Integrating magnetic tweezers and single-molecule FRET: A comprehensive approach to studying local and global conformational changes simultaneously.

This chapter presents the integration of magnetic tweezers with single-molecule FRET technology, a significant advancement in the study of nucleic acids and other biological systems. We detail the technical aspects, challenges, and current status of this hybrid technique, which combines the global manipulation and observation capabilities of magnetic tweezers with the local conformational detection of smFRET. This innovative approach enhances our ability to analyze and understand the molecular mechanics of biological systems. The chapter serves as our first formal documentation of this method, offering insights and methodologies developed in our laboratory over the past decade.

Single-Molecule Methods to Study Z-DNA Mechanics and Dynamics.

Single-molecule methods are powerful in revealing physical and mechanobiological details about biological phenomena. Here, we describe the single-molecule methods applied to study mechanical properties of Z-DNA and dynamics of the B-Z transition.

Cisplatin fastens chromatin irreversibly even at a high chloride concentration.

Cisplatin is one of the most potent anti-cancer drugs developed so far. Recent studies highlighted several intriguing roles of histones in cisplatin's anti-cancer effect. Thus, the effect of nucleosome formation should be considered to give a better account of the anti-cancer effect of cisplatin. Here we investigated this important issue via single-molecule measurements. Surprisingly, the reduced activity of cisplatin under [NaCl] = 180 mM, corresponding to the total concentration of cellular ionic species, is still sufficient to impair the integrity of a nucleosome by retaining its condensed structure firmly, even against severe mechanical and chemical disturbances. Our finding suggests that such cisplatin-induced fastening of chromatin can inhibit nucleosome remodelling required for normal biological functions. The in vitro chromatin transcription assay indeed revealed that the transcription activity was effectively suppressed in the presence of cisplatin. Our direct physical measurements on cisplatin-nucleosome adducts suggest that the formation of such adducts be the key to the anti-cancer effect by cisplatin.

Z-DNA as a Tool for Nuclease-Free DNA Methyltransferase Assay.

Methylcytosines in mammalian genomes are the main epigenetic molecular codes that switch off the repertoire of genes in cell-type and cell-stage dependent manners. DNA methyltransferases (DMT) are dedicated to managing the status of cytosine methylation. DNA methylation is not only critical in normal development, but it is also implicated in cancers, degeneration, and senescence. Thus, the chemicals to control DMT have been suggested as anticancer drugs by reprogramming the gene expression profile in malignant cells. Here, we report a new optical technique to characterize the activity of DMT and the effect of inhibitors, utilizing the methylation-sensitive B-Z transition of DNA without bisulfite conversion, methylation-sensing proteins, and polymerase chain reaction amplification. With the high sensitivity of single-molecule FRET, this method detects the event of DNA methylation in a single DNA molecule and circumvents the need for amplification steps, permitting direct interpretation. This method also responds to hemi-methylated DNA. Dispensing with methylation-sensitive nucleases, this method preserves the molecular integrity and methylation state of target molecules. Sparing methylation-sensing nucleases and antibodies helps to avoid errors introduced by the antibody's incomplete specificity or variable activity of nucleases. With this new method, we demonstrated the inhibitory effect of several natural bio-active compounds on DMT. All taken together, our method offers quantitative assays for DMT and DMT-related anticancer drugs.

Sequence-dependent cost for Z-form shapes the torsion-driven B-Z transition via close interplay of Z-DNA and DNA bubble.

Despite recent genome-wide investigations of functional DNA elements, the mechanistic details about their actions remain elusive. One intriguing possibility is that DNA sequences with special patterns play biological roles, adopting non-B-DNA conformations. Here we investigated dynamics of thymine-guanine (TG) repeats, microsatellite sequences and recurrently found in promoters, as well as cytosine-guanine (CG) repeats, best-known Z-DNA forming sequence, in the aspect of Z-DNA formation. We measured the energy barriers of the B-Z transition with those repeats and discovered the sequence-dependent penalty for Z-DNA generates distinctive thermodynamic and kinetic features in the torque-induced transition. Due to the higher torsional stress required for Z-form in TG repeats, a bubble could be induced more easily, suppressing Z-DNA induction, but facilitate the B-Z interconversion kinetically at the transition midpoint. Thus, the Z-form by TG repeats has advantages as a torsion buffer and bubble selector while the Z-form by CG repeats likely behaves as torsion absorber. Our statistical physics model supports quantitatively the populations of Z-DNA and reveals the pivotal roles of bubbles in state dynamics. All taken together, a quantitative picture for the transition was deduced within the close interplay among bubbles, plectonemes and Z-DNA.

Highly Uniform Resistive Switching Performances Using Two-Dimensional Electron Gas at a Thin-Film Heterostructure for Conductive Bridge Random Access Memory.

This research demonstrates, for the first time, the development of highly uniform resistive switching devices with self-compliance current for conductive bridge random access memory using two-dimensional electron gas (2DEG) at the interface of an Al2O3/TiO2 thin-film heterostructure via atomic layer deposition (ALD). The cell is composed of Cu/Ti/Al2O3/TiO2, where Cu/Ti and Al2O3 overlayers are used as the active/buffer metals and solid electrolyte, respectively, and the 2DEG at the interface of Al2O3/TiO2 heterostructure, grown by the ALD process, is adopted as a bottom electrode. The Cu/Ti/Al2O3/TiO2 device shows reliable resistive switching characteristics with excellent uniformity under a repetitive voltage sweep (direct current sweep). Furthermore, it exhibits a cycle endurance over 107 cycles under short pulse switching. Remarkably, a reliable operation of multilevel data writing is realized up to 107 cycles. The data retention time is longer than 106 s at 85 °C. The uniform resistance switching characteristics are achieved via the formation of small (∼a few nm width) Cu filament with a short tunnel gap (<0.5 nm) owing to the 2DEG at the Al2O3/TiO2 interface. The performance and operation scheme of this device may be appropriate in neuromorphic applications.

Field-Effect Device Using Quasi-Two-Dimensional Electron Gas in Mass-Producible Atomic-Layer-Deposited Al2O3/TiO2 Ultrathin (<10 nm) Film Heterostructures.

We report the field-effect transistors using quasi-two-dimensional electron gas generated at an ultrathin (∼10 nm) Al2O3/TiO2 heterostructure interface grown via atomic layer deposition (ALD) on a SiO2/Si substrate without using a single crystal substrate. The 2DEG at the Al2O3/TiO2 interface originates from oxygen vacancies generated at the surface of the TiO2 bottom layer during ALD of the Al2O3 overlayer. High-density electrons (∼1014 cm-2) are confined within a ∼2.2 nm distance from the Al2O3/TiO2 interface, resulting in a high on-current of ∼12 µA/µm. The ultrathin TiO2 bottom layer is easy to fully deplete, allowing an extremely low off-current, a high on/off current ratio over 108, and a low subthreshold swing of ∼100 mV/decade. Via the implementation of ALD, a mature thin-film process can facilitate mass production as well as three-dimensional integration of the devices.