Oligonucleotide Encapsulated Ag Nanoclusters as Single Molecule Labels

描述（由申请人提供）：本研究的长期目标是通过纳米材料和生物学的整合，开发用于生物系统的超亮单分子标签。不同于集合测量，单分子光谱允许复杂生物活性的机制动力学的分辨率。尽管最近单分子光谱学在生物系统中的应用取得了成功，但研究受到可用荧光团的亮度和光稳定性的限制。创造明亮和极其稳定的荧光团将促进在更快和更持久的时间尺度上理解生物功能的进步。通过三个特定目标，我们将以高荧光银纳米团簇的形式将纳米材料与生物相容性寡核苷酸支架相结合，以创建一类新的生物标签。本文将研究寡核苷酸包封银纳米团簇的体外光物理参数。对溶液中单个分子的闪烁动力学、发射率和光稳定性的表征，将允许鉴定荧光银纳米团簇物种，这些物种作为单分子标记显示出最大的希望。在Specific Aim II中，将通过使用DMA和RNA微阵列来靶向前一目标中确定的纳米材料的封装。通过改变寡核苷酸链长度和碱基序列，可以确定银纳米簇的最佳支架。在Specific Aim III中，将对优化后的寡核苷酸-银纳米簇组合进行生物相容性评估。细胞摄取机制将通过主链修饰和与细胞穿透肽的结合来确定和增强。提出的一类新的单分子荧光团的发展将有助于在解开复杂生物系统的动力学方面取得进展。与公共卫生相关更明亮和更强大的生物标签将允许先进的单分子研究，从而更全面地了解生物分子在生命系统中的功能。解决这些功能和确定致病生物分子和细胞成分之间复杂相互作用的动力学的能力对于理解感染机制至关重要。

DESCRIPTION (provided by applicant): The long term goal of this research is the development of ultra-bright, single molecule labels for biological systems through the integration of nanomaterials and biology. Unlike ensemble measurements, single molecule spectroscopy allows for the resolution of mechanistic dynamics of complex biological activity. Despite recent success in the application of single molecule spectroscopy to biological systems, studies are limited by the brightness and photostability of available fluorophores. The creation of bright and extremely stable fluorophores will facilitate advances in understanding biological function on both much faster and longer lasting timescales. Through the three Specific Aims we will integrate nanomaterials, in the form of highly fluorescent Ag nanoclusters, with biocompatible oligonucleotide scaffolds, to create a new class of biological labels. In Specific Aim I, the in vitro photophysical parameters of oligonucleotide encapsulated Ag nanoclusters will be studied. Characterization of blinking dynamics, emission rates, and photostability of individual molecules in solution will allow for the identification of fluorescent Ag nanocluster species that show the greatest promise as single molecule labels. In Specific Aim II, the encapsulation of the nanomaterials identified in the previous aim will be targeted through the use of DMA and RNA microarrays. By varying oligonucleotide strand length and base sequence, the optimum scaffold for the Ag nanocluster of interest will be determined. In Specific Aim III, the optimized oligonucleotide-Ag nanocluster combination will be evaluated for biocompatibility. Cellular uptake mechanisms will be determined and enhanced through backbone modification and conjugation with cell penetrating peptides. The proposed development of a new class of single molecule fluorophores should help lead to advancements in unraveling the dynamics of complex biological systems. Relevance to Public Health Brighter and more robust biological labels would allow for advanced single molecule studies leading to a more complete understanding of the function of biomolecules within living systems. The ability to resolve these functions and determine the dynamics of complex interactions between disease causing biomolecules and cellular components is paramount to understanding infection mechanisms.