Single-molecule Resolution Research Articles

EpiNano: Detection of m6A RNA Modifications Using Oxford Nanopore Direct RNA Sequencing.

RNA modifications play pivotal roles in the RNA life cycle and RNA fate, and are now appreciated as a major posttranscriptional regulatory layer in the cell. In the last few years, direct RNA nanopore sequencing (dRNA-seq) has emerged as a promising technology that can provide single-molecule resolution maps of RNA modifications in their native RNA context. While native RNA can be successfully sequenced using this technology, the detection of RNA modifications is still challenging. Here, we provide an upgraded version of EpiNano (version 1.2), an algorithm to predict m6A RNA modifications from dRNA-seq datasets. The latest version of EpiNano contains models for predicting m6A RNA modifications in dRNA-seq data that has been base-called with Guppy. Moreover, it can now train models with features extracted from both base-called dRNA-seq FASTQ data and raw FAST5 nanopore outputs. Finally, we describe how EpiNano can be used in stand-alone mode to extract base-calling "error" features and current intensity information from dRNA-seq datasets. In this chapter, we provide step-by-step instructions on how to produce in vitrotranscribed constructs to train EpiNano, as well as detailed information on how to use EpiNano to train, test, and predict m6A RNA modifications in dRNA-seq data.

Nanoplasmonic-Nanofluidic Single-Molecule Biosensors for Ultrasmall Sample Volumes.

Detection of small amounts of biological compounds is of ever-increasing importance but also remains an experimental challenge. In this context, plasmonic nanoparticles have emerged as strong contenders enabling label-free optical sensing with single-molecule resolution. However, the performance of a plasmonic single-molecule biosensor is not only dependent on its ability to detect a molecule but equally importantly on its efficiency to transport it to the binding site. Here, we present a theoretical study of the impact of downscaling fluidic structures decorated with plasmonic nanoparticles from conventional microfluidics to nanofluidics. We find that for ultrasmall picolitre sample volumes, nanofluidics enables unprecedented binding characteristics inaccessible with conventional microfluidic devices, and that both detection times and number of detected binding events can be improved by several orders of magnitude. Therefore, we propose nanoplasmonic–nanofluidic biosensing platforms as an efficient tool that paves the way for label-free single-molecule detection from ultrasmall volumes, such as single cells.

Single-molecule nanopore sequencing reveals extreme target copy number heterogeneity in arylomycin-resistant mutants

Tandem gene amplification is a frequent and dynamic source of antibiotic resistance in bacteria. Ongoing expansions and contractions of repeat arrays during population growth are expected to manifest as cell-to-cell differences in copy number (CN). As a result, a clonal bacterial culture could comprise subpopulations of cells with different levels of antibiotic sensitivity that result from variable gene dosage. Despite the high potential for misclassification of heterogenous cell populations as either antibiotic-susceptible or fully resistant in clinical settings, and the concomitant risk of inappropriate treatment, CN distribution among cells has defied analysis. Here, we use the MinION single-molecule nanopore sequencer to uncover CN heterogeneity in clonal populations of Escherichia coli and Acinetobacter baumannii grown from single cells isolated while selecting for resistance to an optimized arylomycin, a member of a recently discovered class of Gram-negative antibiotic. We found that gene amplification of the arylomycin target, bacterial type I signal peptidase LepB, is a mechanism of unstable arylomycin resistance and demonstrate in E. coli that amplification instability is independent of RecA. This instability drives the emergence of a nonuniform distribution of lepB CN among cells with a range of 1 to at least 50 copies of lepB identified in a single clonal population. In sum, this remarkable heterogeneity, and the evolutionary plasticity it fuels, illustrates how gene amplification can enable bacterial populations to respond rapidly to novel antibiotics. This study establishes a rationale for further nanopore-sequencing studies of heterogeneous cell populations to uncover CN variability at single-molecule resolution.

Smart-ORF: a single-molecule method for accessing ribosome dynamics in both upstream and main open reading frames.

Upstream open reading frame (uORF) translation disrupts scanning 43S flux on mRNA and modulates main open reading frame (mORF) translation efficiency. Current tools, however, have limited access to ribosome dynamics in both upstream and main ORFs of an mRNA. Here, we develop a new two-color in vitro fluorescence assay, Smart-ORF, that monitors individual uORF and mORF translation events in real-time with single-molecule resolution. We demonstrate the utility of Smart-ORF by applying it to uORF-encoded arginine attenuator peptide (AAP)-mediated translational regulation. The method enabled quantification of uORF and mORF initiation efficiencies, 80S dwell time, polysome formation, and the correlation between uORF and mORF translation dynamics. Smart-ORF revealed that AAP-mediated 80S stalling in the uORF stimulates the uORF initiation efficiency and promotes clustering of slower uORF-translating ribosomes. This technology provides a new tool that can reveal previously uncharacterized dynamics of uORF-containing mRNA translation.

Development of a sequencing system for spatial decoding of DNA barcode molecules at single-molecule resolution

Single-cell transcriptome analysis has been revolutionized by DNA barcodes that index cDNA libraries, allowing highly multiplexed analyses to be performed. Furthermore, DNA barcodes are being leveraged for spatial transcriptomes. Although spatial resolution relies on methods used to decode DNA barcodes, achieving single-molecule decoding remains a challenge. Here, we developed an in-house sequencing system inspired by a single-molecule sequencing system, HeliScope, to spatially decode DNA barcode molecules at single-molecule resolution. We benchmarked our system with 30 types of DNA barcode molecules and obtained an average read length of ~20 nt with an error rate of less than 5% per nucleotide, which was sufficient to spatially identify them. Additionally, we spatially identified DNA barcode molecules bound to antibodies at single-molecule resolution. Leveraging this, we devised a method, termed “molecular foot printing”, showing potential for applying our system not only to spatial transcriptomics, but also to spatial proteomics.

Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing.

Probing epigenetic features on DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we use nanopore sequencing to evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA by applying GpC methyltransferase to exogenously label open chromatin. We performed nanopore sequencing of Nucleosome Occupancy and Methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231). The single-molecule resolution allows footprinting of protein and nucleosome binding and determining the combinatorial promoter epigenetic signature on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, allowing us to generate the first fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility. We further apply this to a breast cancer model to evaluate differential methylation and accessibility between cancerous and non-cancerous cells.

Seeing Is Believing: Visualizing Circular RNAs.

Advancement in the RNA sequencing techniques has discovered hundreds of thousands of circular RNAs (circRNAs) in humans. However, the physiological function of most of the identified circRNAs remains unexplored. Recent studies have established that spliceosomal machinery and RNA-binding proteins modulate circRNA biogenesis. Furthermore, circRNAs have been implicated in regulating crucial cellular processes by interacting with various proteins and microRNAs. However, there are several challenges in understanding the mechanism of circRNA biogenesis, transport, and their interaction with cellular factors to regulate cellular events because of their low abundance and sequence similarity with linear RNA. Addressing these challenges requires systematic studies that directly visualize the circRNAs in cells at single-molecule resolution along with the molecular regulators. In this review, we present the design, benefits, and weaknesses of RNA imaging techniques such as single-molecule RNA fluorescence in situ hybridization and BaseScope in fixed cells and fluorescent RNA aptamers in live-cell imaging of circRNAs. Furthermore, we propose the potential use of molecular beacons, multiply labeled tetravalent RNA imaging probes, and Cas-derived systems to visualize circRNAs.

Digital Polymerase Chain Reaction Paired with High-Speed Atomic Force Microscopy for Quantitation and Length Analysis of DNA Length Polymorphisms.

DNA length polymorphisms are found in many serious diseases, and assessment of their length and abundance is often critical for accurate diagnosis. However, measuring their length and frequency in a mostly wild-type background, as occurs in many situations, remains challenging due to their variable and repetitive nature. To overcome these hurdles, we combined two powerful techniques, digital polymerase chain reaction (dPCR) and high-speed atomic force microscopy (HSAFM), to create a simple, rapid, and flexible method for quantifying both the size and proportion of DNA length polymorphisms. In our approach, individual amplicons from each dPCR partition are imaged and sized directly. We focused on internal tandem duplications (ITDs) located within the FLT3 gene, which are associated with acute myeloid leukemia and often indicative of a poor prognosis. In an analysis of over 1.5 million HSAFM-imaged amplicons from cell line and clinical samples containing FLT3-ITDs, dPCR-HSAFM returned the expected variant length and variant allele frequency, down to 5% variant samples. As a high-throughput method with single-molecule resolution, dPCR-HSAFM thus represents an advance in HSAFM analysis and a powerful tool for the diagnosis of length polymorphisms.

Computational Simulations of Nanomechanical Resonators for Understanding their Frequency Dynamics and Sensing Performances

Nanomechanical resonators have received the attention due to their ability to perform as a high-frequency device and/or a (bio)sensor that exhibits the unprecedented detection limit even down to a single-molecule or atomic resolution. For effective design of nanomechanical resonators, it is necessary to quantitatively understand the dynamic behavior of nanomechanical resonators based on theoretical models and/or computational models. In this article, we address the current state-of-arts in the theoretical modeling and/or computational modeling of nanomechanical resonators for characterizing their dynamic behavior and their sensing performances. In particular, we present the different types of models at multiple length scales ranging from continuum models to coarse-grained and atomistic models for characterizing the dynamics and sensing performances of nanomechanical resonators. In addition, we discuss the role of finite size effect, nonlinear dynamics, and stiffness effect in the dynamic behavior and/or sensing performances of nanomechanical resonators. Our article sheds light on the theoretical/computational models, which are able to predict and characterize the dynamics and sensing performances of nanomechanical resonators for effective design of them acting as an actuator and/or a sensor.

Applications of Photo-Responsive Molecules in Nanopore-based Devices

Abstract In biochemistry, it is crucial to explore the conformation, response to external environment and interaction rules of biomacromolecules and small molecules for understanding the correlation of the structures and properties among organic matters. However, it is a challenge for the traditional techniques to observe the conformation heterogeneity and interpret the interaction mechanism of the biomolecules in living cells. As the emerged the third generation single-molecule gene sequencing technology, nanopore is used to monitor the conformation change of the molecules in situ and therefore demonstrate good applications in single-molecule sensing and nucleic acid and protein sequencing. To further improve the detection resolution and precision, the combined photoelectric detection that can achieve higher accuracy via introducing optical-responsive molecules is adopted. In view of the extensive applications and development status of nanopore technique in single-molecule conformation resolution, this paper summarizes the research progress of the nanopore devices and the design and applications of some typical photo-responsive molecules in nanopore sensing, including azobenzenes and their derivatives (Azos), spiropyrane (SP) and diarylethene (DAE). Light regulation is a simple and effective approach for the monitoring of the molecular structures, and the further integration with nanopore devices exhibits great potential in single-molecule detection meanwhile sheds light on the design and applications of multi-responsive nano-devices.

Dynamics of Tpm1.8 domains on actin filaments with single-molecule resolution.

Tropomyosins regulate the dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of the fluorescently labeled tropomyosin isoform Tpm1.8 to unlabeled actin filaments in real time. This approach, in conjunction with mathematical modeling, enabled us to quantify the nucleation, assembly, and disassembly kinetics of Tpm1.8 on single filaments and at the single-molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilized by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented toward the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin–tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

Quantifying the dynamics of IRES and cap translation with single-molecule resolution in live cells.

Viruses use internal ribosome entry sites (IRES) to hijack host ribosomes and promote cap-independent translation. Although they are well-studied in bulk, the dynamics of IRES-mediated translation remain unexplored at the single-molecule level. Here, we developed a bicistronic biosensor encoding distinct repeat epitopes in two open reading frames (ORFs), one translated from the 5' cap, and the other from the encephalomyocarditis virus IRES. When combined with a pair of complementary probes that bind the epitopes cotranslationally, the biosensor lights up in different colors depending on which ORF is translated. Using the sensor together with single-molecule tracking and computational modeling, we measured the kinetics of cap-dependent versus IRES-mediated translation in living human cells. We show that bursts of IRES translation are shorter and rarer than bursts of cap translation, although the situation reverses upon stress. Collectively, our data support a model for translational regulation primarily driven by transitions between translationally active and inactive RNA states.

On-chip protein separation with single-molecule resolution

Accurate identification of both abundant and rare proteins hinges on the development of single-protein sensing methods. Given the immense variation in protein expression levels in a cell, separation of proteins by weight would improve protein classification strategies. Upstream separation facilitates sample binning into smaller groups while also preventing sensor overflow, as may be caused by highly abundant proteins in cell lysates or clinical samples. Here, we scale a bulk analysis method for protein separation, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), to the single-molecule level using single-photon sensitive widefield imaging. Single-molecule sensing of the electrokinetically moving proteins is achieved by in situ polymerization of the PAGE in a low-profile fluidic channel having a depth of only ~ 0.6 µm. The polyacrylamide gel restricts the Brownian kinetics of the proteins, while the low-profile channel ensures that they remain in focus during imaging, allowing video-rate monitoring of single-protein migration. Calibration of the device involves separating a set of Atto647N-covalently labeled recombinant proteins in the size range of 14–70 kDa, yielding an exponential dependence of the proteins’ molecular weights on the measured mobilities, as expected. Subsequently, we demonstrate the ability of our fluidic device to separate and image thousands of proteins directly extracted from a human cancer cell line. Using single-particle image analysis methods, we created detailed profiles of the separation kinetics of lysine and cysteine -labeled proteins. Downstream coupling of the device to single-protein identification sensors may provide superior protein classification and improve our ability to analyze complex biological and medical protein samples.

Clonable EM labeling

Cell Biology Widespread use of the green fluorescent protein for protein localization at the light microscopy level has revolutionized cell biology. The search for a similar genetically encoded label for electron microscopy (EM) that could raise the resolution to the single-molecule level has not yet been successful. Jiang et al. developed a method for synthesizing 2- to 6-nm-diameter gold nanoparticles directly and specifically on individual cysteine-rich tags. This enables the unambiguous visualization of genetically tagged proteins in bacterial, yeast, and mammalian cells with single-molecule resolution. This approach holds promise in circumventing the difficulties of conventional EM immunolabeling and enables biologists to probe the ultrastructure of cells with greater ease. Nat. Methods 17 , 937 (2020).

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide-ribosome-mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

Novel Fluorescent Ligands Enable Single-Molecule Localization Microscopy of the Dopamine Transporter.

The dopamine transporter (DAT) is critical for spatiotemporal control of dopaminergic neurotransmission and is the target for therapeutic agents, including ADHD medications, and abused substances, such as cocaine. Here, we develop new fluorescently labeled ligands that bind DAT with high affinity and enable single-molecule detection of the transporter. The cocaine analogue MFZ2-12 (1) was conjugated to novel rhodamine-based Janelia Fluorophores (JF549 and JF646). High affinity binding of the resulting ligands to DAT was demonstrated by potent inhibition of [3H]dopamine uptake in DAT transfected CAD cells and by competition radioligand binding experiments on rat striatal membranes. Visualization of binding was substantiated by confocal or TIRF microscopy revealing selective binding of the analogues to DAT transfected CAD cells. Single particle tracking experiments were performed with JF549-conjugated DG3-80 (3) and JF646-conjugated DG4-91 (4) on DAT transfected CAD cells enabling quantification and categorization of the dynamic behavior of DAT into four distinct motion classes (immobile, confined, Brownian, and directed). Finally, we show that the ligands can be used in direct stochastic optical reconstruction microscopy (dSTORM) experiments permitting further analyses of DAT distribution on the nanoscale. In summary, these novel fluorescent ligands are promising new tools for studying DAT localization and regulation with single-molecule resolution.

DdRFC: A scalable multiplexed droplet digital nucleic acid amplification test platform

Digital nucleic acid amplification tests (digital NAATs) have emerged as a popular tool for nucleic acid detection due to their high sensitivity and specificity. Most current digital NAAT platforms, however, are limited to a “one-color-one-target” approach wherein each target is encoded with a specific fluorescently-labeled probe for single-plex fluorometric detection. This approach is difficult to multiplex due to spectral overlap between any additional fluorophores, and multiplexability of digital NAATs has therefore been limited. As a means to scale multiplexability, we have developed a multiplexed digital NAAT platform, termed Droplet Digital Ratiometric Fluorescence Coding (ddRFC), via a padlock probe-based nucleic acid detection assay which encodes each nucleic acid target with a unique combination of 2 fluorophores. We detect this encoded two-color fluorescence signature of each target by performing digital amplification in microfluidic droplets. To demonstrate the utility of our platform, we have synthesized 6 distinct padlock probes, each rendering a unique two-color fluorescence signature to a nucleic acid target representing a clinically important sexually transmitted infection (STI). We proceed to demonstrate broad-based, two-plex, four-plex, and six-plex detection of the STI targets with single-molecule resolution. Our design offers a cost-effective approach to scale up multiplexability by simply tuning the number of molecular beacon binding sites on the padlock probe without redesigning amplification primers or fluorescent molecular beacons. With further development, our platform has the potential to enable highly multiplexed detection of nucleic acid targets, with potentially unrestricted multiplexability, and serve as a diagnostic tool for many more diseases in the future.

A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution.

Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.

DNA Fiber Assay for the Analysis of DNA Replication Progression in Human Pluripotent Stem Cells.

Human pluripotent stem cells (PSC) acquire recurrent chromosomal instabilities during prolonged in vitro culture that threaten to preclude their use in cell-based regenerative medicine. The rapid proliferation of pluripotent cells leads to constitutive replication stress, hindering the progression of DNA replication forks and in some cases leading to replication-fork collapse. Failure to overcome replication stress can result in incomplete genome duplication, which, if left to persist into the subsequent mitosis, can result in structural and numerical chromosomal instability. We have recently applied the DNA fiber assay to the study of replication stress in human PSC and found that, in comparison to somatic cells states, these cells display features of DNA replication stress that include slower replication fork speeds, evidence of stalled forks, and replication initiation from dormant replication origins. These findings have expanded on previous work demonstrating that extensive DNA damage in human PSC is replication associated. In this capacity, the DNA fiber assay has enabled the development of an advanced nucleoside-enriched culture medium that increases replication fork progression and decreases DNA damage and mitotic errors in human PSC cultures. The DNA fiber assay allows for the study of replication fork dynamics at single-molecule resolution. The assay relies on cells incorporating nucleotide analogs into nascent DNA during replication, which are then measured to monitor several replication parameters. Here we provide an optimized protocol for the fiber assay intended for use with human PSC, and describe the methods employed to analyze replication fork parameters. © 2020 Wiley Periodicals LLC. Basic Protocol 1: DNA fiber labeling Basic Protocol 2: DNA fiber spreading Basic Protocol 3: Immunostaining Support Protocol 1: Microscopy/data acquisition Support Protocol 2: Data analysis.

Methods for spatial and temporal imaging of the different steps involved in RNA processing at single-molecule resolution.

RNA plays a quintessential role as a messenger of information from genotype (DNA) to phenotype (proteins), as well as acts as a regulatory molecule (noncoding RNAs). All steps in the journey of RNA from synthesis (transcription), splicing, transport, localization, translation, to its eventual degradation, comprise important steps in gene expression, thereby controlling the fate of the cell. This lifecycle refers to the majority of RNAs (primarily mRNAs), but not other RNAs such as tRNAs. Imaging these processes in fixed cells and in live cells has been an important tool in developing an understanding of the regulatory steps in RNAs journey. Single-cell and single-molecule imaging techniques enable a much deeper understanding of cellular biology, which is not possible with bulk studies involving RNA isolated from a large pool of cells. Classic techniques, such as fluorescence in situ hybridization (FISH), as well as more recent aptamer-based approaches, have provided detailed insights into RNA localization, and have helped to predict the functions carried out by many RNA species. However, there are still certain processing steps that await high-resolution imaging, which is an exciting and upcoming area of research. In this review, we will discuss the methods that have revolutionized single-molecule resolution imaging in general, the steps of RNA processing in which these methods have been used, and new emerging technologies. This article is categorized under: RNA Export and Localization > RNA Localization RNA Methods > RNA Analyses in Cells RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions.