Takanori Uzawa

RIKEN, Вако, Saitama, Japan

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Publications (27)147.21 Total impact

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    ABSTRACT: A peptide aptamer that changes fluorescence upon binding to verotoxin was selected in vitro using ribosome display with a tRNA carrying an environment-sensitive fluorescent probe. The aptamer specifically bound to verotoxin with a dissociation constant (K d) of 3.94 ± 1.6 µM, and the fluorescence decreased by 78 % as the verotoxin concentration was increased. The selected peptide can be used for detection of verotoxin.
    Biotechnology Letters 11/2014; · 1.85 Impact Factor
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    ABSTRACT: To increase the inhibitory activity of purvalanol against cyclin-dependent kinase 2, we increased the extent of interaction between the inhibitor and the target by coupling a peptide aptamer to purvalanol. The peptide–purvalanol conjugate, selected using a ribosome display, had a significantly enhanced inhibitory effect compared with purvalanol alone. The technique is useful as another type of fragment-based drug design tool.
    Medicinal Chemistry Communication 08/2014; 5(9). · 2.72 Impact Factor
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    ABSTRACT: Photoresponsive peptide aptamer to glutathione-immobilized microbeads was in vitro selected using ribosome display incorporated with tRNA carrying an amino acid coupled with an azobenzene.
    Journal of Bioscience and Bioengineering 07/2014; · 1.74 Impact Factor
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    ABSTRACT: A peptide that binds and emits fluorescence in response to conformational change in a target protein was developed by in vitro selection using tRNA carrying a fluorogenic amino acid. This technology could prove to be useful for the development of separation-free immunoassays and bio-imaging analyses.
    Chemical Communications 12/2013; · 6.38 Impact Factor
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    ABSTRACT: Ribosome display was used to select peptide aptamers from a random library composed of hydrophilic amino acids for a conducting polymer, poly(3-hexylthiophene-2,5-diyl). Binding of aptamers was measured by quartz crystal microbalance, and the secondary structure of the peptide was investigated by circular dichroism.
    Journal of Bioscience and Bioengineering 11/2013; · 1.74 Impact Factor
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    ABSTRACT: Oligonucleotide-templated reactions are powerful tools for the detection of nucleic acid sequences. One of the major scientific challenges associated with this technique is the rational design of non-enzyme-mediated catalytic templated reactions capable of multiple turnovers that provide high levels of signal amplification. Herein, we report the development of a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe underwent a rapid templated reaction without any of the undesired background reactions. The fluorogenic reaction conducted in the presence of a template provided a 223-fold increase in fluorescence after 30 s compared with the nontemplated reaction. The probe provided an efficient level of signal amplification that ultimately enabled particularly sensitive levels of detection. Assuming a simple model for the templated reactions, it was possible to estimate the rate constants of the chemical reaction in the presence and in the absence of the template. From these kinetic analyses, it was possible to confirm that an efficient turnover cycle had been achieved, on the basis of the dramatic enhancement in the rate of the chemical reaction considered to be the rate-determining step. With maximized turnover efficiency, it was demonstrated that the probe could offer a high turnover number of 1500 times to enable sensitive levels of detection with a detection limit of 0.5 pM in the catalytic templated reactions.
    Journal of the American Chemical Society 09/2013; · 10.68 Impact Factor
  • Takanori Uzawa, Seiichi Tada, Wei Wang, Yoshihiro Ito
    ChemInform 06/2013; 44(23).
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    ABSTRACT: Intramolecular collision dynamics play an essential role in biomolecular folding and function and, increasingly, in the performance of biomimetic technologies. To date, however, the quantitative studies of dynamics of single-stranded nucleic acids have been limited. Thus motivated, here we investigate the sequence composition, chain-length, viscosity, and temperature dependencies of the end-to-end collision dynamics of single-stranded DNAs. We find that both the absolute collision rate and the temperature dependencies of these dynamics are base-composition dependent, suggesting that base stacking interactions are a significant contributor. For example, whereas the end-to-end collision dynamics of poly-thymine exhibit simple, linear Arrhenius behavior, the behavior of longer poly-adenine constructs is more complicated. Specifically, 20- and 25-adenine constructs exhibit biphasic temperature dependencies, with their temperature dependences becoming effectively indistinguishable from that of poly-thymine above 335 K for 20-adenines and 328 K for 25-adenines. The differing Arrhenius behaviors of poly-thymine and poly-adenine and the chain-length dependence of the temperature at which poly-adenine crosses over to behave like poly-thymine can be explained by a barrier friction mechanism in which, at low temperatures, the energy barrier for the local rearrangement of poly-adenine becomes the dominant contributor to its end-to-end collision dynamics.
    Biophysical Journal 06/2013; 104(11):2485-92. · 3.67 Impact Factor
  • Takanori Uzawa, Seiichi Tada, Wei Wang, Yoshihiro Ito
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    ABSTRACT: The possibility of evolving a commonly existing biomolecule into a variety of functional biomolecules has now been realized in the form of aptamers through the development of in vitro selection. In addition to their high affinity and high specificity for the desired targets, aptamers are easily synthesized chemically and can be modified for downstream applications. Although aptamers were originally selected from a library containing only natural components, the past decade has seen a wealth of new aptamers selected from libraries containing unnatural components to provide new aptamer functions artificially. In this review, we highlight this transition (the shift between selection from natural components and selection from unnatural components) and the applications of selected aptamers.
    Chemical Communications 01/2013; · 6.38 Impact Factor
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    ABSTRACT: A ribosome display from a diverse random library was applied for selecting peptide aptamers with high binding affinity to single-wall carbon nanotubes (SWCNTs). The selected peptide aptamer bound to and solubilized SWCNTs more strongly than did the peptide aptamer selected by a phage display method reported previously, and more strongly than other commonly used organic surfactants. The fluorescence spectrum of this aptamer showed a red shift upon interaction with SWCNTs but circular dichroism spectroscopy did not show any significant difference between the presence or absence of SWCNT binding.
    Biotechnology Letters 09/2012; · 1.85 Impact Factor
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    ABSTRACT: The development of rapid, low-cost point-of-care approaches for the quantitative detection of antibodies would drastically impact global health by shortening the delay between sample collection and diagnosis and by improving the penetration of modern diagnostics into the developing world. Unfortunately, however, current methods for the quantitative detection of antibodies, including ELISAs, Western blots, and fluorescence polarization assays, are complex, multiple-step processes that rely on well-trained technicians working in well-equipped laboratories. In response, we describe here a versatile, DNA-based electrochemical "switch" for the rapid, single-step measurement of specific antibodies directly in undiluted whole blood at clinically relevant low-nanomolar concentrations.
    Journal of the American Chemical Society 08/2012; 134(37):15197-200. · 10.68 Impact Factor
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    ABSTRACT: The diagnosis, prevention, and treatment of many illnesses, including infectious and autoimmune diseases, would benefit from the ability to measure specific antibodies directly at the point of care. Thus motivated, we designed a wash-free, electrochemical method for the rapid, quantitative detection of specific antibodies directly in undiluted, unprocessed blood serum. Our approach employs short, contiguous polypeptide epitopes coupled to the distal end of an electrode-bound nucleic acid "scaffold" modified with a reporting methylene blue. The binding of the relevant antibody to the epitope reduces the efficiency with which the redox reporter approaches, and thus exchanges electrons with, the underlying sensor electrode, producing readily measurable change in current. To demonstrate the versatility of the approach, we fabricated a set of six such sensors, each aimed at the detection of a different monoclonal antibody. All six sensors are sensitive (subnanomolar detection limits), rapid (equilibration time constants ∼8 min), and specific (no appreciable cross reactivity with the targets of the other five). When deployed in a millimeter-scale, an 18-pixel array with each of the six sensors in triplicate support the simultaneous measurement of the concentrations of multiple antibodies in a single, submilliliter sample volume. The described sensor platform thus appears be a relatively general approach to the rapid and specific quantification of antibodies in clinical materials.
    Analytical Chemistry 12/2011; 84(2):1098-103. · 5.82 Impact Factor
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    ABSTRACT: Understanding the rate at which various parts of a molecular chain come together to facilitate the folding of a biopolymer (e.g., a protein or RNA) into its functional form remains an elusive goal. Here we use experiments, simulations, and theory to study the kinetics of internal loop closure in disordered biopolymers such as single-stranded oligonucleotides and unfolded proteins. We present theoretical arguments and computer simulation data to show that the relationship between the timescale of internal loop formation and the positions of the monomers enclosing the loop can be recast in a form of a universal master dependence. We also perform experimental measurements of the loop closure times of single-stranded oligonucleotides and show that both these and previously reported internal loop closure kinetics of unfolded proteins are well described by this theoretically predicted dependence. Finally, we propose that experimental deviations from the master dependence can then be used as a sensitive probe of dynamical and structural order in unfolded proteins and other biopolymers.
    Biophysical Journal 12/2010; 99(12):3959-68. · 3.67 Impact Factor
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    ABSTRACT: Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its use has been significantly limited when performed directly in complex, interferant-laced samples. In this work, we report a modification of the TRAP assay that allows the detection of high-fidelity amplification of telomerase products directly from concentrated cell lysates. Briefly, we covalently attached 12 nm gold nanoparticles (AuNPs) to the telomere strand (TS) primer, which is used as a substrate for telomerase elongation. These TS-modified AuNPs significantly reduce polymerase chain reaction (PCR) artifacts (such as primer dimers) and improve the yield of amplified telomerase products relative to the traditional TRAP assay when amplification is performed in concentrated cell lysates. Specifically, because the TS-modified AuNPs eliminate most of the primer-dimer artifacts normally visible at the same position as the shortest amplified telomerase PCR product apparent on agarose gels, the AuNP-modified TRAP assay exhibits excellent sensitivity. Consequently, we observed a 10-fold increase in sensitivity for cancer cells diluted 1000-fold with somatic cells. It thus appears that the use of AuNP-modified primers significantly improves the sensitivity and specificity of the traditional TRAP assay and may be an effective method by which PCR can be performed directly in concentrated cell lysates.
    Journal of the American Chemical Society 10/2010; 132(43):15299-307. · 10.68 Impact Factor
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    ABSTRACT: Electrode-bound, redox-reporter-modified oligonucleotides play roles in the functioning of a number of electrochemical biosensors, and thus the question of electron transfer through or from such molecules has proven of significant interest. In response, we have experimentally characterized the rate with which electrons are transferred between a methylene blue moiety on the distal end of a short, single-stranded polythymine DNA to a monolayer-coated gold electrode to which the other end of the DNA is site-specifically attached. We find that this rate scales with oligonucleotide length to the -1.16 ± 0.09 power. This weak, approximately inverse length dependence differs dramatically from the much stronger dependencies observed for the rates of end-to-end collisions in single-stranded DNA and through-oligonucleotide electron hopping. It instead coincides with the expected length dependence of a reaction-limited process in which the overall rate is proportional to the equilibrium probability that the end of the oligonucleotide chain approaches the surface. Studies of the ionic strength and viscosity dependencies of electron transfer further support this "chain-flexibility" mechanism, and studies of the electron transfer rate of methylene blue attached to the hexanethiol monolayer suggest that heterogeneous electron transfer through the monolayer is rate limiting. Thus, under the circumstances we have employed, the flexibility (i.e., the equilibrium statistical properties) of the oligonucleotide chain defines the rate with which an attached redox reporter transfers electrons to an underlying electrode, an observation that may be of utility in the design of new biosensor architectures.
    Journal of the American Chemical Society 10/2010; 132(45):16120-6. · 10.68 Impact Factor
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    ABSTRACT: We report here an electrochemical approach that offers, for the first time, single-step, room-temperature single nucleotide polymorphism (SNP) detection directly in complex samples (such as blood serum) without the need for target modification, postwashing, or the addition of exogenous reagents. This sensor, which is sensitive, stable, and reusable, is comprised of a single, self-complementary, methylene blue-labeled DNA probe possessing a triple-stem structure. This probe takes advantage of the large thermodynamic changes in enthalpy and entropy that result from major conformational rearrangements that occur upon binding a perfectly matched target, resulting in a large-scale change in the faradaic current. As a result, the discrimination capabilities of this sensor greatly exceed those of earlier single- and double-stem electrochemical sensors and support rapid (minutes), single-step, reagentless, room-temperature detection of single nucleotide substitutions. To elucidate the theoretical basis of the sensor's selectivity, we present a comparative thermodynamic analysis among single-, double-, and triple-stem probes.
    Journal of the American Chemical Society 10/2009; 131(42):15311-6. · 10.68 Impact Factor
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    ABSTRACT: The problem of determining the rate of end-to-end collisions for polymer chains has attracted the attention of theorists and experimentalists for more than three decades. The typical theoretical approach to this problem has focused on the case where a collision is defined as any instantaneous fluctuation that brings the chain ends to within a specific capture distance. In this paper, we study the more experimentally relevant case, where the end-to-end collision dynamics are probed by measuring the excited state lifetime of a fluorophore (or other lumiphore) attached to one chain end and quenched by a quencher group attached to the other end. Under this regime, a "contact" is defined not by the chain ends approach to within some sharp cutoff but, instead, typically by an exponentially distance-dependent process. Previous theoretical models predict that, if quenching is sufficiently rapid, a diffusion-controlled limit is attained, where such measurements report on the probe-independent, intrinsic end-to-end collision rate. In contrast, our theoretical considerations, simulations, and an analysis of experimental measurements of loop closure rates in single-stranded DNA molecules all indicate that no such limit exists, and that the measured effective collision rate has a nontrivial, fractional power-law dependence on both the intrinsic quenching rate of the fluorophore and the solvent viscosity. We propose a simple scaling formula describing the effective loop closure rate and its dependence on the viscosity, chain length, and properties of the probes. Previous theoretical results are limiting cases of this more general formula.
    The Journal of Physical Chemistry B 09/2009; 113(42):14026-34. · 3.61 Impact Factor
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    ABSTRACT: Intramolecular dynamics play an essential role in the folding and function of biomolecules and, increasingly, in the operation of many biomimetic technologies. Thus motivated we have employed both experiment and simulation to characterize the end-to-end collision dynamics of unstructured, single-stranded DNAs ranging from 6 to 26 bases. We find that, because of the size and flexibility of the optical reporters employed experimentally, end-to-end collision dynamics exhibit little length dependence at length scales <11 bases. For longer constructs, however, the end-to-end collision rate exhibits a power-law relationship to polymer length with an exponent of -3.49 +/- 0.13. This represents a significantly stronger length dependence than observed experimentally for unstructured polypeptides or predicted by polymer scaling arguments. Simulations indicate, however, that the larger exponent stems from electrostatic effects that become important over the rather short length scale of these highly charged polymers. Finally, we have found that the end-to-end collision rate also depends linearly on solvent viscosity, with an experimentally significant, nonzero intercept (the extrapolated rate at zero viscosity) that is independent of chain length--n observation that sheds new light on the origins of the "internal friction" observed in the dynamics of many polymer systems.
    Biophysical Journal 08/2009; 97(1):205-10. · 3.67 Impact Factor
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    ABSTRACT: Recent years have seen the emergence of a new class of electrochemical sensors predicated on target binding-induced folding of electrode-bound redox-modified aptamers and directed against targets ranging from small molecules to proteins. Previous studies of the relationship between gain and probe-density for these electrochemical, aptamer-based (E-AB) sensors suggest that signal transduction is linked to binding-induced changes in the efficiency with which the attached redox tag strikes the electrode. This, in turn, suggests that even well folded aptamers may support E-AB signaling if target binding sufficiently alters their flexibility. Here we investigate this using a thrombin-binding aptamer that undergoes binding-induced folding at low ionic strength but can be forced to adopt a folded conformation at higher ionic strength even in the absence of its protein target. We find that, under conditions in which the thrombin aptamer is fully folded prior to target binding, we still obtain a ca. 30% change in E-AB signal upon saturated target levels. In contrast, however, under conditions in which the aptamer is unfolded in the absence of target and thus undergoes binding-induced folding the observed signal change is twice as great. The ability of folded aptamers to support E-AB signaling, however, is not universal: a fully folded anti-IgE aptamer, for example, produces only an extremely small, ca. 2.5% signal change in the presence of target despite the larger steric bulk of this protein. Thus, while it appears that binding-induced changes in the dynamics in fully folded aptamers can support E-AB signaling, this signaling mechanism may not be general, and in order to ensure the design of high-gain sensors binding must be linked to a large-scale conformational change.
    Electroanalysis 06/2009; 21(11):1267-1271. · 2.82 Impact Factor
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    ABSTRACT: The earliest steps in the folding of proteins are complete on an extremely rapid time scale that is difficult to access experimentally. We have used rapid-mixing quench-flow methods to extend the time resolution of folding studies on apomyoglobin and elucidate the structural and dynamic features of members of the ensemble of intermediate states that are populated on a submillisecond time scale during this process. The picture that emerges is of a continuum of rapidly interconverting states. Even after only 0.4 ms of refolding time a compact state is formed that contains major parts of the A, G, and H helices, which are sufficiently well folded to protect amides from exchange. The B, C, and E helix regions fold more slowly and fluctuate rapidly between open and closed states as they search docking sites on this core; the secondary structure in these regions becomes stabilized as the refolding time is increased from 0.4 to 6 ms. No further stabilization occurs in the A, G, H core at 6 ms of folding time. These studies begin to time-resolve a progression of compact states between the fully unfolded and native folded states and confirm the presence an ensemble of intermediates that interconvert in a hierarchical sequence as the protein searches conformational space on its folding trajectory.
    Proceedings of the National Academy of Sciences 10/2008; 105(37):13859-64. · 9.81 Impact Factor

Publication Stats

349 Citations
147.21 Total Impact Points

Institutions

  • 2013–2014
    • RIKEN
      • Nano Medical Engineering Laboratory
      Вако, Saitama, Japan
    • Hokkaido University
      • Division of Chemistry
      Sapporo-shi, Hokkaido, Japan
  • 2009–2012
    • University of California, Santa Barbara
      • • Department of Chemistry and Biochemistry
      • • Department of Physics
      Santa Barbara, CA, United States
  • 2004–2008
    • Kyoto University
      • • Graduate School of Engineering / Faculty of Engineering
      • • Department of Molecular Engineering
      Kyoto, Kyoto-fu, Japan