Biosensing using DNA Nanoreactors that Transform Chromophoric Silver Clusters

使用 DNA 纳米反应器转化发色银簇进行生物传感

• 批准号: 8491043
• 负责人: Jeffrey Thomas Petty
• 金额: $ 32.32万
• 依托单位: FURMAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8491043
• 关键词: 2-Aminopurine; Adenine; Affinity; Base Pair Mismatch; Base Pairing; Binding; Binding Sites; Biochemical; Biocompatible; Biological; Biological Markers; Biological Process; Biosensing Techniques; Chemical Agents; Chemicals; Circular Dichroism; Cleaved cell; Commit; Communities; Complex; Coupled; DNA; DNA Binding; DNA Folding; DNA Structure; Detection; Development; Diagnosis; Diagnostic; Discrimination; Encapsulated; Entropy; Environment; Event; Feedback; Fluorescence; Fluorescent Probes; Foundations; Glean; Goals; Growth; Hybrids; Image; In Vitro; Ionic Strengths; Ligands; Link; Maps; Measurement; Measures; Methodology; MicroRNAs; Molecular; Monitor; Oligonucleotides; Optics; Oxidants; Quantum Dots; Reaction; Relative (related person); Relaxation; Research; Role; Sampling; Scheme; Secondary to; Serum; Shapes; Signal Transduction; Silver; Solvents; Source; Specificity; Spectrum Analysis; Structure; Students; Surveys; Temperature; Thermodynamics; Training; Universities; Variant; Viola; Work; absorption; analog; base; chromophore; computerized data processing; cost; design; enthalpy; experience; high school; human disease; in vivo; insight; instrumentation; nanomaterials; novel; nucleobase; public health relevance; quantum; response; sensor; stoichiometry; tool

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.

描述（由申请人提供）：荧光是生物学分析的有力工具，但是背景干扰可能需要进行广泛的体外分析纯化​​，并且可以严重限制体内研究。我们的目标是使用由〜10个原子组成的小银簇在近红外光谱区域中进行较强的发射，在生物样品相对透明。这些金属配体与寡核苷酸传感器相关联，它们整合了两个不同的功能：形成 通过规定序列内的核碱酶配位的特定银簇的含量，并通过互补的碱基配对识别靶寡核苷酸。当分析物与该传感器的杂交从具有紫罗兰色吸收的非放射性状态转变为具有近红外吸收的高度发射状态时，可以实现自信和高度敏感的检测。这些传感器的区别是它们的强和光稳定的荧光响应及其经济，方便和模块化的合成，从而为多样化的应用开放了这种方法。我们的研究将集中于一系列microRNA序列，这些序列是人类疾病的生物标志物。以下合成，结构和稳定性研究将为发展这种新感测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。与光谱分析结合使用的高度平行序列变化将与簇化学计量和寡核苷酸的测量结果结合到设计传感器，这些传感器适合于生物样品中的直接分析。 ii。这些金属发色团不仅由它们的光谱区分，而且还以它们在塑造传感器中的积极作用而区分。我们已经表明，吸收紫罗兰的DNA宿主折叠，并且将使用生化/化学探针和荧光碱基类似物通过其核碱基的溶剂暴露来绘制DNA宿主的二级结构。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广义传感器。 iii。由于簇坐标并折叠其DNA宿主，因此抑制了靶寡核苷酸的杂交。这种障碍将被利用，以在不匹配碱基对的寡核苷酸之间进行细微的区分，从而减少了对特定传感器的亲和力。温度引起的展开伴随的光谱变化将为区分此类目标提供定量热力学基础。 通过上述研究提供的结构和热力学见解，将开发基于顺序展开的新型信号扩增方案，以检测低丰度物种。