Nanotechnologies / Materials
Medical & healthcare, Drug discovery, Materials

Find rare events at single-molecule level
Department of Bionanotechnology, SANKEN (The Institute of Scientific and Industrial Research)
Assistant Professor Yuki Komoto			

https://researchmap.jp/YukiKomoto?lang=en

Professor Masateru Taniguchi			https://researchmap.jp/read0076413?lang=en

Nanotechnologies / Materials

Abstract
Small-molecule drugs targeting DNA and RNA have recently
been developed. To further develop small-molecule drugs, information on the interactions between the drug ligands and basemolecules is indispensable, determined by the base molecule-ligand binding, selectivity, and hydrogen bonding mode. However,these parameters in the solution are unknown at the single-molecule level. Here we show that single-molecule counting based on
combining single-molecule electrical measurements and artificial
intelligence can quantify the abovementioned parameters. By
analyzing a mixed solution of a ligand and base molecules, thebinding selectivity of each of the five ligands to four different basemolecules was quantitatively evaluated by the ratio of the number of the aggregates. Additionally, single-molecule counting andquantum chemical calculations showed that the mode and numberof microscopic hydrogen bonds are ligand-dependent. Thus, single-molecule counting provided quantitative information about theproperties of the aggregate in the solution without ligand incorporation into the DNA or RNA.

discovery. This new nanotechnology can facilitate quantitative
studies on the binding, selectivity, and hydrogen bonding modes
between the base molecules and small-molecule ligands at thesingle-molecule level without using DNA or RNA.

▍ Background & Results
To date, numerous drugs have been developed by targeting
proteins. However, the development of nucleic acid-targeted molecules aimed at DNA and RNA is expected to open new avenues for
drug discovery. These molecules include not only DNA and RNA
but also small molecules. The first step in drug discovery involves
confirming that small molecules selectively and strongly bind tonucleic acids. However, the mode of hydrogen bonding between
small molecules and nucleic acids has not been explored at thesingle-molecule level. Moreover, the rare binding mode, occurring
at low probability, has never been explored.
In this study, we developed a single-molecule counting method,which identifies molecular species at the single-molecule level.
This method combines a single-molecule measurement technique that identifies single molecules by measuring their electricalconductivity with AI that learns current waveforms obtained fromthe measurement. Additionally, employing this single-molecule
counting method, we successfully quantified the binding strength
between guanosine and ligand. Furthermore, this method canquantitatively evaluate the ligand’s selectivity. By utilizing quantumcalculations and statistical analysis of each signal, we estimatedthe metastable aggregation states. This enabled us to estimate thepotential aggregation states of each signal group in solution at thesingle-molecule level. The estimated aggregation states revealedrare event aggregation states that differed from the predictions.

▍ Significance of the research and Future perspective
Combining nucleic acid-targeted small-molecule drug discovery
and single-molecule counting can create a powerful tool in drug

P a t e n t JP7329806, JP6971499, JP6985687, JP6719773, JP662103
Komoto, Yuki; Taniguchi, Masateru et al. Quantitative microscopic observation of base-ligand interactions via hydrogen bonds by single-molecule
Tr e a t i s e
counting. J. Am. Chem. Soc. 2023, 145(2), 1310-1318. doi: 10.1021/jacs.2c11260
UR
L http://www.bionano.sanken.osaka-u.ac.jp/main.html
K e y w o r d single-molecule counting, single-molecule science, nanogap, ligand, DNA