Design of tailor-made biosensors for cellular and chip applications

采用基于结构的方法设计了两个亚基，并通过组合方法优化了它们的功能。我们设计了一种新型的支架用于定制受体，其中短肽和随机核苷酸区域的RNA形成稳定的特异性复合物。体外从随机“核糖核肽（RNP）”池中选择RNA寡核苷酸，提供特异性的RNP受体。在这项研究中，我们扩大了核糖核肽的用途，以定制荧光生物传感器。构建具有所需特征的荧光生物传感器，即高信噪比，检测波长和配体检测的浓度范围，并不是一项直截了当地的任务。三磷酸核苷酸荧光RNP传感器通过两个文库选择步骤构建。第一步是通过体外选择rna分化的RNP序列来定制特定于配体的RNP受体。在第二步中，将第一步的RNA亚基与一系列荧光团修饰的肽亚基结合，形成荧光RNP文库，从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对荧光RNP文库进行滴定分析，从而可以选择在一定浓度范围内响应的ATP传感器。荧光传感器将是方便测量细胞第二信使的理想工具。我们通过探索pleckstrin同源结构域的IP_3选择性结合特性，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-1,3,4,5-四聚磷酸的荧光传感器。本研究开发的策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质。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