Design, assembly, and evaluation of RNA-protein nanostructures.

The use of RNA-protein interaction motifs (RNP motifs) to design and build nanoscale objects has the potential to expand the field of RNA nanotechnology. In principle, RNP motifs can be integrated easily into RNA nano objects, providing an alternative technique to increase the functional and structural complexities of the RNA. Investigating the design principles of RNP nanostructures will enable the construction of highly sophisticated biomacromolecular complexes such as ribosomes from scratch. As an initial step towards this goal, we designed and constructed triangular-like nanostructures by employing box C/D kink-turn (K-turn)-L7Ae RNP motifs. We showed that the K-turn RNA and the ribosomal protein L7Ae could form a nanostructure shaped like an equilateral triangle that consists of the three proteins attached to the tips of the RNA scaffold. The construction of the complex depends on L7Ae binding to the K-turn motifs in the RNA. The RNP motif allows the RNA to bend by approximately 60° at three positions to form a nanoscale triangle. Functional RNP triangles with desired protein modules at the three tips can be constructed in a modular manner. Here, we describe how to design, construct, and evaluate the RNP nanostructures.

Engineering RNA-protein complexes with different shapes for imaging and therapeutic applications.

Molecular machines composed of RNA­protein (RNP) complexes may expand the fields of molecular robotics, nanomedicine, and synthetic biology. However, constructing and directly visualizing a functional RNP nanostructure to detect and control living cell function remains a challenge. Here we show that RNP nanostructures with modular functions can be designed and visualized at single-RNP resolution in real time. The RNP structural images collected in solution through high-speed atomic force microscopy showed that a single RNP interaction induces a conformational change in the RNA scaffold, which supports the nanostructure formation designed. The specific RNP interaction also improved RNA nanostructure stability in a serum-containing buffer. We developed and visualized functional RNPs (e.g., to detect human cancer cells or knockdown target genes) by attaching a protein or RNA module to the same RNA scaffold of an optimal size. The synthetic RNP architecture may provide alternative materials to detect and control functions in target mammalian cells.

A novel mitochondrial nuclease-associated protein: a major executor of the programmed nuclear death in Tetrahymena thermophila.

BACKGROUND INFORMATION: Programmed nuclear death (PND) in the ciliate Tetrahymena is an apoptosis-like phenomenon that occurs in a restricted space of cytoplasm during conjugation. In the process, only the parental macronucleus is selectively eliminated from the progeny cytoplasm, in conjunction with differentiation of new macronuclei for the next generation. For the last decade, mitochondria have been elucidated to be a crucial executioner like apoptosis: apoptosis-inducing factor and yet-unidentified nucleases localised in mitochondria are major factors for PND. RESULTS: To identify such nucleases, we performed a DNase assay in a PAGE (SDS-DNA-PAGE) using total mitochondrial proteins. Some proteins showed DNase activity, but particularly a 17 kDa protein exhibited the highest and predominant activity. Mass spectrometric analysis revealed a novel mitochondrial nuclease, named TMN1, whose homologue has been discovered only in the ciliate Paramecium tetraurelia, but not in other eukaryotes. Gene disruption of TMN1 led to a drastic reduction of mitochondrial nuclease activity and blocked nuclear degradation during conjugation, but did not affect accumulation of autophagic and lysosomal machinery around the parental macronucleus. CONCLUSIONS: These observations strongly suggest that the mitochondrial nuclease-associated protein plays a key role in PND as a major executor. Taking the novel protein specific to ciliates in consideration, Tetrahymena would have diverted a different protein from common apoptotic factors shared in eukaryotes to PND in the course of ciliate evolution.

Peptide screening using PURE ribosome display.

To demonstrate directed protein evolution or selection of functional polypeptides, ribosome display is one of the most ideal technologies of evolutionary engineering. Intrinsic components, such as nucleases in the cell extract-based cell-free protein synthesis systems, reduce the stability of the messenger RNA-ribosome-polypeptide ternary complex, thereby preventing the attainment of reliable results. To overcome this problem, we have developed an effective and highly controllable ribosome display system using the protein synthesizing using recombinant elements (PURE) system. Since the activities of nucleases and other inhibitory factors are very low in the PURE system, the ternary complex is highly stable and the selected mRNA can be reliably recovered. Using this system, we were able to select peptides that specifically bind to monoclonal antibodies from random peptide libraries. The advantages of the modified PURE system for ribosome display strongly substantiate its usability.

Epitope mapping using ribosome display in a reconstituted cell-free protein synthesis system.

Ribosome display is a powerful technology for selecting ligand-binding peptides or proteins. We demonstrate here that the ribosome display using the reconstituted cell-free protein synthesis system can be applied for the epitope mapping of monoclonal antibodies (mAbs). Using this technology, we selected peptides that specifically bind to three mAbs from random peptide library. When selection was performed against the anti-FLAG M2 antibody, selected peptides contained previously characterized consensus epitope, indicating that the methodology can be applied for the epitope mapping. When the selection was carried out against two anti-beta-Catenin (anti-beta-Cat) mAbs, selected peptides had a homology for the partial peptide sequences of beta-Cat. Western blot analysis showed that these putative epitopes had affinity for the corresponding mAbs and beta-Cat mutants that lack these regions did not bind to the antibodies, indicating we correctly mapped the epitope for these mAbs. The study shown here provides a way for the quick identification of the epitope of mAbs.