Shanyu Zhang

Oligosaccharides, polymers of covalently linked monosaccharides, are essential biomolecules mediating diverse biological and physiological processes. Despite their critical roles, the structural complexity of many oligosaccharides poses a notable challenge for comprehensive characterization, limiting our understanding of their biological functions. Nanopores as emerging single-molecule sensors offer promise for oligosaccharide structural analysis. Here, we demonstrate direct discrimination of diverse mono- and oligosaccharides using a hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore integrated with a single phenylboronic acid (PBA) adapter (MspA-90PBA). The high-resolution event signatures generated by MspA-90PBA enable structural analysis of complex oligosaccharides via hydrolysis into smaller fragments, which can be analyzed for either structure profiling or determination. Using a minimized reference database, we achieve structural determination of raffinose, stachyose, and verbascose through nanopore characterization of its hydrolyzed fragments followed by fragment assembly. This work establishes a framework for nanopore-based oligosaccharide analysis, combining single-molecule sensing with a fragment assembly strategy to address the long-standing challenge of glycan structural elucidation.

N-Acetyl modification, a chemical modification commonly found on biomacromolecules, plays a crucial role in the regulation of cell activities and is related to a variety of diseases. However, due to the instability of N-acetyl modification, accurate and rapid identification of N-acetyl modification with a low measurement cost is still technically challenging. Here, based on hydroxylamine deacetylation and nanopore single molecule chemistry, a universal sensing strategy for N-acetyl modification has been developed. Acetohydroxamic acid (AHA), which is produced by the hydroxylamine deacetylation reaction and serves as a reporter for N-acetylation identification, is specifically sensed by a phenylboronic acid (PBA)-modified Mycobacterium smegmatis porin A (MspA). With this strategy, N-acetyl modifications on RNA, DNA, proteins, and glycans were identified, demonstrating its generality. Specifically, histones can be treated with hydroxylamine deacetylation, from which the generated AHA can represent the amount of N-acetyl modification detected by a nanopore sensor. The unique event features of AHA also demonstrate the robustness of sensing against other interfering analytes in the environment.

Natural herbs, which contain pharmacologically active compounds, have been used historically as medicines. Conventionally, the analysis of chemical components in herbal medicines requires time-consuming sample separation and state-of-the-art analytical instruments. Nanopore, a versatile single molecule sensor, might be suitable to identify bioactive compounds in natural herbs. Here, a phenylboronic acid appended Mycobacterium smegmatis porin A (MspA) nanopore is used as a sensor for herbal medicines. A variety of bioactive compounds based on salvianolic acids, including caffeic acid, protocatechuic acid, protocatechualdehyde, salvianic acid A, rosmarinic acid, lithospermic acid, salvianolic acid A and salvianolic acid B are identified. Using a custom machine learning algorithm, analyte identification is performed with an accuracy of 99.0%. This sensing principle is further used with natural herbs such as Salvia miltiorrhiza, Rosemary and Prunella vulgaris. No complex sample separation or purification is required and the sensing device is highly portable.

Natural fruits contain a large variety of cis-diols. However, due to the lack of a high-resolution sensor that can simultaneously identify all cis-diols without a need of complex sample pretreatment, direct and rapid analysis of fruits in a hand-held device has never been previously reported. Nanopore, a versatile single molecule sensor, can be specially engineered to perform this task. A hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore modified with a sole phenylboronic acid (PBA) adapter is prepared. This engineered MspA accurately recognizes 1,2-diphenols, alditols, α-hydroxy acids and saccharides in prune, grape, lemon, different varieties of kiwifruits and commercial juice products. Assisted with a custom machine learning program, an accuracy of 99.3% is reported and the sample pretreatment is significantly simplified. Enantiomers such as DL-malic acids can also be directly identified, enabling sensing of synthetic food additives. Though demonstrated with fruits, these results suggest wide applications of nanopore in food and drug administration uses.

Thyroid hormones (THs) are a variety of iodine-containing hormones that demonstrate critical physiological impacts on cellular activities. The assessment of thyroid function and the diagnosis of thyroid disorders require accurate measurement of TH levels. However, largely due to their structural similarities, the simultaneous discrimination of different THs is challenging. Nanopores, single-molecule sensors with a high resolution, are suitable for this task. In this paper, a hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore containing a single nickel ion immobilized to the pore constriction has enabled simultaneous identification of five representative THs including l-thyroxine (T4), 3,3',5-triiodo-l-thyronine (T3), 3,3',5'-triiodo-l-thyronine (rT3), 3,5-diiodo-l-thyronine (3,5-T2) and 3,3'-diiodo-l-thyronine (3,3'-T2). To automate event classification and avoid human bias, a machine learning algorithm was also developed, reporting an accuracy of 99.0%. This sensing strategy is also applied in the analysis of TH in a real human serum environment, suggesting its potential use in a clinical diagnosis.

Nucleoside drugs, which are analogues of natural nucleosides, have been widely applied in the clinical treatment of viral infections and cancers. The development of nucleoside drugs, repurposing of existing drugs, and combined use of multiple drug types have made the rapid sensing of nucleoside drugs urgently needed. Nanopores are emerging single-molecule sensors that have high resolution to resolve even minor structural differences between chemical compounds. Here, an engineered Mycobacterium smegmatis porin A hetero-octamer was used to perform general nucleoside drug analysis. Ten nucleoside drugs were simultaneously detected and fully discriminated. An accuracy of >99.9% was consequently reported. This sensing capacity was further demonstrated in direct nanopore analysis of ribavirin buccal tablets, confirming its sensing reliability against complex samples and environments. No sample separation is needed, however, significantly minimizing the complexity of the measurement. This technique may inspire nanopore applications in pharmaceutical production and pharmacokinetics measurements.

Natural proteins are composed of 20 proteinogenic amino acids and their post-translational modifications (PTMs). However, due to the lack of a suitable nanopore sensor that can simultaneously discriminate between all 20 amino acids and their PTMs, direct sequencing of protein with nanopores has not yet been realized. Here, we present an engineered hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore containing a sole Ni²⁺ modification. It enables full discrimination of all 20 proteinogenic amino acids and 4 representative modified amino acids, N^ω,N’^ω-dimethyl-arginine (Me-R), O-acetyl-threonine (Ac-T), N⁴-(β-N-acetyl-d-glucosaminyl)-asparagine (GlcNAc-N) and O-phosphoserine (P-S). Assisted by machine learning, an accuracy of 98.6% was achieved. Amino acid supplement tablets and peptidase-digested amino acids from peptides were also analyzed using this strategy. This capacity for simultaneous discrimination of all 20 proteinogenic amino acids and their PTMs suggests the potential to achieve protein sequencing using this nanopore-based strategy.

Nucleotide sugars, the glycosyl donors in the biosynthesis of carbohydrates, are critical ingredients in the growth and development of all living organisms. A variety of nucleotide sugars simultaneously exist in biological samples. They, however, have only minor structural differences, which make them extremely difficult to discriminate. In this work, a phenylboronic acid (PBA)-modified Mycobacterium smegmatis porin A (MspA) hetero-octamer was applied to sense nucleotide sugars. Five representative nucleotide sugars, including guanosine diphosphate mannose (GDP-Man), adenosine diphosphate glucose (ADP-Glc), uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), uridine diphosphate glucose (UDP-Glc), and uridine diphosphate glucoronic acid (UDP-GlcA), were successfully distinguished. A custom machine learning algorithm was also employed to automatically identify events, reporting a general accuracy of 99.4%. This sensing strategy provides a rapid, direct, and accurate method for identifying different nucleotide sugars. However, single-molecule identification of nucleotide sugars has never been previously reported, to the best of our knowledge.

Isomers of some chemical compounds may be dynamically interconvertible. Due to a lack of sensing methods with a sufficient resolution, however, direct monitoring of such processes can be difficult. Engineered Mycobacterium smegmatis porin A (MspA) nanopores can be applied as nanoreactors so that chemical reactions can be directly monitored. Here, an MspA modified with a phenylboronic acid (PBA) adapter was prepared and was used to observe dynamic interconversion between chiral configurations of boronate esters, which appears as telegraphic switching on top of nanopore events. The mechanism of this behavior was further confirmed by trials with different halogenated catechols, dopamine, adenosine, 1,2-propanediol, and (2R,3R)-2,3-butanediol, and its generality has been demonstrated. These results suggest that an engineered MspA possesses an exceptional resolution in its monitoring of chemical reaction processes and may inspire the future design of nanopore small-molecule sensors.

Ribonucleotides, which widely exist in all living organisms and are essential to both physiological and pathological processes, can naturally appear as ribonucleoside mono-, di-, and triphosphates. Natural ribonucleotides can also dynamically switch between different phosphorylated forms, posing a great challenge for sensing. A specially engineered nanopore sensor is promising for full discrimination of all canonical ribonucleoside mono-, di-, and triphosphates. However, such a demonstration has never been reported, due to the lack of a suitable nanopore sensor that has a sufficient resolution. In this work, we utilized a phenylboronic acid (PBA) modified Mycobacterium smegmatis porin A (MspA) hetero-octamer for ribonucleotide sensing. Twelve types of ribonucleotides, including mono-, di-, and triphosphates of cytidine (CMP, CDP, CTP), uridine (UMP, UDP, UTP), adenosine (AMP, ADP, ATP), and guanosine (GMP, GDP, GTP) were simultaneously discriminated. A machine-learning algorithm was also developed, which achieved a general accuracy of 99.9% for ribonucleotide sensing. This strategy was also further applied to identify ribonucleotide components in ATP tablets and injections. This sensing strategy provides a direct, accurate, easy, and rapid solution to characterize ribonucleotide components in different phosphorylated forms.