A Modular Strategy for Tailoring Fluorescent Biosensors from Ribonucleopeptide Complexes

Abstract

Fluorescent biosensors that facilitate reagentless sensitive detection of small molecules are crucial tools in the areas of therapeutics and diagnostics. 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. An ATP-binding ribonucleopeptide (RNP) receptor was converted to a fluorescent ATP sensor without chemically modifying the nucleotide in the ATP-binding RNA. The RNA subunit of the ATP-binding RNP and a peptide modified with a pyrenyl group formed a stable fluorescent RNP complex that showed an increase in the fluorescence intensity upon binding to ATP. The strategy to convert the ATP-binding RNP receptor to a fluorescent ATP sensor was applied to generate fluorescent ATP-binding RNP libraries by using a pool of RNA subunits obtained from the in vitro selection of ATP-binding RNPs and a series of fluorophore-modified peptide subunits. Simple screening of the fluorescent RNP library based on the fluorescence emission intensity changes in the absence and presence of the ligand afforded fluorescent ATP or GTP sensors with emission wavelengths varying from 390 to 670 nm. Screening of the fluorescence emission intensity changes in the presence of increasing concentrations of ATP allowed titration analysis of the fluorescent RNP library, which provided ATP sensors responding at wide concentration ranges of ATP. The combinatorial strategy using the modular RNP receptor reported here enables tailoring of a fluorescent sensor for a specific ligand without knowledge of detailed structural information for the macromolecular receptor.

A Bioorganic Chemistry Approach to Understanding Molecular Recognition in Protein–Nucleic Acid Complexes

“…31 This RNP complex was chosen as a scaffold for stepwise functionalization. 6,9 In the first step, in vitro selection was applied to construct an RNP receptor with a binding site for the given target molecule, such as ATP, from an RNP pool originating from an RNA library and the Rev peptide ( Figure 5). 5,7 The RNA subunit was constructed from two functionally separated regions: a possible ligand-binding region with randomized nucleotides and an adjacent stem region corresponding to the RRE sequence as the binding site for the Rev peptide.…”

“…8 In vitro selection of the RNP pool originating from an RRE-based RNA library and the Rev peptide affords RNP receptors specific for nucleotide triphosphates 5,6,9 or phosphotyrosine residues in defined amino acid sequences.…”

“…The Rev peptide of the ATP-binding RNP was modified with a fluorophore to afford fluorescent ATP sensors without losing the affinity or selectivity of parent RNP receptor. 9 By taking advantage of the characteristic of RNP as a noncovalent complex, combination of a library of RNA subunits obtained by in vitro selection and a library of fluorophore-modified Rev peptides with a variety of excitation and emission wavelengths affords a pool of fluorescent RNPs with various affinities to the target and optical properties. From this fluorescent RNP library, fluorescent RNP sensors for targets were obtained that respond at detection or excitation wavelengths of interest and with desired ligand affinities.…”

Recent progress in the construction methodology of fluorescent biosensors based on biomolecules

“…By taking the advantage of such the noncovalent nature of the RNP complex, RNP sensors with desired affinity, selectivity and optical sensing properties could be selected in a high-throughput manner by combining a series of RNA subunits derived from each of the library. Actually, a variety of fluorescent biosensors for targeting ATP (Hagihara, M. et al 2006), GTP (Hagihara, M. et al 2006), histamine (Fukuda, M. et al 2009), phosphotyrosine (Hasegawa, T. et al 2005), and phosphotyrosinecontaining peptide fragment (Hasegawa, T. et al 2008) have been produced by the group, showing the generality of the approach. Recently, the group showed that ATP-binding RNP sensor was rationally converted to GTP-binding RNP sensor to have realized the detail of the recognition mechanism (Nakano, S. et al 2011).…”

“…A covalently linking of RNA and peptide subunits without sacrificing the sensing function would overcome such disadvantages. [Hagihara, M. et al 2006]. Combination between the RNA subunit library and several dye-labeled Rev peptide subunits generates combinatorial fluorescent RNP receptor libraries, from which RNP sensors with desired function, such as optical property, affinity and selectivity, are selected.…”