RNA-based cooperative protein labeling that permits direct monitoring of the intracellular concentration change of an endogenous protein.

Imaging the dynamics of proteins in living cells is a powerful means for understanding cellular functions at a deeper level. Here, we report a versatile method for spatiotemporal imaging of specific endogenous proteins in living mammalian cells. The method employs a bifunctional aptamer capable of selective protein recognition and fluorescent probe-binding, which is induced only when the aptamer specifically binds to its target protein. An aptamer for ß-actin protein preferentially recognizes its monomer forms over filamentous forms, resulting in selective G-actin staining in both fixed and living cells. Through actin-drug treatment, the method permitted direct monitoring of the intracellular concentration change of endogenous G-actin. This protein-labeling method, which is highly selective and non-covalent, provides rich insights into the study of spatiotemporal protein dynamics in living cells.

Short-Chain Guide RNA for Site-Directed A-to-I RNA Editing.

Site-directed RNA editing is a promising genetic modification technology for therapeutic and pharmaceutical applications. We previously constructed adenosine deaminases acting on RNA (ADAR)-guiding RNAs (AD-gRNAs) that direct A-to-I RNA editing activity of native human ADAR2 into a programmable target site. In this study, we developed the short-chain AD-gRNA (shAD-gRNA) as a potential basic framework for practical RNA-editing oligonucleotides. Based on knowledge of previous AD-gRNA, shAD-gRNAs were designed to have the shortest possible sequence for the induction of editing activity. In vitro, compared to the original AD-gRNA, the shAD-gRNAs showed similar or superior editing induction activity, depending on the target RNA sequence, and had lower off-target editing activity around the target site, which is predicted to be a hotspot for off-target editing. Moreover, shAD-gRNAs achieved target RNA editing with both exogenous and endogenous human ADARs in cultured cells. Our results present shAD-gRNA as a short basic framework that would be applicable to further development for practical RNA-editing oligonucleotides.

Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing.

As an alternative to DNA mutagenesis, RNA mutagenesis can potentially become a powerful gene-regulation method for fundamental research and applied life sciences. Adenosine-to-inosine (A-to-I) RNA editing alters genetic information at the transcript level and is an important biological process that is commonly conserved in metazoans. Therefore, a versatile RNA-mutagenesis method can be achieved by utilising the intracellular RNA-editing mechanism. Here, we report novel guide RNAs capable of inducing A-to-I mutations by guiding the editing enzyme, human adenosine deaminase acting on RNA (ADAR). These guide RNAs successfully introduced A-to-I mutations into the target-site, which was determined by the reprogrammable antisense region. In ADAR2-over expressing cells, site-directed RNA editing could also be performed by simply introducing the guide RNA. Our guide RNA framework provides basic insights into establishing a generally applicable RNA-mutagenesis method.

Identification of an RNA element for specific coordination of A-to-I RNA editing on HTR2C pre-mRNA.

Adenosine-to-Inosine (A-to-I) RNA editing is an intracellular mechanism in which inosine is specifically substituted against adenosine by the action of adenosine deaminases acting on RNA (ADARs). Serotonin 2C receptor (HTR2C) is encoded through combinatorial A-to-I RNA editing at recoding sites (A - E site) on its pre-mRNA. Although the efficiency of RNA editing at particular sites is known to be critical for modulating the serotonin signaling, the mechanistic details of site-specific editing on HTR2C pre-mRNA are not fully understood. Toward complete understanding of this mechanism, we discovered an RNA element, which coordinates site-specific RNA editing on HTR2C pre-mRNA by an in vitro editing assay and secondary structural analysis of mutant HTR2C RNA fragments. Our results showed that HTR2C pre-mRNA forms a characteristic structure, which was restricted by the internal loop and Watson-Crick base-pair interaction on site E, for intrinsic editing. We suggest that the internal loop would contribute toward adjusting the relative distance and/or geometry between the editing sites and the scaffold for ADAR.

One-pot synthesis of dibenzo[b,h][1,6]naphthyridines from 2-acetylaminobenzaldehyde: application to a fluorescent DNA-binding compound.

Dibenzo[b,h][1,6]naphthyridines were synthesized in one pot by reacting 2-acetylaminobenzaldehyde with methyl ketones under basic conditions via four sequential condensation reactions. This method was also applied to the synthesis of 1,2-dihydroquinolines. 6-Methyl-1,6-dibenzonaphthyridinium triflates showed strong fluorescence, and the fluorescence intensities were changed upon intercalation into double-stranded DNA.

Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing.

Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state-dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites.

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 strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing.

Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.

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.

Structural aspects for the function of ATP-binding ribonucleopeptide receptors.

We describe here analyses of the secondary structure of ATP-binding ribonucleopeptide (RNP) receptors. Mapping of the RNA structure of ATP-binding RNP receptors by using hydrolytic enzymes, chemical probing with dimethyl sulfate (DMS), and in-line probing indicated that ATP-binding RNP receptors take the loop structure at the nucleotide position of the "variable region". In addition, it was evident that a part of the consensus region located next to the variable region directly participated in the binding to ATP. The completely preserved three U nucleotides were essential for the binding of RNP to ATP as revealed by the affinity evaluation and the secondary structure analyses of the U nucleotides mutants of the ATP-binding RNP receptor. Interestingly, two mutants with an adenosine introduced to either of the two U nucleotides showed similar secondary structures to the original ATP-binding RNP. These results imply the possibility that the adenine base introduced at the U position acts just like the substrate ATP, and suggest that the U nucleotides in these positions interact directly to ATP.

Structural analysis of ribonucleopeptide aptamer against ATP.

A ribonucleopeptide aptamer against ATP was obtained by the in vitro selection method. This ribonucleopeptide aptamer comprises a randomized and selected RNA linked to the Rev-responsive element (RRE) in complex with a peptide derived from an HIV Rev protein. The ribonucleopeptide aptamer selectively binds ATP in the presence of the Rev-derived peptide, exclusively. Here, we present the structural analysis of the ribonucleopeptide aptamer with NMR. The secondary structure of the RNA part of the aptamer, the selected RNA region linked to RRE, in the presence of the Rev-derived peptide was determined in an Ado-bound form. G:A and G:G base pairs, together with canonical base pairs, are formed in a duplex of RRE. The selected RNA region plays a crucial role in target binding. It has been found that the two U residues located in the selected RNA region trap Ado through the formation of the U:A:U base triple. This was directly confirmed by the HNN-COSY experiment through the detection of spin-spin couplings across the hydrogen bonds for Watson-Crick and Hoogsteen A:U base pairs in the U:A:U base triple.

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.

Selective recognition of a tetra-amino-acid motif containing phosphorylated tyrosine residue by ribonucleopeptide.

We describe here a ribonucleopeptide (RNP) receptor targeting a tetra-amino-acid motif containing phosphotyrosine, GpYSR. GpYSR-binding RNP receptors were obtained from an RNA-based RNP library by in vitro selection. These receptors have a higher affinity than those of previously obtained pY-binding RNP receptors. One of these RNP receptors exhibited unique specificity to the target GpYSR peptide over other tetra-amino-acid peptides derived from the tyrosine-phosphorylation sites of native proteins. The GpYSR-binding RNP receptor discriminated not only the phosphorylated tyrosine residue, but also its surrounding three amino acid residues. Thus, RNP receptors could target a defined pY-containing amino-acid sequence by expanding the recognition surface within the ligand-binding pocket of RNP.

Development of ribonucleopeptide-based fluorescent sensors for biologically active amines based on the stepwise molding strategy.

General strategy for the development of fluorescent biosensors as a tracer of 'key' molecule in the cellular system would provide important breakthroughs for ubiquitous applications in the field of diagnosis and pharmacology in addition to our understanding of cellular events. The sophisticated design of fluorescent biosensors based on the organic synthesis is one of the promising approaches, but this type of biosensors frequently fail to maintain their performance in the cellular environment despite of laborious protocols. Another procedure for simultaneous preparation of a wide variety of fluorescent biosensors for the optical monitoring of a target molecule represents an especially attractive alternative. In our continuous efforts, we have recently developed a conceptually new strategy for coincidental production of fluorescent biosensors with diverse functions based on a framework of ribonucleopeptide (RNP). RNP-based fluorescent sensors were fabricated with a combination of in vitro selection method and a successive modification of the peptide of RNP with a fluorophore. Each RNP composed of a ligand-binding RNA subunit and a fluorophore-tagged peptide motif facilitated the fluorometric detection of biologically active amines with a unique binding affinity and an inherent fluorescent signal.

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.

Controlling a substrate-binding geometry of ribonucleopeptide receptor.

We describe here a novel strategy to create a ribonucleopeptide (RNP) receptor with defined substrate-binding geometry. RNP library was generated by introducing randomized nucleotide sequences in the RNA subunit of the structurally well-defined complex of RRE-RNA and the Rev peptide by a structure-based design. ATP-binding RNP receptors were selected from the RNP library by in vitro selection. The ATP-binding RNP receptors had RNA sequences varying in the location of the consensus sequence within the randomized nucleotide region. Each RNA subunit was expected to form a ligand-binding pocket with a unique geometry to the N-terminal of the Rev peptide. We have developed a selection strategy to select an ATP-binding receptor with a defined binding geometry by utilizing an ATP-Rev peptide conjugate.

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.