Mehmet C. Tarhan

A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging

Single-molecule localization microscopy is used to construct super-resolution images, but generally requires prior intense laser irradiation and in some cases additives, such as thiols, to induce on-off switching of fluorophores. These requirements limit the potential applications of this methodology. Here, we report a first-in-class spontaneously blinking fluorophore based on an intramolecular spirocyclization reaction. Optimization of the intramolecular nucleophile and rhodamine-based fluorophore (electrophile) provide a suitable lifetime for the fluorescent open form, and equilibrium between the open form and the non-fluorescent closed form. We show that this spontaneously blinking fluorophore is suitable for single-molecule localization microscopy imaging deep inside cells and for tracking the motion of structures in living cells. We further demonstrate the advantages of this fluorophore over existing methodologies by applying it to nuclear pore structures located far above the coverslip with a spinning-disk confocal microscope and for repetitive time-lapse super-resolution imaging of microtubules in live cells for up to 1 h.

Simultaneous and bidirectional transport of kinesin-coated microspheres and dynein-coated microspheres on polarity-oriented microtubules.

Artificial nanotransport systems inspired by intracellular transport processes have been investigated for over a decade using the motor protein kinesin and microtubules. However, only unidirectional cargo transport has been achieved for the purpose of nanotransport in a microfluidic system. Here, we demonstrate bidirectional nanotransport by integrating kinesin and dynein motor proteins. Our molecular system allows microtubule orientation of either polarity in a microfluidic channel to construct a transport track. Each motor protein acts as a nanoactuators that transports microspheres in opposite directions determined by the polarity of the oriented microtubules: kinesin‐coated microspheres move toward the plus end of microtubules, whereas dynein‐coated microspheres move toward the minus end. We demonstrate both unidirectional and bidirectional transport using kinesin‐ and dynein‐coated microspheres on microtubules oriented and glutaraldehyde‐immobilized in a microfluidic channel. Tracking and statistical analysis of microsphere movement demonstrate that 87–98% of microspheres move in the designated direction at a mean velocity of 0.22–0.28 µm/s for kinesin‐coated microspheres and 0.34–0.39 µm/s for dynein‐coated microspheres. This bidirectional nanotransport goes beyond conventional unidirectional transport to achieve more complex artificial nanotransport in vitro. Biotechnol. Biotechnol. Bioeng. 2008;101: 1–8.

DNA molecule manipulation by motor proteins for analysis at the single-molecule level

AbstractMassively parallel and individual DNA manipulation for analysis has been demonstrated by designing a fully self-assembled molecular system using motor proteins. DNA molecules were immobilized by trapping in a polyacrylamide gel replica, and were digested by a restriction enzyme, XhoI, for DNA analysis. One end of the λDNA was modified with biotin and the other end was modified with digoxin molecules by fragment labeling and ligation methods. The digoxin-functionalized end was immobilized on a glass surface coated with anti-digoxigenin antibody. The biotinylated end was freely suspended and experienced Brownian motion in a buffer solution. The free end was attached to a biotinylated microtubule via avidin–biotin biding and the DNA was stretched by a kinesin-based gliding assay. A stretched DNA molecule was fixed between the gel and coverslip to observe the cleavage of the DNA by the enzyme, which was supplied through the gel network structure. This simple process flow from DNA manipulation to analysis offers a new method of performing molecular surgery at the single-molecule scale. FigureDNA molecule manipulation by motor proteins for analysis at the single-molecule level

Specific Transport of Target Molecules by Motor Proteins in Microfluidic Channels

Direct transport powered by motor proteins can alleviate the challenges presented by miniaturization of microfluidic systems. There have been several recent attempts to build motor-protein-driven transport systems based on simple capturing or transport mechanisms. However, to achieve a multifunctional device for practical applications, a more complex sorting/transport system should be realized. Herein, the proof of concept of a sorting device employing selective capture of distinct target molecules and transport of the sorted molecules to different predefined directions is presented. By combining the bottom-up functionality of biological systems with the top-down handling capabilities of micro-electromechanical systems technology, highly selective molecular recognition and motor-protein-based transport is integrated in a microfluidic channel network.

Sorting and direct transportation of target molecules by bio-molecular selectivity and motor function

A bio-hybrid microsystem for sorting and carrying target molecules by bio-functional molecules is presented. Different target molecules, biotin-4-fluorescein and Rabbit anti-mouse IgG labeled with Alexa Fluor 568, were selectively attached on different beads (Streptavidin-coated and protein A-coated beads). Target molecules were separately transported by a motor protein system (kinesin on beads / microtubules on chip) without any liquid manipulation proving the feasibility of multiple target molecules on multiple beads sorter.

Real-time mechanical characterization of DNA degradation under therapeutic X-rays and its theoretical modeling

The killing of tumor cells by ionizing radiation beams in cancer radiotherapy is currently based on a rather empirical understanding of the basic mechanisms and effectiveness of DNA damage by radiation. By contrast, the mechanical behaviour of DNA encompassing sequence sensitivity and elastic transitions to plastic responses is much better understood. A novel approach is proposed here based on a micromechanical Silicon Nanotweezers device. This instrument allows the detailed biomechanical characterization of a DNA bundle exposed to an ionizing radiation beam delivered here by a therapeutic linear particle accelerator (LINAC). The micromechanical device endures the harsh environment of radiation beams and still retains molecular-level detection accuracy. In this study, the first real-time observation of DNA damage by ionizing radiation is demonstrated. The DNA bundle degradation is detected by the micromechanical device as a reduction of the bundle stiffness, and a theoretical model provides an interpretation of the results. These first real-time observations pave the way for both fundamental and clinical studies of DNA degradation mechanisms under ionizing radiation for improved tumor treatment.

A rapid and practical technique for real-time monitoring of biomolecular interactions using mechanical responses of macromolecules

Monitoring biological reactions using the mechanical response of macromolecules is an alternative approach to immunoassays for providing real-time information about the underlying molecular mechanisms. Although force spectroscopy techniques, e.g. AFM and optical tweezers, perform precise molecular measurements at the single molecule level, sophisticated operation prevent their intensive use for systematic biosensing. Exploiting the biomechanical assay concept, we used micro-electro mechanical systems (MEMS) to develop a rapid platform for monitoring bio/chemical interactions of bio macromolecules, e.g. DNA, using their mechanical properties. The MEMS device provided real-time monitoring of reaction dynamics without any surface or molecular modifications. A microfluidic device with a side opening was fabricated for the optimal performance of the MEMS device to operate at the air-liquid interface for performing bioassays in liquid while actuating/sensing in air. The minimal immersion of the MEMS device in the channel provided long-term measurement stability (>10 h). Importantly, the method allowed monitoring effects of multiple solutions on the same macromolecule bundle (demonstrated with DNA bundles) without compromising the reproducibility. We monitored two different types of effects on the mechanical responses of DNA bundles (stiffness and viscous losses) exposed to pH changes (2.1 to 4.8) and different Ag+ concentrations (1 μM to 0.1 M).

Carrying Target Molecules on Beads by Bio Molecular Motors

A motor protein system, kinesin/microtubule, transported target molecules specifically attached on bead surfaces. The target molecule, biotin-4-fluorescein, was specifically immobilized on streptavidin-coated beads, on which kinesin molecules were also attached. The target molecules were successfully transported along microtubules immobilized on a glass surface. The activity of kinesin was not disturbed by co-existing target molecules. The result has significant implication to sort out any target molecule selectively from a sample solution by a nano-scale transport system driven by the kinesin/microtubule system.

Point-of-Care (POC) Devices by Means of Advanced MEMS

Microelectromechanical systems (MEMS) have become an invaluable technology to advance the development of point-of-care (POC) devices for diagnostics and sample analyses. MEMS can transform sophisticated methods into compact and cost-effective microdevices that offer numerous advantages at many levels. Such devices include microchannels, microsensors, etc., that have been applied to various miniaturized POC products. Here we discuss some of the recent advances made in the use of MEMS devices for POC applications.

Pick‐and‐Place Assembly of Single Microtubules

Intracellular transport is affected by the filament network in the densely packed cytoplasm. Biophysical studies focusing on intracellular transport based on microtubule-kinesin system frequently use in vitro motility assays, which are performed either on individual microtubules or on random (or simple) microtubule networks. Assembling intricate networks with high flexibility requires the manipulation of 25 nm diameter microtubules individually, which can be achieved through the use of pick-and-place assembly. Although widely used to assemble tiny objects, pick-and-place is not a common practice for the manipulation of biological materials. Using the high-level handling capabilities of microelectromechanical systems (MEMS) technology, tweezers are designed and fabricated to pick and place single microtubule filaments. Repeated picking and placing cycles provide a multilayered and multidirectional microtubule network even for different surface topographies. On-demand assembly of microtubules forms crossings at desired angles for biophysical studies as well as complex networks that can be used as nanotransport systems.