Biosensing using DNA Nanoreactors that Transform Chromophoric Silver Clusters

描述（由申请人提供）：荧光是生物分析的有力工具，但背景干扰可能需要广泛纯化以进行体外分析，并可能严重限制体内研究。我们的目标是使用小的银簇来推进生物分子诊断和成像，所述小的银簇由在生物样品相对透明的近红外光谱区域中具有强发射的~10个原子组成。这些金属配体与寡核苷酸传感器结合，其整合了两种不同的功能： 特定的银簇通过规定序列内的核碱基配位和通过互补碱基配对识别靶寡核苷酸。当分析物与该传感器的杂交将簇从具有紫色吸收的非发射状态转换为具有近红外吸收的高发射状态时，可以实现可靠和高灵敏度的检测。这些传感器的特点是其强大的和光稳定的荧光响应和其经济，方便和模块化的合成，从而打开这种方法的多种应用。我们的研究将集中在一系列作为人类疾病生物标志物的microRNA序列上。以下合成、结构和稳定性研究将为开发这种新传感策略的全部潜力提供基础。I.优化反应条件和序列环境是形成和转化特定银簇的关键步骤。高度平行的序列变异与光谱分析将与簇化学计量和寡核苷酸形状的测量相结合，以设计适合于直接分析生物样品的传感器。二.这些金属发色团不仅通过它们的光谱来区分，而且通过它们在形成传感器中的积极作用来区分。我们已经表明，一个紫色的吸收集群折叠其DNA主机，和生化/化学探针和荧光碱基类似物将被用来映射二级结构的DNA主机通过溶剂暴露其核碱基。通过识别簇结合的位置以及它们如何影响DNA结构，将开发包含簇诱导折叠的通用传感器。三.因为簇协调和折叠其DNA宿主，所以靶寡核苷酸的杂交被抑制。这种障碍将被利用，使具有错配碱基对的寡核苷酸之间的细微区别，从而降低了对特定传感器的亲和力。伴随温度引起的展开的光谱变化将提供区分这些目标的定量热力学基础。 通过上述研究提供的结构和热力学见解，将开发一种基于顺序展开的新的信号放大方案用于检测低丰度物种。

DESCRIPTION (provided by applicant): Fluorescence is a powerful tool for biological analysis, but background interference can necessitate extensive purification for in vitro analysis and can severely constrain in vivo studies. Our goal is to advance biomolecular diagnosis and imaging using small silver clusters comprised of ~10 atoms with strong emission in the near-infrared spectral region where biological samples are relatively transparent. These metallic ligands associate with oligonucleotide sensors that integrate two distinct functions: the formation of specific silver clusters via nucleobase coordination within prescribed sequences and the recognition of target oligonucleotides through complementary base pairing. Confident and highly sensitive detection is accomplished when hybridization of the analyte with this sensor transforms a cluster from a nonemissive state with a violet absorption to a highly emissive state with near-infrared absorption. These sensors are distinguished by their strong and photostable fluorescence response and their economical, convenient, and modular synthesis, thus opening this methodology to a diversity of applications. Our studies will focus on a range of microRNA sequences that are biomarkers for human diseases. The following synthetic, structural, and stability studies will provide the foundation to develop the full potential of this new sensing strategy. I. Optimizing reaction conditions and the sequence environments are critical steps towards forming and transforming specific silver clusters. Highly parallel sequence variations in conjunction with spectral analysis will be coupled with measurements of the cluster stoichiometry and oligonucleotide shape to design sensors that are appropriate for direct analysis in biological samples. II. These metallic chromophores are not only distinguished by their spectra but also by their active role in shaping the sensors. We have shown that a violet absorbing cluster folds its DNA hosts, and biochemical/chemical probes and fluorescent base analogs will be used to map the secondary structure of DNA hosts through solvent exposure of their nucleobases. By identifying where the clusters bind and how they impact the DNA structure, generalized sensors that incorporate cluster-induced folding will be developed. III. Because the cluster coordinates and folds its DNA hosts, hybridization of the target oligonucleotide is inhibited. This impediment will be exploited to make fine distinctions between oligonucleotides that have mismatched base pairs and thus have reduced affinities for a specific sensor. The spectral changes that accompany temperature induced unfolding will provide the quantitative thermodynamic basis for distinguishing such targets. With the structural and thermodynamic insight provided by the above studies, a novel signal amplification scheme based on sequential unfolding will be developed for the detection of low abundance species.