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1. Nanopores map the acid-base properties of a single site in a single DNA molecule

   https://doi.org/10.1093/nar/gkae518  

   Smith, Drew C.; Thomas, Christopher A.; Craig, Jonathan M.; Brinkerhoff, Henry; Abell, Sarah J.; Franzi, Michaela C.; Carrasco, Jessica D.; Hoshika, Shuichi; Benner, Steven A.; Gundlach, Jens H.; et al (June 2024, Nucleic Acids Research)

   Abstract Nanopores are increasingly powerful tools for single molecule sensing, in particular, for sequencing DNA, RNA and peptides. This success has spurred efforts to sequence non-canonical nucleic acid bases and amino acids. While canonical DNA and RNA bases have pKas far from neutral, certain non-canonical bases, natural RNA modifications, and amino acids are known to have pKas near neutral pHs at which nanopore sequencing is typically performed. Previous reports have suggested that the nanopore signal may be sensitive to the protonation state of an individual moiety. We sequenced ion currents with the MspA nanopore using a single stranded DNA containing a single non-canonical DNA base (Z) at various pH conditions. The Z-base has a near-neutral pKa ∼ 7.8. We find that the measured ion current is remarkably sensitive to the protonation state of the Z-base. We demonstrate how nanopores can be used to localize and determine the pKa of individual moieties along a polymer. More broadly, these experiments provide a path to mapping different protonation sites along polymers and give insight in how to optimize sequencing of polymers that contain moieties with near-neutral pKas. 

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2. Ångström- and Nano-scale Pore-Based Nucleic Acid Sequencing of Current and Emergent Pathogens

   https://doi.org/10.1557/adv.2020.402  

   Shepherd, Britney A.; Tanjil, Md Rubayat-E; Jeong, Yunjo; Baloğlu, Bilgenur; Liao, Jingqiu; Wang, Michael Cai (November 2020, MRS Advances)

   null (Ed.)

   Abstract State-of-the-art nanopore sequencing enables rapid and real-time identification of novel pathogens, which has wide application in various research areas and is an emerging diagnostic tool for infectious diseases including COVID-19. Nanopore translocation enables de novo sequencing with long reads (> 10 kb) of novel genomes, which has advantages over existing short-read sequencing technologies. Biological nanopore sequencing has already achieved success as a technology platform but it is sensitive to empirical factors such as pH and temperature. Alternatively, ångström- and nano-scale solid-state nanopores, especially those based on two-dimensional (2D) membranes, are promising next-generation technologies as they can surpass biological nanopores in the variety of membrane materials, ease of defining pore morphology, higher nucleotide detection sensitivity, and facilitation of novel and hybrid sequencing modalities. Since the discovery of graphene, atomically-thin 2D materials have shown immense potential for the fabrication of nanopores with well-defined geometry, rendering them viable candidates for nanopore sequencing membranes. Here, we review recent progress and future development trends of 2D materials and their ångström- and nano-scale pore-based nucleic acid (NA) sequencing including fabrication techniques and current and emerging sequencing modalities. In addition, we discuss the current challenges of translocation-based nanopore sequencing and provide an outlook on promising future research directions. 

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3. Sequence-to-sequence translation from mass spectra to peptides with a transformer model

   https://doi.org/10.1038/s41467-024-49731-x  

   Yilmaz, Melih; Fondrie, William_E; Bittremieux, Wout; Melendez, Carlo_F; Nelson, Rowan; Ananth, Varun; Oh, Sewoong; Noble, William_Stafford (July 2024, Nature Communications)

   Abstract A fundamental challenge in mass spectrometry-based proteomics is the identification of the peptide that generated each acquired tandem mass spectrum. Approaches that leverage known peptide sequence databases cannot detect unexpected peptides and can be impractical or impossible to apply in some settings. Thus, the ability to assign peptide sequences to tandem mass spectra without prior information—de novo peptide sequencing—is valuable for tasks including antibody sequencing, immunopeptidomics, and metaproteomics. Although many methods have been developed to address this problem, it remains an outstanding challenge in part due to the difficulty of modeling the irregular data structure of tandem mass spectra. Here, we describe Casanovo, a machine learning model that uses a transformer neural network architecture to translate the sequence of peaks in a tandem mass spectrum into the sequence of amino acids that comprise the generating peptide. We train a Casanovo model from 30 million labeled spectra and demonstrate that the model outperforms several state-of-the-art methods on a cross-species benchmark dataset. We also develop a version of Casanovo that is fine-tuned for non-enzymatic peptides. Finally, we demonstrate that Casanovo’s superior performance improves the analysis of immunopeptidomics and metaproteomics experiments and allows us to delve deeper into the dark proteome. 

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4. Nanopore Detection of Metal Ions: Current Status and Future Directions

   https://doi.org/10.1002/smtd.202000266  

   Roozbahani, Golbarg Mohammadi; Chen, Xiaohan; Zhang, Youwen; Wang, Liang; Guan, Xiyun (August 2020, Small Methods)

   Abstract In this review, recent research efforts that aimed at developing nanopore sensors for detection of metal ions, which play a crucial role in environmental safety and human health, are highlighted. Protein pores use three stochastic sensing‐based strategies for metal ion detection. The first strategy is to construct engineered nanopores with metal ion binding sites, so that the interaction between the target analytes and the nanopore can slow the movement of metal ions in the nanochannel. Second, large molecules such as nucleic acids and especially peptides can be utilized as external selective molecular probes to detect metal ions based on the conformational change of the ligand molecules induced by the metal ion–ligand chelation/coordination interaction. Third, enzymatic reactions can also be used as an alternative to the molecule probe strategy in the situation that a sensitive and selective probe molecule for the target analyte is difficult to obtain. On the other hand, by taking advantage of steady‐state analysis, synthetic nanopores mainly use two strategies (modification and modification‐free) to detect metals. Given the advantages of high sensitivity and selectivity, and label‐free detection, nanopore‐based metal ion sensors should find useful application in many fields, including environmental monitoring, medical diagnosis, and so on. 

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5. Ensuring fair assessment of solid-state nanopore sensors with reporting baseline current

   https://doi.org/10.1063/5.0167402  

   Dong, Ming; Tang, Zifan; Guan, Weihua (October 2023, Applied Physics Letters)

   In developing solid-state nanopore sensors for single molecule detection, comprehensive evaluation of the nanopore quality is important. Existing studies typically rely on comparing the noise root mean square or power spectrum density values. Nanopores exhibiting lower noise values are generally considered superior. This evaluation is valid when the single molecule signal remains consistent. However, the signal can vary, as it is strongly related to the solid-state nanopore size, which is hard to control during fabrication consistently. This work emphasized the need to report the baseline current for evaluating solid-state nanopore sensors. The baseline current offers insight into several experimental conditions, particularly the nanopore size. Our experiments show that a nanopore sensor with more noise is not necessarily worse when considering the signal-to-noise ratio (SNR), particularly when the pore size is smaller. Our findings suggest that relying only on noise comparisons can lead to inaccurate evaluations of solid-state nanopore sensors, considering the inherent variability in fabrication and testing setups among labs and measurements. We propose that future studies should include reporting baseline current and sensing conditions. Additionally, using SNR as a primary evaluation tool for nanopore sensors could provide a more comprehensive understanding of their performance. 

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