Unique Electrical Signature of Phosphate for Specific Single-Molecule Detection of Peptide Phosphorylation.

Single-molecule measurements of biomaterials bring novel insights into cellular events. For almost all of these events, post-translational modifications (PTMs), which alter the properties of proteins through their chemical modifications, constitute essential regulatory mechanisms. However, suitable single-molecule methodology to study PTMs is very limited. Here we show single-molecule detection of peptide phosphorylation, an archetypal PTM, based on electrical measurements. We found that the phosphate group stably bridges a nanogap between metal electrodes and exhibited high electrical conductance, which enables specific single-molecule detection of peptide phosphorylation. The present methodology paves the way to single-molecule studies of PTMs, such as single-molecule kinetics for enzymatic modification of proteins as shown here.

Single-molecule junction spontaneously restored by DNA zipper.

The electrical properties of DNA have been extensively investigated within the field of molecular electronics. Previous studies on this topic primarily focused on the transport phenomena in the static structure at thermodynamic equilibria. Consequently, the properties of higher-order structures of DNA and their structural changes associated with the design of single-molecule electronic devices have not been fully studied so far. This stems from the limitation that only extremely short DNA is available for electrical measurements, since the single-molecule conductance decreases sharply with the increase in the molecular length. Here, we report a DNA zipper configuration to form a single-molecule junction. The duplex is accommodated in a nanogap between metal electrodes in a configuration where the duplex is perpendicular to the nanogap axis. Electrical measurements reveal that the single-molecule junction of the 90-mer DNA zipper exhibits high conductance due to the delocalized π system. Moreover, we find an attractive self-restoring capability that the single-molecule junction can be repeatedly formed without full structural breakdown even after electrical failure. The DNA zipping strategy presented here provides a basis for novel designs of single-molecule junctions.

Elementary processes of DNA surface hybridization resolved by single-molecule kinetics: implication for macroscopic device performance.

Direct monitoring of single-molecule reactions has recently become a promising means of mechanistic investigation. However, the resolution of reaction pathways from single-molecule experiments remains elusive, primarily because of interference from extraneous processes such as bulk diffusion. Herein, we report a single-molecule kinetic investigation of DNA hybridization on a metal surface, as an example of a bimolecular association reaction. The tip of the scanning tunneling microscope (STM) was functionalized with single-stranded DNA (ssDNA), and hybridization with its complementary strand on an Au(111) surface was detected by the increase in the electrical conductance associated with the electron transport through the resulting DNA duplex. Kinetic analyses of the conductance changes successfully resolved the elementary processes, which involve not only the ssDNA strands and their duplex but also partially hybridized intermediate strands, and we found an increase in the hybridization efficiency with increasing the concentration of DNA in contrast to the knowledge obtained previously by conventional ensemble measurements. The rate constants derived from our single-molecule studies provide a rational explanation of these findings, such as the suppression of DNA melting on surfaces with higher DNA coverage. The present methodology, which relies on intermolecular conductance measurements, can be extended to a range of single-molecule reactions and to the exploration of novel chemical syntheses.

Kinetic investigation of a chemical process in single-molecule junction.

Herein, we report on the kinetic investigation for the breakdown of single-molecule junctions. Current through the junctions was measured as a function of time to elucidate their lifetimes. The analysis of the lifetimes revealed that the breakdown reaction obeys first-order reaction kinetics, and the rate constants determined from the analysis were found to reflect the stability of the junctions.