Design of tailor-made biosensors for cellular and chip applications

量身定制的受体和酶由基于结构的方法设计的两个亚基组成，并通过组合方法对其功能进行优化。我们设计了一类用于定制受体的新型支架，在这种支架中，一个短肽和具有随机核苷酸区域的RNA形成稳定和特异的复合体，在体外从随机的核糖核肽(RNP)池中选择RNA寡核苷酸，提供针对ATP的RNP受体。在本研究中，我们将核糖核肽的用途扩展到定制荧光生物传感器。构建具有所需特性的荧光生物传感器，即高信噪比、检测波长和用于配体检测的浓度范围，并不是一项简单的任务。通过两个文库选择步骤构建了核苷酸三磷酸的荧光RNP传感器。第一步是通过从rna发散的rnp li…中进行体外选择来定制特定于配体的rnp受体。更多的图书馆。在第二步中，将第一步得到的RNA亚基与一系列荧光团修饰的多肽亚基相结合，得到荧光RNP文库，从其中高通量地选择具有所需光学传感特性的RNP传感器。在ATP浓度增加的情况下筛选荧光发射强度可以滴定分析荧光RNP文库，这使得能够选择在一定浓度范围内响应的ATP传感器。该荧光传感器将是方便地测量细胞第二信使的理想工具。通过研究Pleckstrin同源(PH)结构域与IP_3的选择性结合特性，我们设计了一种基于蛋白质的IP_3传感器。本研究获得了几种肌醇-1，3，4，5-四磷酸肌醇的荧光传感器。这里开发的战略提供了纳米生物技术工具(如生物传感器和蛋白质芯片)所必需的功能性小蛋白。较少

The tailor-made receptors and enzymes are comprised of two subunits designed by the structure-based method and their functions are optimized through the combinatorial approach. We have designed a new class of scaffold for tailor-made receptors, in which a short peptide and RNA with randomized nucleotide region form a stable and specific complex In vitro selection of RNA oligonucleotides from the randomized "ribonucleopeptide (RNP)" pool afforded RNP receptors specific for ATP.In this research, we have expanded the utility of ribonucleopeptides to tailoring fluorescent biosensors. Construction of fluorescent biosensors with desired characteristics, i.e, high signal-to-noise ratios, detection wavelengths and concentration ranges for ligand detection, is not a straight forward task. Fluorescent RNP sensors for nucleotide triphosphates are constructed by two library selection steps. The first step tailors RNP receptors specific for a ligand by in vitro selection from an RNA-diverged RNP li … More brary. In the second step, combination of the RNA subunits from the first step and a series of fluorophore-modified peptide subunits affords fluorescent RNP libraries, from which RNP sensors with desired optical sensing properties are selected in a high-throughput manner. Screening of the fluorescence emission intensities in the presence of increasing concentrations of ATP allowed titration analysis of the fluorescent RNP library, which enabled a selection of ATP sensors responding within certain concentration ranges of ATP.The fluorescent sensor would be an ideal tool for the convenient measurements of cellular second messengers. We have designed a protein based sensor for IP_3 by exploring the selective IP_3 binding properties of pleckstrin homology (PH) domain. Several fluorescent sensors for inositol-1,3,4,5-tetrakisphosphate were obtained in this research. The strategy developed here afford functional small proteins essential for the nano-biotechnology tools such as biosensors and protein chips. Less