Takanori Uzawa

An Electrochemical Sensor for Single Nucleotide Polymorphism Detection in Serum Based on a Triple-Stem DNA Probe

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 sensors selectivity, we present a comparative thermodynamic analysis among single-, double-, and triple-stem probes.

Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR

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.

Detection of Telomerase Activity in High Concentration of Cell Lysates Using Primer-Modified Gold Nanoparticles

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.

Very Rapid DNA-Templated Reaction for Efficient Signal Amplification and Its Steady-State Kinetic Analysis of the Turnover Cycle

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.

In vitro selection of peptide aptamers with affinity to single-wall carbon nanotubes using a ribosome display.

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.

Universality in the Timescales of Internal Loop Formation in Unfolded Proteins and Single-Stranded Oligonucleotides

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.

Sequence and Temperature Dependence of the End-to-End Collision Dynamics of Single-Stranded DNA

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.

In vitro selection of a peptide aptamer that potentiates inhibition of cyclin-dependent kinase 2 by purvalanol

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.

Fluorogenic Enhancement of an in Vitro-Selected Peptide Ligand by Replacement of a Fluorescent Group

To prepare a fluorogenic peptide ligand which binds to an arbitrary target, we previously succeeded in seeking a fluorogenic ligand to calmodulin using in vitro selection. In this study the environment-sensitive fluorescent group in the selected peptide ligand was replaced with other fluorescent groups to find the possibility to increase the fluorogenic activity. Surface plasmon resonance measurement exhibited that the binding affinity was held even after the replacement. However, the replacement significantly affected the fluorogenic activity. It depended on the kind of incorporated fluorophors and linker length. As a result, the incorporation of 4-N,N-dimethylamino-1,8-naphthalimide enhanced the fluorescence intensity over 100-fold in the presence of target calcium-bound calmodulin. This study demonstrated that the functionality of in vitro selected peptide can be tuned with keeping the binding affinity.

In vitro selection of a photoresponsive peptide aptamer to glutathione-immobilized microbeads.

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.