Wendong Jia

Saccharides play critical roles in many forms of cellular activities. Saccharide structures are however complicated and similar, setting a technical hurdle for direct identification. Nanopores, which are emerging single molecule tools sensitive to minor structural differences between analytes, can be engineered to identity saccharides. A hetero-octameric Mycobacterium smegmatis porin A nanopore containing a phenylboronic acid was prepared, and was able to clearly identify nine monosaccharide types, including D-fructose, D-galactose, D-mannose, D-glucose, L-sorbose, D-ribose, D-xylose, L-rhamnose and N-acetyl-D-galactosamine. Minor structural differences between saccharide epimers can also be distinguished. To assist automatic event classification, a machine learning algorithm was developed, with which a general accuracy score of 0.96 was achieved. This sensing strategy is generally suitable for other saccharide types and may bring new insights to nanopore saccharide sequencing.

Biological nanopores are capable of resolving small analytes down to a monoatomic ion. In this research, tetrachloroaurate(III), a polyatomic ion, is discovered to bind to the methionine residue (M113) of a wild-type α-hemolysin by reversible Au(III)-thioether coordination. However, the cylindrical pore geometry of α-hemolysin generates shallow ionic binding events (~5-6 pA) and may have introduced other undesired interactions. Inspired by nanopore sequencing, a Mycobacterium smegmatis porin A (MspA) nanopore, which possesses a conical pore geometry, is mutated to bind tetrachloroaurate(III). Subsequently, further amplified blockage events (up to ~55 pA) are observed, which report the largest single ion binding event from a nanopore measurement. By taking the embedded Au(III) as an atomic bridge, the MspA nanopore is enabled to discriminate between different biothiols from single molecule readouts. These phenomena suggest that MspA is advantageous for single molecule chemistry investigations and has applications as a hybrid biological nanopore with atomic adaptors.

and a systematically larger event amplitude (up to 10 pA) was observed. The measured rate constant suggests that the coordination of a single ion with an amino acid follows hard-soft-acid-base theory, which has never been systematically validated in the case of a single molecule. By adjusting the measurement pH from 6.8 to 8.0, the duration of a single ion binding event could be modified with a ∼46-fold time extension. The phenomena reported suggest MspA to be a superior engineering template for probing a variety of extremely small analytes, such as monatomic and polyatomic ions, small molecules or chemical intermediates, and the principle of hard-soft-acid-base interaction may be instructive in the pore design.

Chemical reactions of single molecules, caused by rapid formation or breaking of chemical bonds, are difficult to observe even with state-of-the-art instruments. A biological nanopore can be engineered into a single molecule reactor, capable of detecting the binding of a monatomic ion or the transient appearance of chemical intermediates. Pore engineering of this type is however technically challenging, which has significantly restricted further development of this technique. We propose a versatile strategy, "programmable nano-reactors for stochastic sensing" (PNRSS), by which a variety of single molecule reactions of hydrogen peroxide, metal ions, ethylene glycol, glycerol, lactic acid, vitamins, catecholamines or nucleoside analogues can be observed directly. PNRSS presents a refined sensing resolution which can be further enhanced by an artificial intelligence algorithm. Remdesivir, a nucleoside analogue and an investigational anti-viral drug used to treat COVID-19, can be distinguished from its active triphosphate form by PNRSS, suggesting applications in pharmacokinetics or drug screening.