Development of A Fluorescent Ribonucleopeptide Sensor for Histamine

Abstract

Biologically active amines play major roles in the regulation of movement and are implicated in the pathophysiology of Parkinson's and Huntington's diseases, psychosis, and drug addiction. Fluorescent biosensors based on the biological macromolecule receptor are useful tool for investigating the function of biologically active amines. Our strategy of the stepwise molding using a ribonucleopeptide (RNP) framework provides fluorescent biosensors with a variety of binding and optical characteristics for small molecules. Here we report fluorescent RNP sensors for histamine with a variety of binding and signal-transducing characteristics. Combination of RNA subunits of histamine-binding RNP receptors obtained by in vitro selection and a Rev peptide modified with 7-methoxycoumarin-3-carboxylic acid afforded a fluorescent RNP sensor with distinct signaling characteristics in the changes of fluorescence emission intensity upon binding to histamine. The fluorescent histamine sensor showed distinct selectivity for histamine over structurally related histamine analogs, such as imidazole, ethylamine and L-histidine. Key words: ribonucleopeptide, aptamer, fluorescent biosensor, biological active amine, in vitro selection Biologically active amines, such as dopamine, histamine, and serotonin, are pivotal molecules in the central nervous system [1-3]. These amines play major roles in the regulation of movement and are implicated in the pathophysiology of Parkinson's and Huntington's diseases, psychosis, and drug addiction [2, 3]. Pharmacotherapy of some major disorders of the central nervous system is based on targeting these biologically active amines. In the process of discovery and evaluation of new central nervous system drugs against these diseases, there is a persistent demand for availability of suitable analytical techniques that enable a highly sensitive detection of biologically active amines. Fluorescent biosensors based on the biological macromolecule receptor [4-8] would serve as useful tools for investigating the function of biologically active amines in the living cell. However, fluorescent biosensors synthesized by the chemical modification of the ligand binding protein domains with fluorophores are not always guaranteed to execute the expected optical signals. We have reported a strategy that enables isolation of fluorescent ribonucleopeptide (RNP) sensors with a variety of binding and signal-transducing characteristics, i.e., a high signal-to-noise ratio, various wavelengths and concentration ranges for the ligand detection [9]. The strategy would provide ideal fluorescent RNP sensors for sensing biologically active amines. Fluorescent RNP sensors for adenosine 5'-triphosphate (ATP) were obtained in a stepwise manner. As the first step to construct a fluorescent RNP sensor for ATP, an RNA-derived RNP pool was constructed by a structure-based design of the Rev Responsive Element (RRE)-HIV Rev peptide complex [10] appended with a randomized nucleotides region, which would provide a ligand binding site, next to the RRE segment. In vitro selection [11, 12] of the RNA-derived pool of RNP afforded ATP-binding RNP receptors with high selectivity and affinity [9]. As the second step, ATP-binding RNP receptors were converted to a pool of fluorescent ATP-binding RNP receptors by chemically modifying with a fluorophore at the N-terminal of the Rev peptide with suitable optical characteristics. From the pool of fluorescent ATP-binding RNP receptors, it was possible to select a fluorescent ATP sensor with desired sensing characteristics. Here we report fluorescent RNP sensors for histamine, one of the biologically active amines, with a variety of affinities and optical sensing characteristics (Fig. 1). By utilizing the RNA subunits of histamine-binding RNPs obtained by in vitro selection and a Rev peptide modified with a fluorophore, a histamine-binding fluorescent RNP library was constructed. Fluorescent histamine sensors with desired optical and binding properties were screened from this library. A fluorescent RNP sensor for histamine modified with 7-methoxycoumarin-3-carboxylic acid showed a change in the fluorescence emission intensity upon binding to histamine. Furthermore, the histamine sensor showed distinct selectivity over histamine analogs, such as imidazole, ethylamine and L-histidine. Fig. 1. A strategy to obtain RNP fluorescent sensors specific for histamine. Combination of the RNA subunits of the histamine-binding RNP receptor and a fluorophore-modified Rev peptide provided a histamine RNP fluorescent sensor.