Ribonucleopeptide Sensors Research Articles

Receptor-based fluorescent sensors constructed from ribonucleopeptide.

Receptor-based fluorescent sensors are the representative tool for quantitative detection of target ligands. The high substrate-selectivity originated from biomacromolecule receptor is one of the advantages of this tool, but a laborious trial and error is usually required to construct sensors showing satisfactory fluorescence intensity changes without diminishing the function of parent receptor. Ribonucleopeptide (RNP) provides a scaffold of fluorescent sensors to improve such issues. RNP receptors for the ligand of interest are constructed by applying in vitro selection for RNA-derived RNP library. Simple modification of the N-terminal of peptide in RNP by an appropriate fluorophore converts the RNP receptor into the fluorescent sensor with retaining the affinity and selectivity for the substrate. In this chapter, we introduce the protocols for construction of fluorescent RNP sensors through selection from a library of fluorophore-modified RNP complex or by a structure-based modular design. Furthermore, we describe the application of covalently linked RNP sensors for simultaneous detection of multiple ligands.

Simultaneous Detection of ATP and GTP by Covalently Linked Fluorescent Ribonucleopeptide Sensors

A noncovalent RNA complex embedding an aptamer function and a fluorophore-labeled peptide affords a fluorescent ribonucleopeptide (RNP) framework for constructing fluorescent sensors. By taking an advantage of the noncovalent properties of the RNP complex, the ligand-binding and fluorescence characteristics of the fluorescent RNP can be independently tuned by taking advantage of the nature of the RNA and peptide subunits, respectively. Fluorescent sensors tailored for given measurement conditions, such as a detection wavelength and a detection concentration range for a ligand of interest can be easily identified by screening of fluorescent RNP libraries. The noncovalent configuration of a RNP becomes a disadvantage when the sensor is to be utilized at very low concentrations or when multiple sensors are applied to the same solution. Here, we report a strategy to convert a fluorescent RNP sensor in the noncovalent configuration into a covalently linked stable fluorescent RNP sensor. This covalently linked fluorescent RNP sensor enabled ligand detection at a low sensor concentration, even in cell extracts. Furthermore, application of both ATP and GTP sensors enabled simultaneous detection of ATP and GTP by monitoring each wavelength corresponding to the respective sensor. Importantly, when a fluorescein-modified ATP sensor and a pyrene-modified GTP sensor were co-incubated in the same solution, the ATP sensor responded at 535 nm only to changes in the concentration of ATP, whereas the GTP sensor detected GTP at 390 nm without any effect on the ATP sensor. Finally, simultaneous monitoring by these sensors enabled real-time measurement of adenosine deaminase enzyme reactions.

A ribonucleopeptide module for effective conversion of an RNA aptamer to a fluorescent sensor

Ribonucleopeptide (RNP) is a new class of scaffold for modular fluorescent sensors. We report here a short RNA motif that induces an efficient communication between the structural changes associated with the ligand-binding event of RNA aptamer and an optical response of a fluorescent RNP module. An optimized short RNA motif was used as a communication module for the rational design of modular RNP sensors. A modular combination of a GTP-binding RNA aptamer, the short RNA motif and the fluorophore-labeled RNP module afforded a fluorescent GTP sensor that retain the ligand-binding affinity of the parent aptamer.

Construction of dopamine sensors by using fluorescent ribonucleopeptide complexes

A facile strategy of stepwise molding of a ribonucleopeptide (RNP) complex affords fluorescent RNP sensors with selective dopamine recognition. In vitro selection of a RNA-derived RNP library, a complex of the Rev peptide and its binding site Rev Responsive Element (RRE) RNA appended with random nucleotides in variable lengths, afforded RNP receptors specific for dopamine. The modular structure of the RNP receptor enables conversion of dopamine-binding RNP receptors to fluorescent dopamine sensors. Application of conditional selection schemes, such as the variation of salt concentrations and application of a counter-selection step by using a competitor ligand norepinephrine resulted in isolation of RNP receptors with defined dopamine-binding characteristics. Increasing the salt condition at the in vitro selection stage afforded RNP receptors with higher dopamine affinity, while addition of norepinephrine in the in vitro selection milieu at the counter-selection step reinforced the selectivity of RNP receptors to dopamine against norepinephrine. Thermodynamic analyses and circular dichroismic studies of the dopamine–RNP complexes suggest that the dopamine-binding RNP with higher selectivity against norepinephrine forms a pre-organized binding pocket and that the dopamine-binding RNP with higher affinity binds dopamine through the induced-fit mechanism. These results indicate that the selection condition controls the ligand-binding mechanism of RNP receptors.

Covalently linked fluorescent ribonucreopeptide sensors

Fluorescent biosensors based on the biological macromolecule are convenient tools for investigating the event occurring in the living cell. As for one of the candidates of such biosensors, we have reported a fluorescent sensor by utilizing a ribonucleopeptide (RNP) framework. Fluorescent RNP sensors are obtained from the fluorescent RNP library constructed by the combination of the RNA subunit, a substrate recognition unit selected by in vitro selection, and a fluorophore-modified peptide subunit. By taking the advantage of the noncovalent nature of fluorescent RNP complexes, RNP sensors with desired optical sensing properties are selected in a high-throughput manner. However, the noncovalent nature of the fluorescent RNP sensor is not suitable for practical applications. We report here a strategy to generate stable covalently linked RNP sensors and demonstrate a multiple ligands sensing system by using the covalently linked RNP sensors to detect biologically active ligands.

Construction of a stable functional ribonucleopeptide complex by the covalent linking method

We describe here a novel strategy to create a stable functional ribonucleopeptide (RNP) complex by the covalent linking method. Adenosine-5'-triphosphate (ATP)-binding RNP receptors were selected from the RNP library by in vitro selection. The RNA subunit of RNP is utilized to construct a ligand-binding cavity, while the peptide subunit can be functionalized independently. By introducing a fluorophore at the N-terminus of the Rev peptide subunit, the ATP-binding RNP receptor is successfully converted to a noncovalent complex of ATP-responsive fluorescent RNP sensor. Such a noncovalent RNP sensor could be covalently linked by the tethering the RNA to the fluorophore-labeled peptide subunit to form a stable RNP sensor without losing the original function.

Context-Dependent Fluorescence Detection of a Phosphorylated Tyrosine Residue by a Ribonucleopeptide

Tools for selective recognition and sensing of specific phosphorylated tyrosine residues on the protein surface are essential for understanding signal transduction cascades in the cell. A stable complex of RNA and peptide, a ribonucleopeptide (RNP), provides effective approaches to tailor RNP receptors and fluorescent RNP sensors for small molecules. In vitro selection of an RNA-derived pool of RNP afforded RNP receptors specific for a phosphotyrosine residue within a defined amino-acid sequence Gly-Tyr-Ser-Arg. The RNP receptor for the specific phosphotyrosine residue was successfully converted to a fluorescent RNP sensor for sequence-specific recognition of a phosphorylated tyrosine by screening a pool of fluorescent phosphotyrosine-binding RNPs generated by a combination of the RNA subunits of phosphotyrosine-binding RNPs and various fluorophore-modified peptide subunits. The phosphotyrosine-binding RNP receptor and fluorescent RNP sensor constructed from the RNP receptor not only discriminated phosphotyrosine against tyrosine, phosphoserine, or phosphothreonine, but also showed specific recognition of amino acid residues surrounding the phosphotyrosine residue. A fluorescent RNP sensor for one of the tyrosine phosphorylation sites of p100 coactivator showed a binding affinity to the target site ~95-fold higher than the other tyrosine phosphorylation site. The fluorescent RNP sensor has an ability to function as a specific fluorescent sensor for the phosphorylated tyrosine residue within a defined amino-acid sequence in HeLa cell extracts.

Stepwise Functionalization of Ribonucleopeptides: Optimization of the Response of Fluorescent Ribonucleopeptide Sensors for ATP

A stable complex of a peptide and RNA, ribonucleopeptide (RNP), provides a new framework to construct a macromolecular receptor for small molecules. The RNP receptor functionalized by a fluorophore-labeled Rev peptide exerts an optical signal associated with the ligand binding events. Replacing the Rev peptide of the ATP-binding RNP with a fluorophore-modified Rev peptide affords a fluorescent ATP sensor.

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.

Design of sensing ribonucleopeptides for small ligands

Here we report a simple method to convert synthetic receptors to fluorescent sensors. An RNA-peptide complex (ribonucleopeptide) with a known three-dimensional structure is used as a framework of the receptor. Artificial ribonucleopeptide sensors were created with a combination of in vitro selection method and successive modification of the peptide with a fluorophore. A ribonucleopeptide complex of the fluorophore labeled peptide showed a remarkable fluorescence emission change upon binding cognate ligands.

Design of a ribonucleopeptide biosensor.

Ribonucleopeptide receptors for ATP have been designed by using a structure-based design and in vitro selection method. The ATP binding ribonucleopeptide receptors revealed submillimolar affinity to ATP and discriminate ATP against other ribonucleotides. In this research, we have developed a simple strategy to convert the ATP-binding ribonucleopeptide receptor into a ribonucleopeptide sensor by introducing a fluorophore in the peptide subunit. Fluorophore labeled ribonucleopeptide complex showed a large change in the fluorescence intensity upon addition of ATP.