Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O-GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis@en

Cyclic oligoadenylates (cOAs) are small second messenger molecules produced by the type III CRISPR-Cas system as part of the prokaryotic immune response. The role of cOAs is to allosterically activate downstream effector proteins that induce dormancy or cell death, and thus abort viral spread through the population. Interestingly, different type III systems have been reported to utilize different cOA stoichiometries (with 3 to 6 adenylate monophosphates). However, so far, their characterization has only been possible in bulk and with sophisticated equipment, while a portable assay with single-molecule resolution has been lacking. Here, we demonstrate the label-free detection of single cOA molecules using a simple protein nanopore assay. It sensitively identifies the stoichiometry of individual cOA molecules and their mixtures from synthetic and enzymatic origin. To achieve this, we trained a convolutional neural network (CNN) and validated it with a series of experiments on mono- and polydisperse cOA samples. Ultimately, we determined the stoichiometric composition of cOAs produced enzymatically by the CRISPR type III-A and III-B variants of Thermus thermophilus and confirmed the results by liquid chromatography-mass spectroscopy (LC-MS). Interestingly, both variants produce cOAs of nearly identical composition (within experimental uncertainties), and we discuss the biological implications of this finding. The presented nanopore-CNN workflow with single cOA resolution can be adapted to many other signaling molecules (including eukaryotic ones), and it may be integrated into portable handheld devices with potential point-of-care applications.

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Understanding the structure of biomolecules is vital for deciphering their roles in biological systems. Single-molecule techniques have emerged as alternatives to conventional ensemble structure analysis methods for uncovering new biology in molecular dynamics and interaction studies, yet only limited structural information could be obtained experimentally. Here, we address this challenge by introducing iMAX FRET, a one-pot method that allows ab initio 3D profiling of individual molecules using two-color FRET measurements. Through the stochastic exchange of fluorescent weak binders, iMAX FRET simultaneously assesses multiple distances on a biomolecule within a few minutes, which can then be used to reconstruct the coordinates of up to four points in each molecule, allowing structure-based inference. We demonstrate the 3D reconstruction of DNA nanostructures, protein quaternary structures, and conformational changes in proteins. With iMAX FRET, we provide a powerful approach to advance the understanding of biomolecular structure by expanding conventional FRET analysis to three dimensions.

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With its candybar form factor and low initial investment cost, the MinION brought affordable portable nucleic acid analysis within reach. However, translating the electrical signal it outputs into a sequence of bases still requires mid-tier computer hardware, which remains a caveat when aiming for deployment of many devices at once or usage in remote areas. For applications focusing on detection of a target sequence, such as infectious disease monitoring or species identification, the computational cost of analysis may be reduced by directly detecting the target sequence in the electrical signal instead. Here, we present baseLess, a computational tool that enables such target-detection-only analysis. BaseLess makes use of an array of small neural networks, each of which efficiently detects a fixed-size subsequence of the target sequence directly from the electrical signal. We show that baseLess can accurately determine the identity of reads between three closely related fish species and can classify sequences in mixtures of 20 bacterial species, on an inexpensive single-board computer.

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Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We test them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

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Single-molecule protein identification is an unrealized concept with potentially ground-breaking applications in biological research. We propose a method called FRET X (Förster Resonance Energy Transfer via DNA eXchange) fingerprinting, in which the FRET efficiency is read out between exchangeable dyes on protein-bound DNA docking strands and accumulated FRET efficiencies constitute the fingerprint for a protein. To evaluate the feasibility of this approach, we simulated fingerprints for hundreds of proteins using a coarse-grained lattice model and experimentally demonstrated FRET X fingerprinting on model peptides. Measured fingerprints are in agreement with our simulations, corroborating the validity of our modeling approach. In a simulated complex mixture of >300 human proteins of which only cysteines, lysines, and arginines were labeled, a support vector machine was able to identify constituents with 95% accuracy. We anticipate that our FRET X fingerprinting approach will form the basis of an analysis tool for targeted proteomics.

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Förster resonance energy transfer (FRET) is a useful phenomenon in biomolecular investigations, as it can be leveraged for nanoscale measurements. The optical signals produced by such experiments can be analyzed by fitting a statistical model. Several software tools exist to fit such models in an unsupervised manner but lack the flexibility to adapt to different experimental setups and require local installations. Here, we propose to fit models to optical signals more intuitively by adopting a semisupervised approach, in which the user interactively guides the model to fit a given data set, and introduce FRETboard, a web tool that allows users to provide such guidance. We show that our approach is able to closely reproduce ground truth FRET statistics in a wide range of simulated single-molecule scenarios and correctly estimate parameters for up to 11 states. On in vitro data, we retrieve parameters identical to those obtained by laborious manual classification in a fraction of the required time. Moreover, we designed FRETboard to be easily extendable to other models, allowing it to adapt to future developments in FRET measurement and analysis.

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