Publications by authors named "Takashi Morii"

91 Publications

Stabilization and structural changes of 2D DNA origami by enzymatic ligation.

Nucleic Acids Res 2021 Aug;49(14):7884-7900

Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.

The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures melt below 60°C due to the presence of discontinuities in the phosphate backbone (i.e., nicks) of the staple strands. In molecular biology, enzymatic ligation is commonly used to seal the nicks in the duplex DNA. However, in DNA nanotechnology, the ligation procedures are neither optimized for the DNA origami nor routinely applied to link the nicks in it. Here, we report a detailed analysis and optimization of the conditions for the enzymatic ligation of the staple strands in four types of 2D square lattice DNA origami. Our results indicated that the ligation takes overnight, efficient at 37°C rather than the usual 16°C or room temperature, and typically requires much higher concentration of T4 DNA ligase. Under the optimized conditions, up to 10 staples ligation with a maximum ligation efficiency of 55% was achieved. Also, the ligation is found to increase the thermal stability of the origami as low as 5°C to as high as 20°C, depending on the structure. Further, our studies indicated that the ligation of the staple strands influences the globular structure/planarity of the DNA origami, and the origami is more compact when the staples are ligated. The globular structure of the native and ligated origami was also found to be altered dynamically and progressively upon ethidium bromide intercalation in a concentration-dependent manner.
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http://dx.doi.org/10.1093/nar/gkab611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373134PMC
August 2021

Dynamic Shape Transformation of a DNA Scaffold Applied for an Enzyme Nanocarrier.

Front Chem 2021 24;9:697857. Epub 2021 Jun 24.

Institute of Advanced Energy, Kyoto University, Kyoto, Japan.

Structural programmability and accurate addressability of DNA nanostructures are ideal characteristics for the platform of arranging enzymes with the nanoscale precision. In this study, a three-dimensional DNA scaffold was designed to enable a dynamic shape transition from an open plate-like structure to its closed state of a hexagonal prism structure. The two domains in the open state were folded together to transform into the closed state by hybridization of complementary short DNA closing keys at both of the facing edges in over 90% yield. The shape transformation of the DNA scaffold was extensively studied by means of the fluorescence energy transfer measurement, atomic force microscope images, and agarose gel electrophoretic analyses. A dimeric enzyme xylitol dehydrogenase was assembled on the DNA scaffold in its open state in a high-loading yield. The enzyme loaded on the scaffold was subsequently transformed to its closed state by the addition of short DNA closing keys. The enzyme encapsulated in the closed state displayed comparable activity to that in the open state, ensuring that the catalytic activity of the enzyme was well maintained in the DNA nanocarrier. The nanocarrier with efficient encapsulation ability is potentially applicable for drug delivery, biosensing, biocatalytic, and diagnostic tools.
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http://dx.doi.org/10.3389/fchem.2021.697857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8263910PMC
June 2021

A facile combinatorial approach to construct a ratiometric fluorescent sensor: application for the real-time sensing of cellular pH changes.

Chem Sci 2021 May 8;12(23):8231-8240. Epub 2021 May 8.

Institute of Advanced Energy, Kyoto University Kyoto Japan

Realtime monitoring of the cellular environment, such as the intracellular pH, in a defined cellular space provides a comprehensive understanding of the dynamics processes in a living cell. Considering the limitation of spatial resolution in conventional microscopy measurements, multiple types of fluorophores assembled within that space would behave as a single fluorescent probe molecule. Such a character of microscopic measurements enables a much more flexible combinatorial design strategy in developing fluorescent probes for given targets. Nanomaterials with sizes smaller than the microscopy spatial resolution provide a scaffold to assemble several types of fluorophores with a variety of optical characteristics, therefore providing a convenient strategy for designing fluorescent pH sensors. In this study, fluorescein (CF) and tetramethylrhodamine (CR) were assembled on a DNA nanostructure with controlling the number of each type of fluorophore. By taking advantage of the different responses of CF and CR emissions to the pH environment, an appropriate assembly of both CF and CR on DNA origami enabled a controlled intensity of fluorescence emission and ratiometric pH monitoring within the space defined by DNA origami. The CF and CR-assembled DNA origami was successfully applied for monitoring the intracellular pH changes.
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http://dx.doi.org/10.1039/d1sc01575cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8208317PMC
May 2021

Enhanced enzymatic activity exerted by a packed assembly of a single type of enzyme.

Chem Sci 2020 Jul 27;11(34):9088-9100. Epub 2020 Jul 27.

Institute of Advanced Energy, Kyoto University Uji Kyoto 611-0011 Japan

In contrast to the dilute conditions employed for biochemical studies, enzymes are spatially organized at high density in cellular micro-compartments. In spite of being crucial for cellular functions, enzymatic reactions in such highly packed states have not been fully addressed. Here, we applied a protein adaptor to assemble a single type of monomeric enzyme on a DNA scaffold in the packed or dispersed states for carbonic anhydrase. The enzymatic reactions proceeded faster in the packed than in the dispersed state. Acceleration of the reaction in the packed assembly was more prominent for substrates with higher hydrophobicity. In addition, carbonic anhydrase is more tolerant of inhibitors in the packed assembly. Such an acceleration of the reaction in the packed state over the dispersed state was also observed for xylose reductase. We propose that the entropic force of water increases local substrate or cofactor concentration within the domain confined between enzyme surfaces, thus accelerating the reaction. Our system provides a reasonable model of enzymes in a packed state; this would help in engineering artificial metabolic systems.
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http://dx.doi.org/10.1039/d0sc03498cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8161546PMC
July 2020

Evaluation of the role of the DNA surface for enhancing the activity of scaffolded enzymes.

Chem Commun (Camb) 2021 Apr 24;57(32):3925-3928. Epub 2021 Mar 24.

Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan.

The catalytic enhancements of enzymes loaded on DNA nanostructures have been attributed to the characteristics provided by highly negative charges on the surface of the DNA scaffold, such as the modulation of the local pH near enzymes. In this study, two types of enzymes with optimal activity at pH 6 and 8 equally displayed significant catalytic enhancements on the DNA scaffold surface. By using a ratiometric pH indicator, a lower local pH shift of 0.8 was observed near the DNA scaffold surface. The postulated local pH change near the DNA scaffold surface is unlikely to play a general role in enhancing the activity of the scaffolded enzymes.
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http://dx.doi.org/10.1039/d1cc00276gDOI Listing
April 2021

Inositol 1,4,5-trisphosphate 3-kinase B promotes Ca mobilization and the inflammatory activity of dendritic cells.

Sci Signal 2021 Mar 30;14(676). Epub 2021 Mar 30.

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy.

Innate immune responses to Gram-negative bacteria depend on the recognition of lipopolysaccharide (LPS) by a receptor complex that includes CD14 and TLR4. In dendritic cells (DCs), CD14 enhances the activation not only of TLR4 but also that of the NFAT family of transcription factors, which suppresses cell survival and promotes the production of inflammatory mediators. NFAT activation requires Ca mobilization. In DCs, Ca mobilization in response to LPS depends on phospholipase C γ2 (PLCγ2), which produces inositol 1,4,5-trisphosphate (IP). Here, we showed that the IP receptor 3 (IPR3) and ITPKB, a kinase that converts IP to inositol 1,3,4,5-tetrakisphosphate (IP), were both necessary for Ca mobilization and NFAT activation in mouse and human DCs. A pool of IPR3 was located on the plasma membrane of DCs, where it colocalized with CD14 and ITPKB. Upon LPS binding to CD14, ITPKB was required for Ca mobilization through plasma membrane-localized IPR3 and for NFAT nuclear translocation. Pharmacological inhibition of ITPKB in mice reduced both LPS-induced tissue swelling and the severity of inflammatory arthritis to a similar extent as that induced by the inhibition of NFAT using nanoparticles that delivered an NFAT-inhibiting peptide specifically to phagocytic cells. Our results suggest that ITPKB may represent a promising target for anti-inflammatory therapies that aim to inhibit specific DC functions.
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http://dx.doi.org/10.1126/scisignal.aaz2120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091591PMC
March 2021

Cryogenic Far-Field Fluorescence Nanoscopy: Evaluation with DNA Origami.

J Phys Chem B 2020 09 24;124(35):7525-7536. Epub 2020 Aug 24.

Department of Physics, Tokyo Institute of Technology, Meguro, Tokyo 152-8550, Japan.

Far-field fluorescence localization nanoscopy of individual fluorophores at a temperature of 1.8 K was demonstrated using DNA origami as a one-nanometer-accurate scaffold. Red and near-infrared fluorophores were modified to the scaffold, and the fluorophores were 11 or 77 nm apart. We performed the localization nanoscopy of these two fluorophores at 1.8 K with a far-field fluorescence microscope. Under the cryogenic conditions, the fluorophores were perfectly immobilized and their photobleaching was drastically suppressed; consequently, the lateral spatial precision (a measure of reproducibility) was increased to 1 nm. However, the lateral spatial accuracy (a measure of trueness) remained tens of nanometers. We observed that the fluorophore centroids were laterally shifted as a function of the axial position. Because the orientation of the transition dipole of the fluorophores was fixed under cryogenic conditions, the anisotropic emission from the single fixed dipole had led to the lateral shift. This systematic error due to the dipole-orientation effect could be corrected by the three-dimensional localization of the individual fluorophores with spatial precisions of (lateral) 1 nm and (axial) 17 nm. In addition, the -error arising from the three-dimensional (3D) orientation of the scaffold with the two fluorophores 11 nm apart was estimated to be 0.3 nm. As a result, the individual fluorophores on the DNA origami were localized at the designed position, and the lateral spatial accuracy was quantified to be 4 nm in the standard error.
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http://dx.doi.org/10.1021/acs.jpcb.0c04721DOI Listing
September 2020

Receptor-based fluorescent sensors constructed from ribonucleopeptide.

Methods Enzymol 2020 17;641:183-223. Epub 2020 Jun 17.

Institute of Advanced Energy, Kyoto University, Uji, Kyoto, Japan. Electronic address:

Receptor-based fluorescent sensors are the representative tool for quantitative detection of target ligands. The high substrate-selectivity originated from biomacromolecule receptor is one of the advantages of this tool, but a laborious trial and error is usually required to construct sensors showing satisfactory fluorescence intensity changes without diminishing the function of parent receptor. Ribonucleopeptide (RNP) provides a scaffold of fluorescent sensors to improve such issues. RNP receptors for the ligand of interest are constructed by applying in vitro selection for RNA-derived RNP library. Simple modification of the N-terminal of peptide in RNP by an appropriate fluorophore converts the RNP receptor into the fluorescent sensor with retaining the affinity and selectivity for the substrate. In this chapter, we introduce the protocols for construction of fluorescent RNP sensors through selection from a library of fluorophore-modified RNP complex or by a structure-based modular design. Furthermore, we describe the application of covalently linked RNP sensors for simultaneous detection of multiple ligands.
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http://dx.doi.org/10.1016/bs.mie.2020.04.041DOI Listing
June 2021

Fluorescence detection of the nitric oxide-induced structural change at the putative nitric oxide sensing segment of TRPC5.

Bioorg Med Chem 2020 04 17;28(8):115430. Epub 2020 Mar 17.

Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan. Electronic address:

The plausible nitric oxide (NO)-sensing module of TRPC5 was incorporated in a enhanced green fluorescent protein (EGFP) to evaluate its conformational change as an optical response upon the reaction with NO. Two cysteine residues located in the NO-sensing module have been proposed to form a disulfide bond through S-nitrosylation of the thiol group by NO. Modification of the cysteine residues by NO resulted a ratiometric change of EGFP emission through transducing the conformational change of NO-sensing module to the EGFP chromophore. The oxidized form of NO-sensing module fused EGFP changed the intensity of emission spectra upon reduction of the disulfide bond at the NO-reactive module. The NO-sensing module fused EGFP in its reduced form avidly reacted with NO and realized the ratiometric fluorescence intensity changes depending on the formation of disulfide bond. These results support the notion that NO induces a conformational change at the putative NO-sensing segment of TRPC5, and provide a prototype for the genetically encoded cellular NO sensors.
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http://dx.doi.org/10.1016/j.bmc.2020.115430DOI Listing
April 2020

Rational design of a DNA sequence-specific modular protein tag by tuning the alkylation kinetics.

Chem Sci 2019 Oct 20;10(40):9315-9325. Epub 2019 Aug 20.

Institute of Advanced Energy , Kyoto University , Uji , Kyoto 611-0011 , Japan . Email:

Sequence-selective chemical modification of DNA by synthetic ligands has been a long-standing challenge in the field of chemistry. Even when the ligand consists of a sequence-specific DNA binding domain and reactive group, sequence-selective reactions by these ligands are often accompanied by off-target reactions. A basic principle to design DNA modifiers that react at specific sites exclusively governed by DNA sequence recognition remains to be established. We have previously reported selective DNA modification by a self-ligating protein tag conjugated with a DNA-binding domain, termed as a modular adaptor, and orthogonal application of modular adaptors by relying on the chemoselectivity of the protein tag. The sequence-specific crosslinking reaction by the modular adaptor is thought to proceed in two steps: the first step involves the formation of a DNA-protein complex, while in the second step, a proximity-driven intermolecular crosslinking occurs. According to this scheme, the specific crosslinking reaction of a modular adaptor would be driven by the DNA recognition process only when the dissociation rate of the DNA complex is much higher than the rate constant for the alkylation reaction. In this study, as a proof of principle, a set of combinations for modular adaptors and their substrates were utilized to evaluate the reactions. Three types of modular adaptors consisting of a single type of self-ligating tag and three types of DNA binding proteins fulfill the kinetic requirements for the reaction of the self-ligating tag with a substrate and the dissociation of the DNA-protein complex. These modular adaptors actually undergo sequence-specific crosslinking reactions exclusively driven by the recognition of a specific DNA sequence. The design principle of sequence-specific modular adaptors based on the kinetic aspects of complex formation and chemical modification is applicable for developing recognition-driven selective modifiers for proteins and other biological macromolecules.
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http://dx.doi.org/10.1039/c9sc02990gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7006624PMC
October 2019

Detection of Inositol Phosphates by Split PH Domains.

Methods Mol Biol 2020 ;2091:47-57

Institute of Advanced Energy, Kyoto University, Uji, Kyoto, Japan.

The pleckstrin homology (PH) domain is a family of structurally conserved proteins which can bind inositol phosphate derivatives. Some proteins involved in cellular signaling and cytoskeletal organization possess split PH domains that assemble into a structure which can bind specific inositol phosphates. Here we describe the design of split PH domain from a structurally well-characterized PH domain of phospholipase C (PLC) δ and Bruton's tyrosine kinase (Btk), which selectively bind Ins(1,4,5)P and Ins(1,3,4,5)P, respectively. The PH domains fold into a functional structure when the split halves are brought to close proximity, and can be utilized to detect specific inositol phosphate of interest.
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http://dx.doi.org/10.1007/978-1-0716-0167-9_4DOI Listing
January 2021

Reaction of ribulose biphosphate carboxylase/oxygenase assembled on a DNA scaffold.

Bioorg Med Chem 2019 11 14;27(22):115120. Epub 2019 Sep 14.

Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan. Electronic address:

Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO), an enzyme in the Calvin-Benson-Bassham cycle of photosynthesis, catalyzes the first step of CO fixation in plants, algae, and photosynthetic bacteria. Despite of the important function in the global carbon cycle, RuBisCO suffers from a slow reaction rate and a competing reaction with O which draw attentions to improve the enzyme efficiency. In this study, a RuBisCO dimer from Rhodospirillum rubrum was assembled on a DNA scaffold using a dimeric DNA binding protein as an adaptor. The enzyme assembly was characterized by atomic force microscopy and RuBisCO assembled on the DNA scaffold showed avid enzymatic activity with retaining its parent carboxylase function. To mimic the environment of the natural microcompartment in cyanobacterial carboxysome that encapsulate the second enzyme carbonic anhydrase (CA) with RuBisCO, RuBisCO was next co-assembled with CA on the DNA scaffold. Although the natural carboxysome assembly is believed to enhance the RuBisCO activity, the co-assembly of RuBisCO and CA reduced the RuBisCO activity, suggesting that the preferential CO dehydration by CA reduced the RuBisCO reaction rate. In line with the recent study, our results suggest that the proximity in the interenzyme distance of RuBisCO and CA is not the crucial determinant for the enhanced RuBisCO activity in carboxysome. The assembly of RuBisCO and CA on DNA scaffold provides a platform for further study on the spatial control of RuBisCO and associating enzymes.
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http://dx.doi.org/10.1016/j.bmc.2019.115120DOI Listing
November 2019

Protein adaptors assemble functional proteins on DNA scaffolds.

Chem Commun (Camb) 2019 Oct;55(83):12428-12446

Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan.

DNA is an attractive molecular building block to construct nanoscale structures for a variety of applications. In addition to their structure and function, modification the DNA nanostructures by other molecules opens almost unlimited possibilities for producing functional DNA-based architectures. Among the molecules to functionalize DNA nanostructures, proteins are one of the most attractive candidates due to their vast functional variations. DNA nanostructures loaded with various types of proteins hold promise for applications in the life and material sciences. When loading proteins of interest on DNA nanostructures, the nanostructures by themselves act as scaffolds to specifically control the location and number of protein molecules. The methods to arrange proteins of interest on DNA scaffolds at high yields while retaining their activity are still the most demanding task in constructing usable protein-modified DNA nanostructures. Here, we provide an overview of the existing methods applied for assembling proteins of interest on DNA scaffolds. The assembling methods were categorized into two main classes, noncovalent and covalent conjugation, with both showing pros and cons. The recent advance of DNA-binding adaptor mediated assembly of proteins on the DNA scaffolds is highlighted and discussed in connection with the future perspectives of protein assembled DNA nanoarchitectures.
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http://dx.doi.org/10.1039/c9cc04661eDOI Listing
October 2019

Nanometer Accuracy in Cryogenic Far-Field Localization Microscopy of Individual Molecules.

J Phys Chem Lett 2019 Oct 19;10(19):5841-5846. Epub 2019 Sep 19.

Department of Physics , Tokyo Institute of Technology , Meguro , Tokyo 152-8550 , Japan.

We demonstrate the nanometer accuracy of far-field fluorescence localization microscopy at a temperature of 1.8 K using near-infrared and red fluorophores bonded to double-stranded DNA molecules (10.2 nm length). Although each fluorophore was localized with a 1 nm lateral precision by acquiring an image at one axial position within the focal depth of ±0.7 μm, the distance between the two fluorophores on the lateral plane () was distributed from 0 to 50 nm. This systematic error was mainly due to detecting with the large focal depth the dipole emission from orientationally fixed fluorophores. Each fluorophore was localized with precisions of ±1 nm (lateral) and simultaneously ±11 nm (axial) by acquiring images every 100 nm in the axial direction from -900 to 900 nm. By correcting the dipole orientation effects, the distribution of was centered around the DNA length. The average and standard deviation of were 10 and 5 nm.
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http://dx.doi.org/10.1021/acs.jpclett.9b02184DOI Listing
October 2019

Contribution of Coiled-Coil Assembly to Ca/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes.

J Am Soc Nephrol 2019 09 2;30(9):1587-1603. Epub 2019 Jul 2.

Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,

Background: TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved.

Methods: We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes.

Results: Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca elevations and a disorganized cytoskeleton.

Conclusions: The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC's coiled-coil assembly which is essential for CaM binding. The resulting excess Ca may contribute to structural damage in the podocytes.
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http://dx.doi.org/10.1681/ASN.2018070756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727271PMC
September 2019

DNA binding adaptors to assemble proteins of interest on DNA scaffold.

Methods Enzymol 2019 13;617:287-322. Epub 2019 Feb 13.

Institute of Advanced Energy, Kyoto University, Uji, Kyoto, Japan. Electronic address:

DNA nanostructures serve as the ideal scaffolds to assemble materials of interest. Among these, proteins are of particularly interesting class of molecules to assemble because of their huge functional variability. Sequence-specific DNA binding proteins have been applied as adaptors to stably locate the fused proteins at defined positions of DNA scaffold in high loading yields. The strategy allows to control the number of enzyme molecules and to maintain the catalytic activity. By fusing a chemoselective self-ligating protein tag to the DNA binding protein, the modular adaptors formed covalent bonds at respective sequences on DNA scaffold with fast reaction kinetics. Application of a set of orthogonal modular adaptors enables spatial organization of multiple types of enzymes.
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http://dx.doi.org/10.1016/bs.mie.2018.12.014DOI Listing
November 2019

Highly selective dual sensing of ATP and ADP using fluorescent ribonucleopeptide sensors.

Chem Commun (Camb) 2019 Jan;55(11):1611-1614

Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.

Highly selective fluorescent sensors for ATP and ADP were constructed from RNA aptamers by applying a modular design of a ribonucleopeptide scaffold. These sensors allow facile and quantitative detection of ATP and ADP simultaneously in a solution and enable monitoring of the time-course changes of ATP and ADP concentrations in an enzymatic reaction.
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http://dx.doi.org/10.1039/c8cc09934kDOI Listing
January 2019

DNA Origami Scaffolds as Templates for Functional Tetrameric Kir3 K Channels.

Angew Chem Int Ed Engl 2018 03 1;57(10):2586-2591. Epub 2018 Feb 1.

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyotodaigakukatsura, Nishikyo-ku, Kyoto, 615-8510, Japan.

In native systems, scaffolding proteins play important roles in assembling proteins into complexes to transduce signals. This concept is yet to be applied to the assembly of functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami has the potential to serve as scaffolds that arrange proteins at specific positions in complexes. Herein, we report that Kir3 K channel proteins are assembled through zinc-finger protein (ZFP)-adaptors at specific locations on DNA origami scaffolds. Specific binding of the ZFP-fused Kir3 channels and ZFP-based adaptors on DNA origami were confirmed by atomic force microscopy and gel electrophoresis. Furthermore, the DNA origami with ZFP binding sites nearly tripled the K channel current activity elicited by heterotetrameric Kir3 channels in HEK293T cells. Thus, our method provides a useful template to control the oligomerization states of membrane protein complexes in vitro and in living cells.
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http://dx.doi.org/10.1002/anie.201709982DOI Listing
March 2018

Facile conversion of ATP-binding RNA aptamer to quencher-free molecular aptamer beacon.

Bioorg Med Chem Lett 2018 01 6;28(2):77-80. Epub 2017 Dec 6.

Department of Chemistry, Division of Advanced Materials Science, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea. Electronic address:

We have developed RNA-based quencher-free molecular aptamer beacons (RNA-based QF-MABs) for the detection of ATP, taking advantage of the conformational changes associated with ATP binding to the ATP-binding RNA aptamer. The RNA aptamer, with its well-defined structure, was readily converted to the fluorescence sensors by incorporating a fluorophore into the loop region of the hairpin structure. These RNA-based QF-MABs exhibited fluorescence signals in the presence of ATP relative to their low background signals in the absence of ATP. The fluorescence emission intensity increased upon formation of a RNA-based QF-MAB·ATP complex.
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http://dx.doi.org/10.1016/j.bmcl.2017.12.008DOI Listing
January 2018

A Diversity-Oriented Library of Fluorophore-Modified Receptors Constructed from a Chemical Library of Synthetic Fluorophores.

Chembiochem 2017 11 2;18(22):2212-2216. Epub 2017 Oct 2.

Institute of Advanced Energy, Kyoto University, Uji, Kyoto, 611-0011, Japan.

The practical application of biosensors can be determined by evaluating the sensing ability of fluorophore-modified derivatives of a receptor with appropriate recognition characteristics for target molecules. One of the key determinants for successfully obtaining a useful biosensor is wide variation in the fluorophores attached to a given receptor. Thus, using a larger fluorophore-modified receptor library provides a higher probability of obtaining a practically useful biosensor. However, no effective method has yet been developed for constructing such a diverse library of fluorophore-modified receptors. Herein, we report a method for constructing fluorophore-modified receptors by using a chemical library of synthetic fluorophores with a thiol-reactive group. This library was converted into a library of fluorophore-modified adenosine-binding ribonucleopeptide (RNP) receptors by introducing the fluorophores to the Rev peptide of the RNP complex by alkylation of the thiol group. This method enabled the construction of 263 fluorophore-modified ATP-binding RNP receptors and allowed the selection of suitable receptor-based fluorescent sensors that target ATP.
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http://dx.doi.org/10.1002/cbic.201700403DOI Listing
November 2017

Design of Modular Protein Tags for Orthogonal Covalent Bond Formation at Specific DNA Sequences.

J Am Chem Soc 2017 06 14;139(25):8487-8496. Epub 2017 Jun 14.

Institute of Advanced Energy, Kyoto University , Uji, Kyoto 611-0011, Japan.

Simultaneous formation of specific covalent linkages at nucleotides in given DNA sequences demand distinct orthogonal reactivity of DNA modification agents. Such highly specific reactions require well-balanced reactivity and affinity of the DNA modification agents. Conjugation of a sequence-specific DNA binding zinc finger protein and a self-ligating protein tag provides a modular adaptor that expedites formation of a covalent bond between the protein tag and a substrate-modified nucleotide at a specific DNA sequence. The modular adaptor stably locates a protein of interest fused to it at the target position on DNA scaffold in its functional form. Modular adaptors with orthogonal selectivity and fast reaction kinetics to specific DNA sequences enable site-specific location of different protein molecules simultaneously. Three different modular adaptors consisting of zinc finger proteins with distinct DNA sequence specificities and self-ligating protein tags with different substrate specificities achieved orthogonal covalent bond formation at respective sequences on the same DNA scaffold with an overall coassembly yield over 90%. Application of this unique set of orthogonal modular adaptors enabled construction of a cascade reaction of three enzymes from xylose metabolic pathway on DNA scaffold.
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http://dx.doi.org/10.1021/jacs.7b01640DOI Listing
June 2017

Construction of a library of structurally diverse ribonucleopeptides with catalytic groups.

Bioorg Med Chem 2017 03 10;25(6):1881-1888. Epub 2017 Feb 10.

Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bmc.2017.02.007DOI Listing
March 2017

Nucleic-Acid-Templated Enzyme Cascades.

Chembiochem 2017 04 22;18(8):696-716. Epub 2017 Mar 22.

Institute of Advanced Energy, Kyoto University, Uji, Kyoto, 611-0011, Japan.

Cellular metabolism involves complex sequences of organized enzymatic reactions, known as metabolic pathways, that convert substrates into readily usable materials. In nature, these enzymatic complexes are organized in a well-defined manner so that the cascade reactions are more rapid and efficient than they would be if the enzymes were randomly distributed in the cytosol. Development of artificial enzyme cascades that resemble nature's organization of sequentially assembled enzymes is of current interest due to its potential applications, from diagnostics to the production of high-value chemicals. Nucleic acids and their nanostructures have been used to organize enzyme cascades and have been shown to enhance the efficiencies and rates of sequential reactions. Here we summarize the recent progress in the development of artificial enzyme cascades and sequential reactions by arranging enzymes on various DNA/RNA templates and discuss the future directions of this research endeavour.
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http://dx.doi.org/10.1002/cbic.201600703DOI Listing
April 2017

Latent pH-responsive ratiometric fluorescent cluster based on self-assembled photoactivated SNARF derivatives.

Sci Technol Adv Mater 2016 2;17(1):431-436. Epub 2016 Aug 2.

Institute of Advanced Energy, Kyoto University , Uji , Kyoto , Japan.

We have developed a self-assembled fluorescent cluster comprising a seminaphthorhodafluor (SNARF) derivative protected by a photoremovable -nitrobenzyl group. Prior to UV irradiation, a colorless and nonfluorescent cluster was spontaneously assembled in aqueous solution. After UV irradiation, the self-assembled cluster remained intact and showed a large enhancement in pH-responsive fluorescence. The unique pH responsive fluorescent cluster could be used as a dual-emissive ratiometric fluorescent pH probe not only in the test tube but also in HeLa cell cultures.
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http://dx.doi.org/10.1080/14686996.2016.1204888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5101900PMC
August 2016

Spatially Organized Enzymes Drive Cofactor-Coupled Cascade Reactions.

J Am Chem Soc 2016 Mar 29;138(9):3012-21. Epub 2016 Feb 29.

Institute of Advanced Energy, Kyoto University , Uji, Kyoto 611-0011, Japan.

We report the construction of an artificial enzyme cascade based on the xylose metabolic pathway. Two enzymes, xylose reductase and xylitol dehydrogenase, were assembled at specific locations on DNA origami by using DNA-binding protein adaptors with systematic variations in the interenzyme distances and defined numbers of enzyme molecules. The reaction system, which localized the two enzymes in close proximity to facilitate transport of reaction intermediates, resulted in significantly higher yields of the conversion of xylose into xylulose through the intermediate xylitol with recycling of the cofactor NADH. Analysis of the initial reaction rate, regenerated amount of NADH, and simulation of the intermediates' diffusion indicated that the intermediates diffused to the second enzyme by Brownian motion. The efficiency of the cascade reaction with the bimolecular transport of xylitol and NAD(+) likely depends more on the interenzyme distance than that of the cascade reaction with unimolecular transport between two enzymes.
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http://dx.doi.org/10.1021/jacs.5b10198DOI Listing
March 2016

Validating subcellular thermal changes revealed by fluorescent thermosensors.

Nat Methods 2015 Sep;12(9):801-2

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan.

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http://dx.doi.org/10.1038/nmeth.3548DOI Listing
September 2015

A modular zinc finger adaptor accelerates the covalent linkage of proteins at specific locations on DNA nanoscaffolds.

Chem Commun (Camb) 2015 Jan;51(6):1016-9

Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan.

A modular adaptor consisting of a sequence-specific DNA binding zinc finger protein and a self-ligating protein-tag was developed to expedite efficient formation of a covalent linkage between an individual protein molecule and the programmed address modified with a tag-substrate on the DNA nanostructure.
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http://dx.doi.org/10.1039/c4cc08167fDOI Listing
January 2015

Phosphorylation regulates fibrillation of an aggregation core peptide in the second repeat of microtubule-binding domain of human tau.

Bioorg Med Chem 2014 Nov;22(22):6471-80

Hyperphosphorylation of the microtubule-associated protein tau is believed to play a crucial role in the neurofibrillary tangles formation in Alzheimer’s disease brain. In this study, fibril formation of peptides containing the critical sequences for tau aggregation VQIINK and a plausible serine phosphorylation site of tau at its C-terminal was investigated. All the peptides formed fibrils with the typical cross-b structural core. However, stability of the fibrils was highly sensitive to the pH conditions for the phosphorylated VQIINK peptide, suggesting a regulatory role of phosphorylation for the amyloid-formation of tau.
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http://dx.doi.org/10.1016/j.bmc.2014.09.032DOI Listing
November 2014

A neutron dynamic therapy with a boron tracedrug UTX-51 using a compact neutron generator.

Anticancer Res 2014 Aug;34(8):4557-60

Institute of Advanced Energy, Kyoto University, Uji, Kyoto, Japan.

Background/aim: We are developing a neutron dynamic therapy (NDT) with boron tracedrugs for a new mechanical-clearance treatment of pathotoxic misfolded, aggregated, and self-propagating prion-associated disease proteins. We present a compact neutron generator-based NDT using a boron tracedrug UTX-51. Our NDT is based on the weak thermal neutron-bombarded destructive action of UTX-51 on bovine serum albumin (BSA) using the neutron beams produced from a compact inertial electrostatic confinement fusion (IECF) neutron generator.

Results: BSA as an NDT molecular target was subjected to thermal neutron irradiation for eight hours using a compact neutron generator. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern showed no protein band when 2 nmoles of BSA were irradiated with more than 100 nmoles of UTX-51, while BSA was not affected when irradiated without UTX-51.

Conclusion: For the first time, we have succeeded in the molecular destruction of a prion-disease model protein, BSA, by NDT with a boron tracedrug, UTX-51, using a compact neutron generator.
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August 2014

A protein adaptor to locate a functional protein dimer on molecular switchboard.

Methods 2014 May 1;67(2):142-50. Epub 2013 Nov 1.

Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan; CREST, JST, Uji, Kyoto 611-0011, Japan. Electronic address:

The addressable DNA nanostructures offer ideal platforms to construct organized assemblies of multiple protein molecules. Sequence-specific DNA binding proteins that target defined sites on DNA nanostructures would act as orthogonal adaptors to carry individual protein molecules to the programmed addresses. We have recently developed a protein-based adaptor by utilizing the sequence-specific DNA binding zinc finger protein to locate a monomeric protein of interest at specific positions on DNA origami, which serves as a molecular switchboard. We herein report a new adaptor to locate a protein dimer on the DNA origami scaffold based on a homodimeric basic-leucine zipper protein GCN4. Specific binding of GCN4 to programmed addresses on DNA origami and orthogonal targeting by GCN4- and zinc finger protein-based adaptors to the respective addresses on DNA origami were confirmed by gel electrophoretic and AFM analyses. Furthermore, a GCN4-fused homodimeric enzyme showed even higher activity than the wild type enzyme, and exhibited avid reactivity when assembled at the specific site of DNA origami. Thus, GCN4 serves as an ideal adaptor to locate homodimeric proteins in the functional form on DNA origami.
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http://dx.doi.org/10.1016/j.ymeth.2013.10.014DOI Listing
May 2014
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