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Structure-based design of fluorescent biosensors from ribonucleopeptide complexes.

Fluorescent biosensors are crucial tools to facilitate sensitive detection of small molecules. However, construction of fluorescent biosensors with desired characteristics, that is, detection wavelengths and concentration ranges for ligand detection, from macromolecular receptors is not a straightforward task. Previously, we reported a modular strategy (Hagihara, et al., J. Am. Chem. Soc., 2006, 128, 12932) for constructing fluorescent ATP sensors from ribonucleopeptide (RNP) complexes. These RNP sensors had a variety of emission wavelengths and/or responding ligand concentration ranges. The modular strategy allows choosing a fluorescent RNP sensor with appropriate optical properties, but does not allow us to design optimal properties of fluorescent RNP sensors. Here we report a design strategy to optimize the response of fluorescent RNP sensors based on the secondary structural analyses of ATP-binding RNPs. We analyzed the RNA secondary structure of RNP sensors and examined the relationship between the secondary structure and fluorescent characteristics. We have discovered that fluorescent ATP sensors with high value of I/I0 ratio tend to contain interior loop and to record low fluorescence intensity in the absence of ATP. Indeed, ATP sensors, which was inserted sequence that construct an interior loop of ATP sensor with large fluorescence intensity change, were induced increase of fluorescent intensity change. This result suggests that use of the secondary structural elements would allow a rational functional design of RNP.