Nanopore signal deviations from pseudouridine modifications in RNA are sequence-specific: quantification requires dedicated synthetic controls.

Chemical modifications to mRNA respond dynamically to environmental cues and are important modulators of gene expression. Nanopore direct RNA sequencing has been applied for assessing the presence of pseudouridine (ψ) modifications through basecalling errors and signal analysis. These approaches strongly depend on the sequence context around the modification, and the occupancies derived from these measurements are not quantitative. In this work, we combine direct RNA sequencing of synthetic RNAs bearing site-specific modifications and supervised machine learning models (ModQuant) to achieve near-analytical, site-specific ψ quantification. Our models demonstrate that the ionic current signal features important for accurate ψ classification are sequence dependent and encompass information extending beyond n + 2 and n - 2 nucleotides from the ψ site. This is contradictory to current models, which assume that accurate ψ classification can be achieved with signal information confined to the 5-nucleotide k-mer window (n + 2 and n - 2 nucleotides from the ψ site). We applied our models to quantitatively profile ψ occupancy in five mRNA sites in datasets from seven human cell lines, demonstrating conserved and variable sites. Our study motivates a wider pipeline that uses ground-truth RNA control sets with site-specific modifications for quantitative profiling of RNA modifications. The ModQuant pipeline and guide are freely available at https://github.com/wanunulab/ModQuant .

The first Chromosomal-level genome assembly of Sageretia thea using Nanopore long reads and Pore-C technology.

Sageretia thea, a notable species within the mock buckthorn genus, is recognized for its intriguing biogeographical distribution and diverse medicinal properties. Despite this significance, genomic studies on S. thea are still in the nascent stages. We present the first chromosome-level genome assembly of S. thea that was generated using a combination of Oxford Nanopore long-read and Illumina short-read sequencing technologies complemented by Pore-C chromatin conformation capture. The genome assembly had a size of 197.8 Mb with 12 chromosomal scaffolds and a scaffold N50 length of 15.9 Mb. A total of 25,434 protein-coding genes were identified and functionally annotated, and the gene model indicated 96.5% complete eukaryotic BUSCOs. Additionally, orthologous gene profiling and synteny analysis were performed to elucidate the evolutionary relationships within the Rhamnaceae family and Rosales. This high-quality chromosomal genome is the first genomic view of S. thea, which will serve as the basis for future studies on its biological and medicinal properties, and evolutionary history.

Registration of activity of a single molecule of horseradish peroxidase using a detector based on a solid-state nanopore.

This work demonstrates the use of a solid-state nanopore detector to monitor the activity of a single molecule of a model enzyme, horseradish peroxidase (HRP). This detector includes a measuring cell, which is divided into cis- and trans- chambers by a silicon nitride chip (SiN structure) with a nanopore of 5 nm in diameter. To entrap a single HRP molecule into the nanopore, an electrode had been placed into the cis-chamber; HRP solution was added into this chamber after application of a negative voltage. The reaction of the HRP substrate, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), oxidation by the enzyme molecule was performed in the presence of hydrogen peroxide. During this reaction, the functioning of a single HRP molecule, entrapped in the nanopore, was monitored by recording the time dependence of the ion current flowing through the nanopore. The approach proposed in our work is applicable for further studies of functioning of various enzymes at the level of single molecules, and this is an important step in the development of single-molecule enzymology.

Multi-pass, single-molecule nanopore reading of long protein strands.

The ability to sequence single protein molecules in their native, full-length form would enable a more comprehensive understanding of proteomic diversity. Current technologies, however, are limited in achieving this goal1,2. Here, we establish a method for the long-range, single-molecule reading of intact protein strands on a commercial nanopore sensor array. By using the ClpX unfoldase to ratchet proteins through a CsgG nanopore3,4, we provide single-molecule evidence that ClpX translocates substrates in two-residue steps. This mechanism achieves sensitivity to single amino acids on synthetic protein strands hundreds of amino acids in length, enabling the sequencing of combinations of single-amino-acid substitutions and the mapping of post-translational modifications, such as phosphorylation. To enhance classification accuracy further, we demonstrate the ability to reread individual protein molecules multiple times, and we explore the potential for highly accurate protein barcode sequencing. Furthermore, we develop a biophysical model that can simulate raw nanopore signals a priori on the basis of residue volume and charge, enhancing the interpretation of raw signal data. Finally, we apply these methods to examine full-length, folded protein domains for complete end-to-end analysis. These results provide proof of concept for a platform that has the potential to identify and characterize full-length proteoforms at single-molecule resolution.

Blobs form during the single-file transport of proteins across nanopores.

The transport of biopolymers across nanopores is an important biological process currently under investigation for the rapid analysis of DNA and proteins. While the transport of DNA is generally understood, methods to induce unfolded protein translocation have only recently been discovered (Yu et al., 2023, Sauciuc et al., 2023). Here, we found that during electroosmotically driven translocation of polypeptides, blob-like structures typically form inside nanopores, often obstructing their transport and preventing addressing individual amino acids. This is in contrast with the electrophoretic transport of DNA, where the formation of such structures has not been reported. Comparisons between different nanopore sizes and shapes and modifications by different surface chemistries allowed formulating a mechanism for blob formation. We also show that single-file transport can be achieved by using 1) nanopores that have an entry and an internal diameter smaller than the persistence length of the polymer, 2) nanopores with a nonsticky (i.e., nonaromatic) inner surface, and 3) moderate translocation velocities. These experiments provide a basis for understanding polypeptide transport under confinement and for improving the design and engineering of nanopores for protein analysis.

Seasonal dynamics, Leishmania diversity, and nanopore-based metabarcoding of blood meal origins in Culicoides spp. in the newly emerging focus of leishmaniasis in Northern Thailand.

BACKGROUND: Clinical cases of leishmaniasis caused by Leishmania (Mundinia) parasites have been increasingly reported in Southeast Asia, particularly Thailand. Recent evidence has shown that Leishmania (Mundinia) parasites successfully developed into infective metacyclic promastigotes in Culicoides biting midges, strongly supporting their putative role in disease transmission. However, Culicoides diversity, host preference, and Leishmania prevalence in endemic areas remain largely unknown. METHODS: We investigated the seasonal dynamics, infection prevalence, and blood meal identification of Culicoides collected from the emerging focus of visceral leishmaniasis in Lampang Province, Northern Thailand, during 2021-2023. Midge samples were molecularly screened for Leishmania using SSU rRNA-qPCR and ITS1-PCR, followed by Sanger plasmid sequencing, and parasite haplotype diversity was analyzed. Host blood meal origins were comparatively identified using host-specific Cytb-PCRs and a nanopore-based metabarcoding approach. RESULTS: A total of 501 parous and gravid females and 46 blood-engorged ones belonging to at least 17 species of five subgenera (Remmia, Trithecoides, Avaritia, Hoffmania, and Meijerehelea) and two species groups (Shortti and Calvipalpis) were collected with temporal differences in abundance. Leishmania was detected by SSU rRNA-qPCR in 31 samples of at least 11 midge species, consisting of Culicoides oxystoma, C. guttifer, C. orientalis, C. mahasarakhamense, C (Trithecoides) spp., C. innoxius, C. shortti, C. arakawae, C. sumatrae, C. actoni, and C. fulvus, with the overall infection prevalence of 5.7%. The latter six species represent the new records as putative leishmaniasis vectors in Northern Thailand. The ITS1-PCR and plasmid sequencing revealed that Leishmania martiniquensis was predominantly identified in all qPCR-positive species, whereas L. orientalis was identified only in three C. oxystoma samples. The most dominant haplotype of L. martiniquensis in Thailand was genetically intermixed with those from other geographical regions, confirming its globalization. Neutrality test statistics were also significantly negative on regional and country-wide scales, suggesting rapid population expansion or selective sweeps. Nanopore-based blood meal analysis revealed that most Culicoides species are mammalophilic, with peridomestic and wild mammals (cow, pig, deer, and goat-like species) and humans as hosts, while C. guttifer and C. mahasarakhamense fed preferentially on chickens. CONCLUSIONS: This study revealed seasonal dynamics and sympatric circulation of L. martiniquensis and L. orientalis in different species of Culicoides. Evidence of human blood feeding was also demonstrated, implicating Culicoides as putative vectors of human leishmaniasis in endemic areas. Further research is therefore urgently needed to develop vector control strategies and assess the infection status of their reservoir hosts to effectively minimize disease transmission.

Simultaneous Identification of Vitamins B1, B3, B5, and B6 by an Engineered Nanopore.

Vitamin Bs, a group of water-soluble compounds, are essential nutrients for almost all living organisms. However, due to their structural heterogeneity, rapid and simultaneous analysis of multiple vitamin Bs is still challenging. In this paper, it is discovered that a hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore containing a sole nickel ion-bound nitrilotriacetic acid (NTA-Ni) adapter at its pore constriction is suitable for the simultaneous sensing of different vitamin Bs, including vitamin B1 (thiamine), vitamin B3 (nicotinic acid and nicotinamide), vitamin B5 (pantothenic acid), and vitamin B6 (pyridoxine, pyridoxal, and pyridoxamine). Assisted by a custom machine learning algorithm, all seven vitamin Bs can be fully distinguished, reporting a general accuracy of 99.9%. This method was further validated in the rapid analysis of commercial cosmetics and natural food, suggesting its potential uses in food and drug administration.

Nucleosomal DNA unwinding pathway through canonical and non-canonical histone disassembly.

The nucleosome including H2A.B, a mammalian-specific H2A variant, plays pivotal roles in spermatogenesis, embryogenesis, and oncogenesis, indicating unique involvement in transcriptional regulation distinct from canonical H2A nucleosomes. Despite its significance, the exact regulatory mechanism remains elusive. This study utilized solid-state nanopores to investigate DNA unwinding dynamics, applying local force between DNA and histones. Comparative analysis of canonical H2A and H2A.B nucleosomes demonstrated that the H2A.B variant required a lower voltage for complete DNA unwinding. Furthermore, synchronization analysis and molecular dynamics simulations indicate that the H2A.B variant rapidly unwinds DNA, causing the H2A-H2B dimer to dissociate from DNA immediately upon disassembly of the histone octamer. In contrast, canonical H2A nucleosomes unwind DNA at a slower rate, suggesting that the H2A-H2B dimer undergoes a state of stacking at the pore. These findings suggest that nucleosomal DNA in the H2A.B nucleosomes undergoes a DNA unwinding process involving histone octamer disassembly distinct from that of canonical H2A nucleosomes, enabling smoother unwinding. The integrated approach of MD simulations and nanopore measurements is expected to evolve into a versatile tool for studying molecular interactions, not only within nucleosomes but also through the forced dissociation of DNA-protein complexes.

Estimates of Sequences with Ultralong and Short CDR3s in the Bovine IgM B Cell Receptor Repertoire Using the Long-read Oxford Nanopore MinION Platform.

Cattle produce Abs with an H chain ultralong CDR3 (40-70 aa). These Abs have been shown to have features such as broad neutralization of viruses and are investigated as human therapeutics. A common issue in sequencing the bovine BCR repertoire is the sequence length required to capture variable (V) and isotype gene information. This study aimed to assess the use of Oxford Nanopore Technologies' MinION platform to perform IgM BCR repertoire sequencing to assess variation in the percentage of ultralong CDR3s among dairy cattle. Blood was collected from nine Holstein heifers. B cells were isolated using magnetic bead-based separation, RNA was extracted, and IgM+ transcripts were amplified using PCR and sequenced using a MinION R10.4 flow cell. The distribution of CDR3 lengths was trimodal, and the percentage of ultralong CDR3s ranged among animals from 2.32 to 20.13% in DNA sequences and 1.56% to 17.02% in productive protein sequences. V segment usage varied significantly among heifers. Segment IGHV1-7, associated with ultralong CDR3s, was used in 5.8-24.2% of sequences; usage was positively correlated with ultralong CDR3 production (r = 0.99, p < 0.01). To our knowledge, this is the first study to sequence the bovine BCR repertoire using Oxford Nanopore Technologies and demonstrates the potential for cost-efficient long-read repertoire sequencing in cattle without assembly. Findings from this study support literature describing the distribution of length and percentage of ultralong CDR3s. Future studies will investigate changes in the bovine BCR repertoire associated with age, antigenic exposure, and genetics.

Primed and ready: nanopore metabarcoding can now recover highly accurate consensus barcodes that are generally indel-free.

BACKGROUND: DNA metabarcoding applies high-throughput sequencing approaches to generate numerous DNA barcodes from mixed sample pools for mass species identification and community characterisation. To date, however, most metabarcoding studies employ second-generation sequencing platforms like Illumina, which are limited by short read lengths and longer turnaround times. While third-generation platforms such as the MinION (Oxford Nanopore Technologies) can sequence longer reads and even in real-time, application of these platforms for metabarcoding has remained limited possibly due to the relatively high read error rates as well as the paucity of specialised software for processing such reads. RESULTS: We show that this is no longer the case by performing nanopore-based, cytochrome c oxidase subunit I (COI) metabarcoding on 34 zooplankton bulk samples, and benchmarking the results against conventional Illumina MiSeq sequencing. Nanopore R10.3 sequencing chemistry and super accurate (SUP) basecalling model reduced raw read error rates to ~ 4%, and consensus calling with amplicon_sorter (without further error correction) generated metabarcodes that were ≤ 1% erroneous. Although Illumina recovered a higher number of molecular operational taxonomic units (MOTUs) than nanopore sequencing (589 vs. 471), we found no significant differences in the zooplankton communities inferred between the sequencing platforms. Importantly, 406 of 444 (91.4%) shared MOTUs between Illumina and nanopore were also found to be free of indel errors, and 85% of the zooplankton richness could be recovered after just 12-15 h of sequencing. CONCLUSION: Our results demonstrate that nanopore sequencing can generate metabarcodes with Illumina-like accuracy, and we are the first study to show that nanopore metabarcodes are almost always indel-free. We also show that nanopore metabarcoding is viable for characterising species-rich communities rapidly, and that the same ecological conclusions can be obtained regardless of the sequencing platform used. Collectively, our study inspires confidence in nanopore sequencing and paves the way for greater utilisation of nanopore technology in various metabarcoding applications.

Nanopore ion sources deliver individual ions of amino acids and peptides directly into high vacuum.

Electrospray ionization is widely used to generate vapor phase ions for analysis by mass spectrometry in proteomics research. However, only a small fraction of the analyte enters the mass spectrometer due to losses that are fundamentally linked to the use of a background gas to stimulate the generation of ions from electrosprayed droplets. Here we report a nanopore ion source that delivers ions directly into high vacuum from aqueous solutions. The ion source comprises a pulled quartz pipette with a sub-100 nm opening. Ions escape an electrified meniscus by ion evaporation and travel along collisionless trajectories to the ion detector. We measure mass spectra of 16 different amino acid ions, post-translationally modified variants of glutathione, and the peptide angiotensin II, showing that these analytes can be emitted as desolvated ions. The emitted current is composed of ions rather than charged droplets, and more than 90% of the current can be recovered in a distant collector.

ReadCurrent: a VDCNN-based tool for fast and accurate nanopore selective sequencing.

Nanopore selective sequencing allows the targeted sequencing of DNA of interest using computational approaches rather than experimental methods such as targeted multiplex polymerase chain reaction or hybridization capture. Compared to sequence-alignment strategies, deep learning (DL) models for classifying target and nontarget DNA provide large speed advantages. However, the relatively low accuracy of these DL-based tools hinders their application in nanopore selective sequencing. Here, we present a DL-based tool named ReadCurrent for nanopore selective sequencing, which takes electric currents as inputs. ReadCurrent employs a modified very deep convolutional neural network (VDCNN) architecture, enabling significantly lower computational costs for training and quicker inference compared to conventional VDCNN. We evaluated the performance of ReadCurrent across 10 nanopore sequencing datasets spanning human, yeasts, bacteria, and viruses. We observed that ReadCurrent achieved a mean accuracy of 98.57% for classification, outperforming four other DL-based selective sequencing methods. In experimental validation that selectively sequenced microbial DNA from human DNA, ReadCurrent achieved an enrichment ratio of 2.85, which was higher than the 2.7 ratio achieved by MinKNOW using the sequence-alignment strategy. In summary, ReadCurrent can rapidly classify target and nontarget DNA with high accuracy, providing an alternative in the toolbox for nanopore selective sequencing. ReadCurrent is available at https://github.com/Ming-Ni-Group/ReadCurrent.

Highly Sensitive Detection of Tumor Cell-Derived Exosomes Using Solid-State Nanopores Assisted with a Slight Salt Gradient.

As an important biomarker, tumor cell-derived exosomes have substantial application prospects in early cancer screening and diagnosis. However, the unsatisfactory sensitivity and complicated sample pretreatment processes of conventional detection approaches have limited their use in clinical diagnosis. Nanopore sensors, as a highly sensitive, label-free, single-molecule technology, are widely utilized in molecule and bioparticle detection. Nevertheless, the exosome capture rate through nanopores is extremely low due to the low surface charge densities of exosomes and the effects of electrolyte concentration on their structural stability, thereby reducing the detection throughput. Here, we report an approach to improve the capture rate of exosome translocations using silicon nitride (SiNx) nanopores assisted by a slight salt electrolyte gradient. Improvements in exosome translocation event frequency are assessed in electrolyte solutions with different concentration gradients. In the case of asymmetric electrolytes (cis1× PBS and trans0.2 M NaCl, 1× PBS), the event frequency of tumor cell (HepG2)-derived exosome translocations is enhanced by nearly 2 orders of magnitude while maintaining vesicle structure stability. Furthermore, benefiting from the salt gradient effect, tumor cell (AsPC-1 and HCT116)-derived exosome translocations could be discriminated from those of HepG2 cell-derived exosomes. The developed highly sensitive detection method for tumor cell-derived exosomes at the single-particle level provides an approach for early cancer diagnosis.

Evaluation of Commercial RNA Extraction Protocols for Avian Influenza Virus Using Nanopore Metagenomic Sequencing.

Avian influenza virus (AIV) is a significant threat to the poultry industry, necessitating rapid and accurate diagnosis. The current AIV diagnostic process relies on virus identification via real-time reverse transcription-polymerase chain reaction (rRT-PCR). Subsequently, the virus is further characterized using genome sequencing. This two-step diagnostic process takes days to weeks, but it can be expedited by using novel sequencing technologies. We aim to optimize and validate nucleic acid extraction as the first step to establishing Oxford Nanopore Technologies (ONT) as a rapid diagnostic tool for identifying and characterizing AIV from clinical samples. This study compared four commercially available RNA extraction protocols using AIV-known-positive clinical samples. The extracted RNA was evaluated using total RNA concentration, viral copies as measured by rRT-PCR, and purity as measured by a 260/280 absorbance ratio. After NGS testing, the number of total and influenza-specific reads and quality scores of the generated sequences were assessed. The results showed that no protocol outperformed the others on all parameters measured; however, the magnetic particle-based method was the most consistent regarding CT value, purity, total yield, and AIV reads, and it was less error-prone. This study highlights how different RNA extraction protocols influence ONT sequencing performance.

Identification of non-model mammal species using the MinION DNA sequencer from Oxford Nanopore.

Background: The Neotropics harbors the largest species richness of the planet; however, even in well-studied groups, there are potentially hundreds of species that lack a formal description, and likewise, many already described taxa are difficult to identify using morphology. Specifically in small mammals, complex morphological diagnoses have been facilitated by the use of molecular data, particularly from mitochondrial sequences, to obtain accurate species identifications. Obtaining mitochondrial markers implies the use of PCR and specific primers, which are largely absent for non-model organisms. Oxford Nanopore Technologies (ONT) is a new alternative for sequencing the entire mitochondrial genome without the need for specific primers. Only a limited number of studies have employed exclusively ONT long-reads to assemble mitochondrial genomes, and few studies have yet evaluated the usefulness of such reads in multiple non-model organisms. Methods: We implemented fieldwork to collect small mammals, including rodents, bats, and marsupials, in five localities in the northern extreme of the Cordillera Central of Colombia. DNA samples were sequenced using the MinION device and Flongle flow cells. Shotgun-sequenced data was used to reconstruct the mitochondrial genome of all the samples. In parallel, using a customized computational pipeline, species-level identifications were obtained based on sequencing raw reads (Whole Genome Sequencing). ONT-based identifications were corroborated using traditional morphological characters and phylogenetic analyses. Results: A total of 24 individuals from 18 species were collected, morphologically identified, and deposited in the biological collection of Universidad EAFIT. Our different computational pipelines were able to reconstruct mitochondrial genomes from exclusively ONT reads. We obtained three new mitochondrial genomes and eight new molecular mitochondrial sequences for six species. Our species identification pipeline was able to obtain accurate species identifications for up to 75% of the individuals in as little as 5 s. Finally, our phylogenetic analyses corroborated the identifications from our automated species identification pipeline and revealed important contributions to the knowledge of the diversity of Neotropical small mammals. Discussion: This study was able to evaluate different pipelines to reconstruct mitochondrial genomes from non-model organisms, using exclusively ONT reads, benchmarking these protocols on a multi-species dataset. The proposed methodology can be applied by non-expert taxonomists and has the potential to be implemented in real-time, without the need to euthanize the organisms and under field conditions. Therefore, it stands as a relevant tool to help increase the available data for non-model organisms, and the rate at which researchers can characterize life specially in highly biodiverse places as the Neotropics.

Ten Thousand Recaptures of a Single DNA Molecule in a Nanopore and Variance Reduction of Translocation Characteristics.

Nanopores have emerged as highly sensitive biosensors operating at the single-molecule level. However, the majority of nanopore experiments still rely on averaging signals from multiple molecules, introducing systematic errors. To overcome this limitation and obtain accurate information from a single molecule, the molecular ping-pong methodology provides a precise approach involving repeated captures of a single molecule. In this study, we have enhanced the molecular ping-pong technique by incorporating a customized electronic system and control algorithm, resulting in a recapture number exceeding 10,000. During the ping-pong process, we observed a significant reduction in the variance of translocation characteristics, providing fresh insights into single-molecule translocation dynamics. An inhomogeneous translocation velocity of folded DNA has been revealed, illustrating a strong interaction between the molecule and the solid-state nanopore. The results not only promise heightened experimental efficiency with reduced sample volume but also increase the precision in statistical analysis of translocation events, marking a significant stride toward authentic single-molecule nanopore sensing.

Photothermally Heated Asymmetric Thin Nanopores Suggest the Influence of Temperature on the Intermediate Conformational State of Cytochrome c in an Electric Field.

Nanopore sensing is a label-free single-molecule technique that enables the study of the dynamical structural properties of proteins. Here, we detect the translocation of cytochrome c (Cyt c) through an asymmetric thin nanopore with photothermal heating to evaluate the influence of temperature on Cyt c conformation during its translocation in an electric field. Before Cyt c translocates through an asymmetric thin SiNx nanopore, ∼1 ms trapping events occur due to electric field-induced denaturation. These trapping events were corroborated by a control analysis with a transmission electron microscopy-drilled pore and denaturant buffer. Cyt c translocation events exhibited markedly greater broad current blockade when the pores were photothermally heated. Collectively, our molecular dynamics simulation predicted that an increased temperature facilitates denaturation of the α-helical structure of Cyt c, resulting in greater blockade current during Cyt c trapping. Our photothermal heating method can be used to study the influence of temperature on protein conformation at the single-molecule level in a label-free manner.

Facile roll-to-roll production of nanoporous fiber coatings for advanced wound care sutures.

Theranostic sutures are derived from innovative ideas to enhance wound healing results by adding wound diagnostics and therapeutics to typical sutures by functionalizing them with additional materials. Here, we present a new direct electrospinning method for the fast, continuous, inexpensive, and high-throughput production of versatile nanofibrous-coated suture threads, with precise control over various essential microstructural and physical characteristics. The thickness of the coating layer and the alignment of nanofibers with the thread's direction can be adjusted by the user by varying the spooling speed and the displacement between the spinneret needle and thread. To show the flexibility of our method for a range of different materials selected, gelatin, polycaprolactone, silk fibroin, and PEDOT:PSS (poly(3,4-ethylene dioxythiophene):poly(styrene sulfonate)) were the resultant nanofibers characterized by scanning electron microscopy (SEM) imaging and conductivity tests. In a series of in vitro and ex vivo tests (pig skin), sutures were successfully tested for their flexibility and mechanical properties when used as weaving and knotting sutures, and their biocompatibility with a keratinocyte cell line. For temperature-based drug-releasing tests, two fluorescent molecules as drug models with high and low molecular weight, namely fluorescein isothiocyanate-dextran (20 kDa) and rhodamine B (470 Da), were used, and their steady release with incremental increase of temperature to 37 °C over 120 min was seen, which is appropriate for bacterial treatment drugs. Given the advantages of the presented technique, it seems to have promising potential to be used in future medical applications for wound closure and bacterial infection treatment via a temperature-triggered drug release strategy.

Single-Molecule Identification and Quantification of Steviol Glycosides with a Deep Learning-Powered Nanopore Sensor.

Steviol glycosides (SGs) are a class of high-potency noncalorie natural sweeteners made up of a common diterpenoid core and varying glycans. Thus, the diversity of glycans in composition, linkage, and isomerism results in the tremendous structural complexity of the SG family, which poses challenges for the precise identification and leads to the fact that SGs are frequently used in mixtures and their variances in biological activity remain largely unexplored. Here we show that a wild-type aerolysin nanopore can detect and discriminate diverse SG species through the modulable electro-osmotic flow effect at varied applied voltages. At low voltages, the neutral SG molecule was drawn and stuck in the pore entrance due to an energy barrier around R220 sites. The ensuing binding events enable the identification of the majority of SG species. Increasing the voltage can break the barrier and cause translocation events, allowing for the unambiguous identification of several pairs of SGs differing by only one hydroxyl group through recognition accumulation from multiple sensing regions and sites. Based on nanopore data of 15 SGs, a deep learning-based artificial intelligence (AI) model was created to process the individual blockage events, achieving the rapid, automated, and precise single-molecule identification and quantification of SGs in real samples. This work highlights the value of nanopore sensing for precise structural analysis of complex glycans-containing glycosides, as well as the potential for sensitive and rapid quality assurance analysis of glycoside products with the use of AI.