Construction of a library of structurally diverse ribonucleopeptides with catalytic groups

Abstract

Functional screening of structurally diverse libraries consisting of proteins or nucleic acids is an effective method to obtain receptors or aptamers with unique molecular recognition characteristics. However, further modification of these selected receptors to exert a newly desired function is still a challenging task. We have constructed a library of structurally diverse ribonucleopeptides (RNPs) that are modified with a catalytic group, in which the catalytic group aligns with various orientations against the ATP binding pocket of RNA subunit. As a proof-of-principle, the screening of the constructed RNP library for the catalytic reaction of ester hydrolysis was successfully carried out. The size of both the substrate-binding RNA library and the catalytic group modified peptide library are independently expandable, and thus, the size of RNPs library could be enlarged by a combination of these two subunits. We anticipate that the library of functionalized and structurally diverse RNPs would be expanded for various other catalytic reactions.

Introduction

Biomacromolecular receptors provide useful frameworks for constructing sensors with high specificity for a particular ligand. Efforts have been taken to construct biosensors through modification of proteins and nucleic acids receptors with appropriate fluorophores1, 2, 3, 4, 5 or artificial enzymes with a catalytic group to achieve proximity effect between the substrate and the catalytic group.6, 7 However, naturally occurring receptors do not always provide the recognition characteristics for the molecule of interest, and rational design of a receptor with desired specificity is still a challenging task. A library of RNA molecules that differ in their three-dimensional structures by means of randomized nucleotide sequences has been applied for the selection of receptors for the target ligands.8, 9, 10 Although the library-based selection offers one of the promising methods to obtain aptamers with the specific recognition characteristics for the molecule of interest, further modification of the selected aptamers to exert a newly desired function is often difficult due to the lack of their structural information. In addition, the original substrate binding characteristics of the aptamer could be impaired by the introduction of a new functional group. Synthetic functional groups were incorporated to biomacromolecules in the library by modification of proteins or nucleic acids via chemical modification or genetic mutation.11, 12 These chemical or genetic methods require laborious tasks to generate a library with limited size. Therefore, an alternative method is required to construct a large size library to increase the possibility of selecting a “hit” biomacromolecule with the function of interest.

Ribonucleopeptide (RNP) is one of the appropriate scaffolds to construct a library with structurally diverse RNA-peptide complexes. We have reported a method to obtain ATP-binding RNP receptors by in vitro selection of a library of RNP with randomized RNA sequences.¹³ Because the RNA subunit works as a receptor for the substrate, the selected ATP-binding RNP receptor was further converted into a new RNP library by complexation of the RNA subunit and a library of Rev peptides, in which a peptide loop with randomized amino acid residues was incorporated at the N-terminus of Rev peptide. An ATP-binding RNP receptor selected from this peptide-based RNP library exerted higher ATP-binding specificity than the original RNP (Fig. 1a).¹⁴ A fluorescent RNP library was also constructed by combining the RNA subunit library and a library containing fluorophore-modified Rev peptides (Fig. 1a).15, 16, 17 The original substrate-binding ability of the RNP receptor was maintained in the selected RNP sensor even upon modification of the Rev peptide by a fluorophore. By taking advantage of the noncovalent complex formation of RNP, combination of the RNA library and the peptide library would dramatically increase the size of the RNP library. Therefore, RNP is a good candidate of scaffolds to construct a library of structurally diverse biomolecular assemblies containing a substrate-binding pocket and a synthetic functional group.

In this study, we have constructed a library of structurally diverse RNP receptors equipped with a synthetic functional group. A series of RNA subunits obtained by the in vitro selection of ATP-binding RNP receptors15, 17 afforded a library of structurally diverse RNAs consisting of the ATP-binding region and the RRE (Rev Responsive Element) nucleotide sequence. A library of peptides was constructed by modification of the Rev peptide with catalytic groups through various peptide linkers (Fig. 1b). Combination of these two types of libraries by the complex formation between the RRE RNA and the Rev peptide generated a library of functionalized and structurally diverse RNPs, in which the catalytic group would align with various orientations against the ATP binding pocket of RNA subunits.

The functionalized and structurally diverse RNPs were expected to exhibit the ATP-binding ability. An adenosine derivative with an ester group was utilized to select the RNPs that could accelerate the ester hydrolysis reaction by the proximity effect between the ATP binding domain and the catalytic group on the peptide (Fig. 1a). Screening of the RNP library was successfully performed for the hydrolysis reaction of the ester derivative of adenosine to obtain catalytic RNPs.