Design of tailor-made biosensors for cellular and chip applications

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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