两种类型的结合基序-单链和发夹寡核苷酸-将被用作模板合成的银簇。这种方法也将被用来考虑如何反应条件，如pH值的影响集群的形成。二.在利用DNA芯片快速筛选的基础上，将对特定复合物的物理化学特性进行评价。有限的光稳定性是其他发色团的特征。例如，与氧的反应导致发色团的光破坏。此外，在自旋禁止激发态的长停留时间导致间歇性荧光，这降低了净荧光产率并抑制了单分子检测。初步的研究表明，银团簇是不太容易受到这些问题，我们的研究将集中在序列依赖性的光稳定性使用的序列确定使用的阵列。三.阵列研究提供了评估簇如何形成的基础，因此将研究与这些DNA序列的银阳离子络合物。等温滴定量热法将表征阳离子-DNA缀合物的热力学参数。Ag+与单个碱的结合位点的相互作用将使用溶液的pH和离子强度的热力学参数的变化进行研究。此外，Ag+对单链寡核苷酸构象的影响将通过测量阳离子诱导的碱基溶剂暴露的变化来研究。光谱学将用于确定复合物的结构和稳定性。 公共卫生相关性：荧光是一种强大的医学工具，应用于分子诊断和体内成像。我们正在开发一类新的生物相容性和生物功能的荧光探针基于DNA封装银簇。它们良好的光稳定性、明亮的荧光和独特的光谱表明了这类新的荧光标记物的前景。

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.