Folding RNA-Protein Complex into Designed Nanostructures.

RNA-protein (RNP) complexes are promising biomaterials for the fields of nanotechnology and synthetic biology. Protein-responsive RNA sequences (RNP motifs) can be integrated into various RNAs, such as messenger RNA, short-hairpin RNA, and synthetic RNA nano-objects for a variety of purposes. Direct observation of RNP interaction in solution at high resolution is important in the design and construction of RNP-mediated nanostructures. Here we describe a method to construct and visualize RNP nanostructures that precisely arrange a target protein on the RNA scaffold with nanometer scale. High-speed AFM (HS-AFM) images of RNP nanostructures show that the folding of RNP complexes of defined sizes can be directly visualized at single RNP resolution in solution.

TectoRNP: self-assembling RNAs with peptide recognition motifs as templates for chemical peptide ligation.

TectoRNA, an artificial RNA with self-assembling ability, has been employed as a structural platform for RNA nanotechnology and RNA synthetic biology. In this study, tectoRNA was applied as a specific template for chemical peptide ligation. On the basis of a self-assembling tectoRNA, we designed and constructed a template RNA that facilitates peptide ligation depending on controlled dimer formation. Two RNA-binding peptides were recognized by two peptide-binding RNA motifs embedded in the template RNA, and chemical ligation was promoted because of the entropic effect of Mg(2+) -dependent dimerization. In a series of biochemical analyses, we determined the relationship between the structures of the tectoRNA-based templates and the extent of acceleration in peptide ligation.

Fully automated sequential solid phase approach towards viral RNA-nucleopeptides.

The synthesis of two ribonucleoprotein fragments of unprecedented complexity is reported. These hybrid biomolecules are prepared combining the use of an automated solid phase peptide and oligonucleotide synthesizer on a single solid support.

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.

RNA and RNP as new molecular parts in synthetic biology.

Synthetic biology has a promising outlook in biotechnology and for understanding the self-organizing principle of biological molecules in life. However, synthetic biologists have been looking for new molecular "parts" that function as modular units required in designing and constructing new "devices" and "systems" for regulating cell function because the number of such parts is strictly limited at present. In this review, we focus on RNA/ribonucleoprotein (RNP) architectures that hold promise as new "parts" for synthetic biology. They are constructed with molecular design and an experimental evolution technique. So far, designed self-folding RNAs, RNA (RNP) enzymes, and nanoscale RNA architectures have been successfully constructed by utilizing Watson-Crick base-pairs together with specific RNA-RNA or RNA-protein binding motifs of known defined 3D structures. Riboregulators for regulating targeted gene expression have also been designed and produced in vitro as well as in vivo. Lately, RNA and ribonucleoprotein complexes have been strongly attracting the attention of molecular biologists because a variety of noncoding RNAs discovered in nature perform spatiotemporal gene expressions. Thus we hope that newly accumulating knowledge on naturally occurring RNAs and RNP complexes will provide a variety of new parts, devices and systems for synthetic biology.

A modular strategy for tailoring fluorescent biosensors from ribonucleopeptide complexes.

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.

Stepwise molding of a highly selective ribonucleopeptide receptor.

The structural characteristics of RNA-peptide (RNP) complexes are suitable for molding of a ligand-binding pocket of the RNP complex in a stepwise manner. The first step involves molding of the RNA subunit by in vitro selection of an RNP pool originating from an RNA library and the peptide, as previously reported for the construction of an ATP-binding RNP complex from an RRE RNA-Rev peptide complex. The second step involves selection from an RNP library consisting of Rev peptides with randomized amino acid residues and the RNA subunit selected in the first molding. The ATP-binding pocket produced by sequential molding of RNA and peptide subunits shows higher affinity to ATP and a distinct specificity for ATP versus dATP as compared to the ATP-binding RNP receptor in which only the RNA subunit has been molded. The second step selection from the peptide-based RNP library allows expansion of the ATP recognition surface, consisting of both RNA and peptide subunits, to enhance the affinity and selectivity to discriminate ATP against dATP. Our approach of stepwise molding offers the advantage of increasing the diversity of the RNP library by utilizing characteristics of different biopolymers. The ribonucleopeptide-based, multi-subunit approach is also extendable to other biomacromolecular assemblies, which may yield artificial receptors and enzymes with increased specificity and more diverse chemical activities.

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.

Recognition of small molecules by a ribonucleopeptide.

In this research, we have design an ATP binding domain that consists of an RNA subunit and a peptide subunit by means of structure-based design approach and in vitro selection method. The RNA subunit is designed to consist of two functional domains; an ATP binding domain with 20 randomized nucleotides and an adjacent stem region that serves as a binding site for the RNA-binding peptide. RNA-peptide receptors to ATP were isolated from a pool of different RNAs by selection with affinity column and enzymatic amplification. Effects of the RNA-binding peptide for the specific ATP binding to the selected RNA-peptide receptors are discussed.

Solid-phase synthesis of an RNA nucleopeptide fragment from the nucleoprotein of poliovirus.

The naturally occurring RNA-nucleopeptide H-Ala-Tyr[5'-pUUAAAAC-3']-NH2 is prepared via a solid-phase phosphite triester approach using N-SiOMB/O-TBDMS-protected nucleosides. Preliminary 1H-NMR studies show that the peptidyl unit has a remarkable effect on the conformational behaviour of the RNA moiety in the nucleopeptide.