Single-molecule detection of modified amino acid regulating transcriptional activity.

Acetylation of lysine, a component of histones, regulates transcriptional activity. Simple detection methods for acetyl lysine are essential for early diagnosis of diseases and understanding of the physiological effects. We have detected and recognized acetyl lysine at the single-molecule level by combining MCBJ measurement and machine learning.

Solid-State Single-Molecule Sensing with the Electronic Life-Detection Instrument for Enceladus/Europa (ELIE).

Growing evidence of the potential habitability of Ocean Worlds across our solar system is motivating the advancement of technologies capable of detecting life as we know it-sharing a common ancestry or physicochemical origin with life on Earth-or don't know it, representing a distinct emergence of life different than our one known example. Here, we propose the Electronic Life-detection Instrument for Enceladus/Europa (ELIE), a solid-state single-molecule instrument payload that aims to search for life based on the detection of amino acids and informational polymers (IPs) at the parts per billion to trillion level. As a first proof-of-principle in a laboratory environment, we demonstrate the single-molecule detection of the amino acid L-proline at a 10 μM concentration in a compact system.

Single-Molecule Identification of Nucleotides Using a Quantum Computer.

Genomic information is essential for human health. Due to its large volume, genomic information can be potentially computed using quantum computers, which are rapidly developing. Genome analysis using quantum computers can accelerate the development of personalized medicine, innovative drugs, and novel diagnostics based on genomic information.

Direct biomolecule discrimination in mixed samples using nanogap-based single-molecule electrical measurement.

In single-molecule measurements, metal nanogap electrodes directly measure the current of a single molecule. This technique has been actively investigated as a new detection method for a variety of samples. Machine learning has been applied to analyze signals derived from single molecules to improve the identification accuracy.

Quantitative Microscopic Observation of Base-Ligand Interactions via Hydrogen Bonds by Single-Molecule Counting.

Chemical properties have been based on statistical averages since the introduction of Avogadro's number. The lack of suitable methods for counting identified single molecules has posed challenges to counting statistics. The selectivity, affinity, and mode of hydrogen bonding between base and small molecules that make up DNA, which is vital for living organisms, have not yet been revealed at the single molecule level.

Direct observation of DNA alterations induced by a DNA disruptor.

DNA alterations, such as base modifications and mutations, are closely related to the activity of transcription factors and the corresponding cell functions; therefore, detection of DNA alterations is important for understanding their relationships. Particularly, DNA alterations caused by exposure to exogenous molecules, such as nucleic acid analogues for cancer therapy and the corresponding changes in cell functions, are of interest in medicine for drug development and diagnosis purposes. However, detection of comprehensive direct evidence for the relationship of DNA modifications/mutations in genes, their effect on transcription factors, and the corresponding cell functions have been limited.

Single-Molecule Classification of Aspartic Acid and Leucine by Molecular Recognition through Hydrogen Bonding and Time-Series Analysis.

Amino acid detection/identification methods are important for understanding biological systems. In this study, we developed single-molecule measurements for investigating quantum tunneling enhancement by chemical modification and carried out machine learning-based time series analysis for developing accurate amino acid discrimination. We performed single-molecule measurement of L-aspartic acid (Asp) and L-leucine (Leu) with a mercaptoacetic acid (MAA) chemical modified nano-gap.

Review of the use of nanodevices to detect single molecules.

The development of methodologies to identify single molecules and/or to detect/monitor molecular behavior at the single-molecule level is one of the important research topics in chemistry and biology. In this review, we summarized the state-of-the-art of single molecule measurement methods and its latest applications using nanodevices integrated with molecular-size functional nanostructures, nanopores, nanogaps, and nanofluidic channels, which detect differences in chemical species, presence or absence of translational modifications, changes in steric structure, and changes in interactions between molecules. Besides these fundamental analytical achievements of molecular identification abilities, the latest applications include the single-molecule electrical sequencing, disease diagnosis, viral testing, single-molecule drug screening, and environmental monitoring.

Single-molecule RNA sequencing for simultaneous detection of m6A and 5mC.

Epitranscriptomics is the study of RNA base modifications involving functionally relevant changes to the transcriptome. In recent years, epitranscriptomics has been an active area of research. However, a major issue has been the development of sequencing methods to map transcriptome-wide RNA base modifications.

Key aurophilic motif for robust quantum-tunneling-based characterization of a nucleoside analogue marker.

A quantum sequencer offers a scalable electrical platform for single-molecule analysis of genomic events. A thymidine (dT) analog exhibiting uniquely high single-molecule conductance is a key element in capturing DNA synthesis dynamics by serving as a decodable marker for enzymatic labeling of nascent strands. However, the current design strategies of dT analogs that focus on their molecular orbital energy levels require bulky chemical modifications to extend the π-conjugation, which hinders polymerase recognition.

Development of Single-Molecule Electrical Identification Method for Cyclic Adenosine Monophosphate Signaling Pathway.

Cyclic adenosine monophosphate (cAMP) is an important research target because it activates protein kinases, and its signaling pathway regulates the passage of ions and molecules inside a cell. To detect the chemical reactions related to the cAMP intracellular signaling pathway, cAMP, adenosine triphosphate (ATP), adenosine monophosphate (AMP), and adenosine diphosphate (ADP) should be selectively detected. This study utilized single-molecule quantum measurements of these adenosine family molecules to detect their individual electrical conductance using nanogap devices.

Length Discrimination of Homo-oligomeric Nucleic Acids with Single-molecule Measurement.

Single-molecule DNA/RNA sequencing based on single-molecule measurement is a prominent method for higher throughput sequencing. In a previous report, the single-molecule DNA/RNA sequencing method has applied to detect each base-conductance difference in the tunneling current time profiles, and determined the sequence. However, discrimination of identical base lengths has not yet been achieved.

Single-Molecule Counting of Nucleotide by Electrophoresis with Nanochannel-Integrated Nano-Gap Devices.

We utilized electrophoresis to control the fluidity of sample biomolecules in sample aqueous solutions inside the nanochannel for single-molecule detection by using a nanochannel-integrated nanogap electrode, which is composed of a nano-gap sensing electrode, nanochannel, and tapered focusing channel. In order to suppress electro-osmotic flow and thermal convection inside this nanochannel, we optimized the reduction ratios of the tapered focusing channel, and the ratio of inlet 10 μm to outlet 0.5 μm was found to be high performance of electrophoresis with lower concentration of 0.

Detection of an alcohol-associated cancer marker by single-molecule quantum sequencing.

Alcoholic beverages are a well-known risk factor for cancer. N-Ethyl-2'-deoxyguanosine (N-Et-dG) is a promising biomarker for alcohol-associated cancers. However, the lack of a convenient detection method for N-Et-dG hinders the development of practical DNA damage markers.

Time-resolved neurotransmitter detection in mouse brain tissue using an artificial intelligence-nanogap.

The analysis of neurotransmitters in the brain helps to understand brain functions and diagnose Parkinson's disease. Pharmacological inhibition experiments, electrophysiological measurement of action potentials, and mass analysers have been applied for this purpose; however, these techniques do not allow direct neurotransmitter detection with good temporal resolution by using nanometre-sized electrodes. Hence, we developed a method for direct observation of a single neurotransmitter molecule with a gap width of ≤ 1 nm and on the millisecond time scale.

Chemical-Labeling-Assisted Detection of Nucleobase Modifications by Quantum-Tunneling-Based Single-Molecule Sensing.

Quantum-tunneling-based DNA sensing is a single-molecule technique that promises direct mapping of nucleobase modifications. However, its applicability is seriously limited because of the small difference in conductivity between modified and unmodified nucleobases. Herein, a chemical labeling strategy is presented that facilitates the detection of modified nucleotides by quantum tunneling.

Highly Conductive Nucleotide Analogue Facilitates Base-Calling in Quantum-Tunneling-Based DNA Sequencing.

Quantum-tunneling-based DNA sequencing is a single molecular technology that has great potential for achieving facile and high-throughput DNA sequencing. In principle, the sequence of DNA could be read out by the time trace of the tunnel current that can be changed according to molecular conductance of nucleobases passing through nanosized gap electrodes. However, efficient base-calling of four genetic alphabets has been seriously impeded due to the similarity of molecular conductance among canonical nucleotides.

Direct Analysis of Incorporation of an Anticancer Drug into DNA at Single-Molecule Resolution.

Identifying positions at which anticancer drug molecules incorporate into DNA is essential to define mechanisms underlying their activity, but current methodologies cannot yet achieve this. The thymidine fluorine substitution product trifluridine (FTD) is a DNA-damaging anticancer agent thought to incorporate into thymine positions in DNA. This mechanism, however, has not been directly confirmed.

Quantitative analysis of DNA with single-molecule sequencing.

Cancer can be diagnosed by identifying DNA and microRNA base sequences that have the same base length yet differ in a few base sequences, if the abundance ratios of these slightly deviant base sequences can be determined. However, such quantitative analyses cannot be performed using the current DNA sequencers. Here we determine entire base sequences of four types of DNA corresponding to the let-7 microRNA, which is a 22-base cancer marker.

Detection of post-translational modifications in single peptides using electron tunnelling currents.

Post-translational modifications alter the properties of proteins through the cleavage of peptide bonds or the addition of a modifying group to one or more amino acids. These modifications allow proteins to perform their primary biological functions, but single-protein studies of post-translational modifications have been hindered by a lack of suitable analysis methods. Here, we show that single amino acids can be identified using electron tunnelling currents measured as the individual molecules pass through a nanoscale gap between electrodes.

Nonequilibrium Ionic Response of Biased Mechanically Controllable Break Junction (MCBJ) Electrodes.

Novel experimental techniques allow for the manipulation and interrogation of biomolecules between metallic probes immersed in micro/nanofluidic channels. The behavior of ions in response to applied fields is a major issue in the use of these techniques in sensing applications. Here, we experimentally and theoretically elucidate the behavior of background currents in these systems.

Single-molecule electrical random resequencing of DNA and RNA.

Two paradigm shifts in DNA sequencing technologies-from bulk to single molecules and from optical to electrical detection-are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals.

Synthesis, structure, and biological activity of dumbbell-shaped nanocircular RNAs for RNA interference.

RNA interference (RNAi) is one of the most promising new approaches for disease therapy. The design of a dumbbell-shaped nanocircular RNA allows it to act as a short interfering RNA (siRNA) precursor. To optimize the design, we studied the relationship between the nanostructure and RNAi activity by synthesizing various RNA dumbbells.