Yasuko Osakada

Sequence-independent and rapid long-range charge transfer through DNA

Interest in using DNA as a building block for nanoelectronic sensors and devices stems from its efficient hole-conducting properties and the relative ease with which it can be organized into predictable nanometre-sized two- and three-dimensional structures. However, because a hole migrates along DNA through the highest occupied molecular orbital of the guanine bases, its conductivity decreases as the adenine-thymine base-pair content increases. This means that there are limitations on what sequences can be used to construct functional nanoelectronic circuits, particularly those rich in adenine-thymine pairs. Here we show that the charge-transfer efficiency can be dramatically increased in a manner independent of guanine-cytosine content by adjusting the highest occupied molecular orbital level of the adenine-thymine base pair to be closer to that of the guanine-cytosine pair. This is achieved by substituting the N7 nitrogen atom of adenine with a C-H group to give 7-deazaadenine, which does not disturb the complementary base pairing observed in DNA.

Defective axonal transport of Rab7 GTPase results in dysregulated trophic signaling.

Retrograde trophic signaling of nerve growth factor (NGF) supports neuronal survival and differentiation. Dysregulated trophic signaling could lead to various neurological disorders. Charcot-Marie-Tooth type 2B (CMT2B) is one of the most common inherited peripheral neuropathies characterized by severe terminal axonal loss. Genetic analysis of human CMT2B patients has revealed four missense point mutations in Rab7, a small GTPase that regulates late endosomal/lysosomal pathways, but the exact pathological mechanism remains poorly understood. Here, we show that these Rab7 mutants dysregulated axonal transport and diminished the retrograde signaling of NGF and its TrkA receptor. We found that all CMT2B Rab7 mutants were transported significantly faster than Rab7wt in the anterograde direction, accompanied with an increased percentile of anterograde Rab7-vesicles within axons of rat E15.5 dorsal root ganglion (DRG) neurons. In PC12M cells, the CMT2B Rab7 mutants drastically reduced the level of surface TrkA and NGF binding, presumably by premature degradation of TrkA. On the other hand, siRNA knock-down of endogenous Rab7 led to the appearance of large TrkA puncta in enlarged Rab5-early endosomes within the cytoplasm, suggesting delayed TrkA degradation. We also show that CMT2B Rab7 mutants markedly impaired NGF-induced Erk1/2 activation and differentiation in PC12M cells. Further analysis revealed that CMT2B Rab7 mutants caused axonal degeneration in rat E15.5 DRG neurons. We propose that Rab7 mutants induce premature degradation of retrograde NGF-TrkA trophic signaling, which may potentially contribute to the CMT2B disease.

Iridium oxide nanotube electrodes for sensitive and prolonged intracellular measurement of action potentials

Intracellular recording of action potentials is important to understand electrically-excitable cells. Recently, vertical nanoelectrodes have been developed to achieve highly sensitive, minimally invasive, and large scale intracellular recording. It has been demonstrated that the vertical geometry is crucial for the enhanced signal detection. Here we develop nanoelectrodes made up of nanotubes of iridium oxide. When cardiomyocytes are cultured upon those nanotubes, the cell membrane not only wraps around the vertical tubes but also protrudes deep into the hollow center. We show that this geometry enhances cell-electrode coupling and results in measuring much larger intracellular action potentials. The nanotube electrodes afford much longer intracellular access and are minimally invasive, making it possible to achieve stable recording up to an hour in a single session and more than 8 days of consecutive daily recording. This study suggests that the electrode performance can be significantly improved by optimizing the electrode geometry.

Charge transfer through DNA nanoscaled assembly programmable with DNA building blocks

DNA nanostructures based on programmable DNA molecular recognition have been developed, but the nanoelectronics of using DNA is still challenging. A more rapid charge-transfer (CT) process through the DNA nanoassembly is required for further development of programmable DNA nanoelectronics. In this article, we present direct absorption measurements of the long-range CT over a 140-Å DNA assembly based on a GC repetitive sequence constructed by simply mixing DNA building blocks. We show that a CT through DNA nanoscale assembly is possible and programmable with the designed DNA sequence.

Single-Molecule Imaging of NGF Axonal Transport in Microfluidic Devices

Nerve growth factor (NGF) signaling begins at the nerve terminal, where it binds and activates membrane receptors and subsequently carries the cell-survival signal to the cell body through the axon. A recent study revealed that the majority of endosomes contain a single NGF molecule, which makes single-molecule imaging an essential tool for NGF studies. Despite being an increasingly popular technique, single-molecule imaging in live cells is often limited by background fluorescence. Here, we employed a microfluidic culture platform to achieve background reduction for single-molecule imaging in live neurons. Microfluidic devices guide the growth of neurons and allow separately controlled microenvironment for cell bodies or axon termini. Designs of microfluidic devices were optimized and a three-compartment device successfully achieved direct observation of axonal transport of single NGF when quantum dot labeled NGF (Qdot-NGF) was applied only to the distal-axon compartment while imaging was carried out exclusively in the cell-body compartment. Qdot-NGF was shown to move exclusively toward the cell body with a characteristic stop-and-go pattern of movements. Measurements at various temperatures show that the rate of NGF retrograde transport decreased exponentially over the range of 36-14 degrees C. A 10 degrees C decrease in temperature resulted in a threefold decrease in the rate of NGF retrograde transport. Our successful measurements of NGF transport suggest that the microfluidic device can serve as a unique platform for single-molecule imaging of molecular processes in neurons.

Kinetics of charge transfer in DNA containing a mismatch

Charge transfer (CT) in DNA offers a unique approach for the detection of a single-base mismatch in a DNA molecule. While the single-base mismatch would significantly affect the CT in DNA, the kinetic basis for the drastic decrease in the CT efficiency through DNA containing mismatches still remains unclear. Recently, we determined the rate constants of the CT through the fully matched DNA, and we can now estimate the CT rate constant for a certain fully matched sequence. We assumed that further elucidating of the kinetics in mismatched sequences can lead to the discrimination of the DNA single-base mismatch based on the kinetics. In this study, we investigated the detailed kinetics of the CT through DNA containing mismatches and tried to discriminate a mismatch sequence based on the kinetics of the CT in DNA containing a mismatch.

Automated image analysis for tracking cargo transport in axons

The dynamics of cargo movement in axons encodes crucial information about the underlying regulatory mechanisms of the axonal transport process in neurons, a central problem in understanding many neurodegenerative diseases. Quantitative analysis of cargo dynamics in axons usually includes three steps: (1) acquiring time‐lapse image series, (2) localizing individual cargos at each time step, and (3) constructing dynamic trajectories for kinetic analysis. Currently, the later two steps are usually carried out with substantial human intervention. This article presents a method of automatic image analysis aiming for constructing cargo trajectories with higher data processing throughput, better spatial resolution, and minimal human intervention. The method is based on novel applications of several algorithms including 2D kymograph construction, seed points detection, trajectory curve tracing, back‐projection to extract spatial information, and position refining using a 2D Gaussian fitting. This method is sufficiently robust for usage on images with low signal‐to‐noise ratio, such as those from single molecule experiments. The method was experimentally validated by tracking the axonal transport of quantum dot and DiI fluorophore‐labeled vesicles in dorsal root ganglia neurons. Microsc. Res. Tech., 2011.

Hard X-ray-induced optical luminescence via biomolecule-directed metal clusters.

Here, we demonstrate that biomolecule-directed metal clusters are applicable in the study of hard X-ray excited optical luminescence, promising a new direction in the development of novel X-ray-activated imaging probes.

Mechanism of Charge Separation in DNA by Hole Transfer through Consecutive Adenines

To investigate the mechanism of charge separation in DNA with consecutive adenines adjacent to a photosensitizer (Sens), a series of naphthalimide (NI) and 5-bromouracil ((br)U)-modified DNAs were prepared, and the quantum yields of formation of the charge-separated states (Phi) upon photo-excitation of the Sens NI in DNA were measured. The Phi was modulated by the incorporation site of (br)U, which changes the oxidation potential of its complementary A through hydrogen bonding and the hole-transfer rates between adenines. The results were interpreted as charge separation by means of the initial charge transfer between NI in the singlet excited state and the second- and third-nearest adenine to the NI. In addition, the oxidation of the A nearest to NI leads to the rapid charge recombination within a contact ion pair. This suggests that the charge-separation process can be refined to maximize the Phi by putting a redox-inactive spacer base pair between a photosensitizer and an A-T stretch.

Charge separation and photosensitized damage in DNA mediated by naphthalimide, naphthaldiimide, and anthraquinone.

Charge-separation and charge-recombination dynamics and oxidative DNA degradation were investigated for DNA modified with a photosensitizer (Sens) naphthalimide (NI), naphthaldiimide (ND), or anthraquinone (AQ). In all three Sens-modified DNA systems, the formation of long-lived charge-separated states was observed in which the lifetime increased with increasing numbers of A-T base pairs between Sens and the neighboring G-C base pair. The lifetime of the charge-separated state correlated well with the DNA damage yield, indicating that the charge-separated state provides time for the irreversible DNA oxidative damage to occur. The quantum yield of DNA damage was the lowest for ND-modified DNA due to the slow reaction of ND radical anion with molecular oxygen; the process needed to preclude charge recombination. The AQ-modified DNA resulted in the highest charge separation and subsequent DNA damage yield, which would be partly explained by the formation of the spin-forbidden triplet radical ion pairs during charge separation.