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We used fluorescence microscopy of Madin-Darby Canine Kidney (MDCK) cells grown on polycarbonate filters to study a possible link between plasma membrane electrical potential (delta psi pm) and infectivity of vesicular stomatitis virus (VSV). Complete substitution of K+ for extracellular Na+ blocks VSV infection of MDCK cells as well as baby hamster kidney (BHK) cells. When we independently perfused the apical and basal-lateral surfaces of high resistance monolayers, high K+ inhibited VSV infection of MDCK cells only when applied to the basal-lateral side; high K+ applied apically had no effect on VSV infection. This morphological specificity correlates with a large decrease in delta psi pm of MDCK cells when high K+ buffer is perfused across the basal-lateral surface. Depolarization of the plasma membrane by 130 mM basal K+ causes a sustained increase of cytosol pH in MDCK cells from 7.3 to 7.5 as reported by the fluorescent dye BCECF. Depolarization also causes a transient increase of cytosol Ca2+ from 70 to 300 nM as reported by the dye Fura-2. Neither increase could explain the block of VSV infectivity by plasma membrane depolarization. One alternative hypothesis is that delta psi pm facilitates membrane translocation of viral macromolecules as previously described for colicins, mitochondrial import proteins, and proteins secreted by Escherichia coli. [ABSTRACT FROM AUTHOR]

Transfer RNAs (tRNAs) contain dozens of chemical modifications. These modifications are critical for maintaining tRNA tertiary structure and optimizing protein synthesis. Here we advance the use of Nanopore direct RNA-sequencing (DRS) to investigate the synergy between modifications that are known to stabilize tRNA structure. We sequenced the 42 cytosolic tRNA isoacceptors from wild-type yeast and five tRNA-modifying enzyme knockout mutants. These data permitted comprehensive analysis of three neighboring and conserved modifications in T-loops: 5-methyluridine (m5U54), pseudouridine (Ψ55), and 1-methyladenosine (m1A58). Our results were validated using direct measurements of chemical modifications by mass spectrometry. We observed concerted T-loop modification circuits-the potent influence of Ψ55 for subsequent m1A58 modification on more tRNA isoacceptors than previously observed. Growing cells under nutrient depleted conditions also revealed a novel condition-specific increase in m1A58 modification on some tRNAs. A global and isoacceptor-specific classification strategy was developed to predict the status of T-loop modifications from a user-input tRNA DRS dataset, applicable to other conditions and tRNAs in other organisms. These advancements demonstrate how orthogonal technologies combined with genetics enable precise detection of modification landscapes of individual, full-length tRNAs, at transcriptome-scale., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional direct RNA nanopore sequencing, the 5' and 3' ends of poly(A) RNA cannot be identified unambiguously. This is due in part to RNA degradation in vivo and in vitro that can obscure transcription start and end sites. In this study, we aimed to identify individual full-length human RNA isoforms among ∼4 million nanopore poly(A)-selected RNA reads. First, to identify RNA strands bearing 5' m 7 G caps, we exchanged the biological cap for a modified cap attached to a 45-nt oligomer. This oligomer adaptation method improved 5' end sequencing and ensured correct identification of the 5' m 7 G capped ends. Second, among these 5'-capped nanopore reads, we screened for features consistent with a 3' polyadenylation site. Combining these two steps, we identified 294,107 individual high-confidence full-length RNA scaffolds from human GM12878 cells, most of which (257,721) aligned to protein-coding genes. Of these, 4876 scaffolds indicated unannotated isoforms that were often internal to longer, previously identified RNA isoforms. Orthogonal data for m 7 G caps and open chromatin, such as CAGE and DNase-HS seq, confirmed the validity of these high-confidence RNA scaffolds., (© 2022 Mulroney et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Understanding transcriptomes requires documenting the structures, modifications, and abundances of RNAs as well as their proximity to other molecules. The methods that make this possible depend critically on enzymes (including mutant derivatives) that act on nucleic acids for capturing and sequencing RNA. We tested two 3' nucleotidyl transferases, Saccharomyces cerevisiae poly(A) polymerase and Schizosaccharomyces pombe Cid1, for the ability to add base and sugar modified rNTPs to free RNA 3' ends, eventually focusing on Cid1. Although unable to polymerize ΨTP or 1meΨTP, Cid1 can use 5meUTP and 4thioUTP. Surprisingly, Cid1 can use inosine triphosphate to add poly(I) to the 3' ends of a wide variety of RNA molecules. Most poly(A) mRNAs efficiently acquire a uniform tract of about 50 inosine residues from Cid1, whereas non-poly(A) RNAs acquire longer, more heterogeneous tails. Here we test these activities for use in direct RNA sequencing on nanopores, and find that Cid1-mediated poly(I)-tailing permits detection and quantification of both mRNAs and non-poly(A) RNAs simultaneously, as well as enabling the analysis of nascent RNAs associated with RNA polymerase II. Poly(I) produces a different current trace than poly(A), enabling recognition of native RNA 3' end sequence lost by in vitro poly(A) addition. Addition of poly(I) by Cid1 offers a broadly useful alternative to poly(A) capture for direct RNA sequencing on nanopores., (© 2021 Vo et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

We describe a method for direct tRNA sequencing using the Oxford Nanopore MinION. The principal technical advance is custom adapters that facilitate end-to-end sequencing of individual transfer RNA (tRNA) molecules at subnanometer precision. A second advance is a nanopore sequencing pipeline optimized for tRNA. We tested this method using purified E. coli tRNA fMet , tRNA Lys , and tRNA Phe samples. 76-92% of individual aligned tRNA sequence reads were full length. As a proof of concept, we showed that nanopore sequencing detected all 43 expected isoacceptors in total E. coli MRE600 tRNA as well as isodecoders that further define that tRNA population. Alignment-based comparisons between the three purified tRNAs and their synthetic controls revealed systematic nucleotide miscalls that were diagnostic of known modifications. Systematic miscalls were also observed proximal to known modifications in total E. coli tRNA alignments, including a highly conserved pseudouridine in the T loop. This work highlights the potential of nanopore direct tRNA sequencing as well as improvements needed to implement tRNA sequencing for human healthcare applications.

The accurate identification and quantitation of RNA isoforms present in the cancer transcriptome is key for analyses ranging from the inference of the impacts of somatic variants to pathway analysis to biomarker development and subtype discovery. The ICGC-TCGA DREAM Somatic Mutation Calling in RNA (SMC-RNA) challenge was a crowd-sourced effort to benchmark methods for RNA isoform quantification and fusion detection from bulk cancer RNA sequencing (RNA-seq) data. It concluded in 2018 with a comparison of 77 fusion detection entries and 65 isoform quantification entries on 51 synthetic tumors and 32 cell lines with spiked-in fusion constructs. We report the entries used to build this benchmark, the leaderboard results, and the experimental features associated with the accurate prediction of RNA species. This challenge required submissions to be in the form of containerized workflows, meaning each of the entries described is easily reusable through CWL and Docker containers at https://github.com/SMC-RNA-challenge. A record of this paper's transparent peer review process is included in the supplemental information., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Determining the layers of gene regulation within the innate immune response is critical to our understanding of the cellular responses to infection and dysregulation in disease. We identified a conserved mechanism of gene regulation in human and mouse via changes in alternative first exon (AFE) usage following inflammation, resulting in changes to the isoforms produced. Of these AFE events, we identified 95 unannotated transcription start sites in mice using a de novo transcriptome generated by long-read native RNA-sequencing, one of which is in the cytosolic receptor for dsDNA and known inflammatory inducible gene, Aim2 . We show that this unannotated AFE isoform of Aim2 is the predominant isoform expressed during inflammation and contains an iron-responsive element in its 5'UTR enabling mRNA translation to be regulated by iron levels. This work highlights the importance of examining alternative isoform changes and translational regulation in the innate immune response and uncovers novel regulatory mechanisms of Aim2 ., Competing Interests: ER, PJ, SC, MC, VS, RS, BS, RA, MJ, SC, SC No competing interests declared, MA holds options in Oxford Nanopore Technologies (ONT), is a paid consultant to ONT, received reimbursement for travel, accommodation and conference fees to speak at events organized by ONT,received research funding from ONT and is an inventor on 11 UC patents licensed to ONT (6,267,872, 6,465,193, 6,746,594, 6,936,433, 7,060,50, 8,500,982, 8,679,747, 9,481,908, 9,797,013, 10,059,988, and 10,081,835), AB received reimbursement for travel, accommodation and conference fees to speak at events organized by Oxford Nanopore Technologies (ONT), (© 2021, Robinson et al.)

Among the large, diverse set of mammalian long noncoding RNAs (lncRNAs), long noncoding primary microRNAs (lnc-pri-miRNAs) are those that host miRNAs. Whether lnc-pri-miRNA loci have important biological function independent of their cognate miRNAs is poorly understood. From a genome-scale lncRNA screen, lnc-pri-miRNA loci were enriched for function in cell proliferation, and in glioblastoma (i.e., GBM) cells with DGCR8 or DROSHA knockdown, lnc-pri-miRNA screen hits still regulated cell growth. To molecularly dissect the function of a lnc-pri-miRNA locus, we studied LOC646329 (also known as MIR29HG ), which hosts the miR-29a/b1 cluster. In GBM cells, LOC646329 knockdown reduced miR-29a/b1 levels, and these cells exhibited decreased growth. However, genetic deletion of the miR-29a/b1 cluster ( LOC646329-miR29Δ ) did not decrease cell growth, while knockdown of LOC646329-miR29Δ transcripts reduced cell proliferation. The miR-29a/b1-independent activity of LOC646329 corresponded to enhancer-like activation of a neighboring oncogene ( MKLN1 ), regulating cell propagation. The LOC646329 locus interacts with the MKLN1 promoter, and antisense oligonucleotide knockdown of the lncRNA disrupts these interactions and reduces the enhancer-like activity. More broadly, analysis of genome-wide data from multiple human cell types showed that lnc-pri-miRNA loci are significantly enriched for DNA looping interactions with gene promoters as well as genomic and epigenetic characteristics of transcriptional enhancers. Functional studies of additional lnc-pri-miRNA loci demonstrated cognate miRNA-independent enhancer-like activity. Together, these data demonstrate that lnc-pri-miRNA loci can regulate cell biology via both miRNA-dependent and miRNA-independent mechanisms., Competing Interests: The authors declare no competing interest.

For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of limitations, with the most significant being bias towards the detection of culturable organisms and the inherent delay between sample collection and ground-based analysis. In recent years, portable and easy-to-use molecular-based tools, such as Oxford Nanopore Technologies' MinION™ sequencer and miniPCR bio's miniPCR™ thermal cycler, have been validated onboard the ISS. Here, we report on the development, validation, and implementation of a swab-to-sequencer method that provides a culture-independent solution to real-time microbial profiling onboard the ISS. Method development focused on analysis of swabs collected in a low-biomass environment with limited facility resources and stringent controls on allowed processes and reagents. ISS-optimized procedures included enzymatic DNA extraction from a swab tip, bead-based purifications, altered buffers, and the use of miniPCR and the MinION. Validation was conducted through extensive ground-based assessments comparing current standard culture-dependent and newly developed culture-independent methods. Similar microbial distributions were observed between the two methods; however, as expected, the culture-independent data revealed microbial profiles with greater diversity. Protocol optimization and verification was established during NASA Extreme Environment Mission Operations (NEEMO) analog missions 21 and 22, respectively. Unique microbial profiles obtained from analog testing validated the swab-to-sequencer method in an extreme environment. Finally, four independent swab-to-sequencer experiments were conducted onboard the ISS by two crewmembers. Microorganisms identified from ISS swabs were consistent with historical culture-based data, and primarily consisted of commonly observed human-associated microbes. This simplified method has been streamlined for high ease-of-use for a non-trained crew to complete in an extreme environment, thereby enabling environmental and human health diagnostics in real-time as future missions take us beyond low-Earth orbit.

Proteins present a significant challenge for nanopore-based sequence analysis. This is partly due to their stable tertiary structures that must be unfolded for linear translocation, and the absence of regular charge density. To address these challenges, here we describe how ClpXP, an ATP-dependent protein unfoldase, can be harnessed to unfold and processively translocate multi-domain protein substrates through an alpha-hemolysin nanopore sensor. This process results in ionic current patterns that are diagnostic of protein sequence and structure at the single-molecule level.

Historically, RNA has been sequenced as cDNA copies derived from reverse transcription of cellular RNA followed by PCR amplification. Recently, RNA sequencing using nanopores has emerged as an alternative. Using this technology, individual cellular RNA strands are read directly as they are driven through nanoscale pores by an applied voltage. The speed of translocation is regulated by a helicase that is loaded onto each RNA strand by an adapter that also facilitates capture by the nanopore electric field. Here we describe a technique for adapting human ribosomal RNA subunits for nanopore sequencing. Using this strategy, a single Oxford Nanopore MinION run delivered 470,907 sequence reads of which 396,048 aligned to ribosomal RNA, with 28S, 18S, 5.8S, and 5S coverage of 6053, 369,472, 16,058, and 4465 reads, respectively. Example alignments that reveal putative nucleotide modifications are provided.

De novo assembly of a human genome using nanopore long-read sequences has been reported, but it used more than 150,000 CPU hours and weeks of wall-clock time. To enable rapid human genome assembly, we present Shasta, a de novo long-read assembler, and polishing algorithms named MarginPolish and HELEN. Using a single PromethION nanopore sequencer and our toolkit, we assembled 11 highly contiguous human genomes de novo in 9 d. We achieved roughly 63× coverage, 42-kb read N50 values and 6.5× coverage in reads >100 kb using three flow cells per sample. Shasta produced a complete haploid human genome assembly in under 6 h on a single commercial compute node. MarginPolish and HELEN polished haploid assemblies to more than 99.9% identity (Phred quality score QV = 30) with nanopore reads alone. Addition of proximity-ligation sequencing enabled near chromosome-level scaffolds for all 11 genomes. We compare our assembly performance to existing methods for diploid, haploid and trio-binned human samples and report superior accuracy and speed.

Background: Long non-coding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer treatments. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients., Results: We use CRISPR interference (CRISPRi) to screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modify cell growth in the presence of clinically relevant doses of fractionated radiation. Thirty-three of these lncRNA hits sensitize cells to radiation, and based on their expression in adult and pediatric gliomas, nine of these hits are prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1, a primate-conserved, nuclear-enriched lncRNA, inhibits the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a three-dimensional tissue substrate to model the invasive growth of glioma, we find that antisense oligonucleotides targeting lncGRS-1 selectively decrease tumor growth and sensitize glioma cells to radiation therapy., Conclusions: These studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome to enhance radiation therapy.

The MinION sequencer has made in situ sequencing feasible in remote locations. Following our initial demonstration of its high performance off planet with Earth-prepared samples, we developed and tested an end-to-end, sample-to-sequencer process that could be conducted entirely aboard the International Space Station (ISS). Initial experiments demonstrated the process with a microbial mock community standard. The DNA was successfully amplified, primers were degraded, and libraries prepared and sequenced. The median percent identities for both datasets were 84%, as assessed from alignment of the mock community. The ability to correctly identify the organisms in the mock community standard was comparable for the sequencing data obtained in flight and on the ground. To validate the process on microbes collected from and cultured aboard the ISS, bacterial cells were selected from a NASA Environmental Health Systems Surface Sample Kit contact slide. The locations of bacterial colonies chosen for identification were labeled, and a small number of cells were directly added as input into the sequencing workflow. Prepared DNA was sequenced, and the data were downlinked to Earth. Return of the contact slide to the ground allowed for standard laboratory processing for bacterial identification. The identifications obtained aboard the ISS, Staphylococcus hominis and Staphylococcus capitis , matched those determined on the ground down to the species level. This marks the first ever identification of microbes entirely off Earth, and this validated process could be used for in-flight microbial identification, diagnosis of infectious disease in a crewmember, and as a research platform for investigators around the world.

High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes.

The ribosome small subunit is expressed in all living cells. It performs numerous essential functions during translation, including formation of the initiation complex and proofreading of base-pairs between mRNA codons and tRNA anticodons. The core constituent of the small ribosomal subunit is a ~1.5 kb RNA strand in prokaryotes (16S rRNA) and a homologous ~1.8 kb RNA strand in eukaryotes (18S rRNA). Traditional sequencing-by-synthesis (SBS) of rRNA genes or rRNA cDNA copies has achieved wide use as a 'molecular chronometer' for phylogenetic studies, and as a tool for identifying infectious organisms in the clinic. However, epigenetic modifications on rRNA are erased by SBS methods. Here we describe direct MinION nanopore sequencing of individual, full-length 16S rRNA absent reverse transcription or amplification. As little as 5 picograms (~10 attomole) of purified E. coli 16S rRNA was detected in 4.5 micrograms of total human RNA. Nanopore ionic current traces that deviated from canonical patterns revealed conserved E. coli 16S rRNA 7-methylguanosine and pseudouridine modifications, and a 7-methylguanosine modification that confers aminoglycoside resistance to some pathological E. coli strains., Competing Interests: MA holds options in Oxford Nanopore Technologies (ONT). MA is a paid consultant to ONT. MA is an inventor on 11 University of California patents licensed to ONT (6,267,872, 6,465,193, 6,746,594, 6,936,433, 7,060,50, 8,500,982, 8,679,747, 9,481,908, 9,797,013, 10,059,988, and 10,081,835). DRG, who contributed to each facet of the paper, is an employee of Oxford Nanopore Technologies. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The human genome reference sequence remains incomplete owing to the challenge of assembling long tracts of near-identical tandem repeats in centromeres. We implemented a nanopore sequencing strategy to generate high-quality reads that span hundreds of kilobases of highly repetitive DNA in a human Y chromosome centromere. Combining these data with short-read variant validation, we assembled and characterized the centromeric region of a human Y chromosome.

Understanding gene regulation and function requires a genome-wide method capable of capturing both gene expression levels and isoform diversity at the single-cell level. Short-read RNAseq is limited in its ability to resolve complex isoforms because it fails to sequence full-length cDNA copies of RNA molecules. Here, we investigate whether RNAseq using the long-read single-molecule Oxford Nanopore MinION sequencer is able to identify and quantify complex isoforms without sacrificing accurate gene expression quantification. After benchmarking our approach, we analyse individual murine B1a cells using a custom multiplexing strategy. We identify thousands of unannotated transcription start and end sites, as well as hundreds of alternative splicing events in these B1a cells. We also identify hundreds of genes expressed across B1a cells that display multiple complex isoforms, including several B cell-specific surface receptors. Our results show that we can identify and quantify complex isoforms at the single cell level.

DNA chemical modifications regulate genomic function. We present a framework for mapping cytosine and adenosine methylation with the Oxford Nanopore Technologies MinION using this nanopore sequencer's ionic current signal. We map three cytosine variants and two adenine variants. The results show that our model is sensitive enough to detect changes in genomic DNA methylation levels as a function of growth phase in Escherichia coli.

Nanopore DNA strand sequencing has emerged as a competitive, portable technology. Reads exceeding 150 kilobases have been achieved, as have in-field detection and analysis of clinical pathogens. We summarize key technical features of the Oxford Nanopore MinION, the dominant platform currently available. We then discuss pioneering applications executed by the genomics community.

Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both translators of the genetic code and regulators of protein synthesis. As such, numerous methods have focused on studying tRNA abundance and regulation, with the most widely used methods being RNA-seq and microarrays. Though revolutionary to transcriptomics, these assays are limited by an inability to encode tRNA modifications in the requisite cDNA. These modifications are abundant in tRNA and critical to their function. Here, we describe proof-of-concept experiments where individual tRNA molecules are examined as linear strands using a biological nanopore. This method utilizes an enzymatically ligated synthetic DNA adapter to concentrate tRNA at the lipid bilayer of the nanopore device and efficiently denature individual tRNA molecules, as they are pulled through the α-hemolysin (α-HL) nanopore. Additionally, the DNA adapter provides a loading site for ϕ29 DNA polymerase (ϕ29 DNAP), which acts as a brake on the translocating tRNA. This increases the dwell time of adapted tRNA in the nanopore, allowing us to identify the region of the nanopore signal that is produced by the translocating tRNA itself. Using adapter-modified Escherichia coli tRNA(fMet) and tRNA(Lys), we show that the nanopore signal during controlled translocation is dependent on the identity of the tRNA. This confirms that adapter-modified tRNA can translocate end-to-end through nanopores and provide the foundation for future work in direct sequencing of individual transfer RNA with a nanopore-based device.

Speed, single-base sensitivity and long read lengths make nanopores a promising technology for high-throughput sequencing. We evaluated and optimized the performance of the MinION nanopore sequencer using M13 genomic DNA and used expectation maximization to obtain robust maximum-likelihood estimates for insertion, deletion and substitution error rates (4.9%, 7.8% and 5.1%, respectively). Over 99% of high-quality 2D MinION reads mapped to the reference at a mean identity of 85%. We present a single-nucleotide-variant detection tool that uses maximum-likelihood parameter estimates and marginalization over many possible read alignments to achieve precision and recall of up to 99%. By pairing our high-confidence alignment strategy with long MinION reads, we resolved the copy number for a cancer-testis gene family (CT47) within an unresolved region of human chromosome Xq24.

Previously we showed that the protein unfoldase ClpX could facilitate translocation of individual proteins through the α-hemolysin nanopore. This results in ionic current fluctuations that correlate with unfolding and passage of intact protein strands through the pore lumen. It is plausible that this technology could be used to identify protein domains and structural modifications at the single-molecule level that arise from subtle changes in primary amino acid sequence (e.g., point mutations). As a test, we engineered proteins bearing well-characterized domains connected in series along an ∼700 amino acid strand. Point mutations in a titin immunoglobulin domain (titin I27) and point mutations, proteolytic cleavage, and rearrangement of beta-strands in green fluorescent protein (GFP), caused ionic current pattern changes for single strands predicted by bulk phase and force spectroscopy experiments. Among these variants, individual proteins could be classified at 86-99% accuracy using standard machine learning tools. We conclude that a ClpXP-nanopore device can discriminate among distinct protein domains, and that sequence-dependent variations within those domains are detectable.

Individual DNA molecules can be read at single nucleotide precision using nanopores coupled to processive enzymes. Discrimination among the four canonical bases has been achieved, as has discrimination among cytosine, 5-methylcytosine (mC), and 5-hydroxymethylcytosine (hmC). Two additional modified cytosine bases, 5-carboxylcytosine (caC) and 5-formylcytosine (fC), are produced during enzymatic conversion of hmC to cytosine in mammalian cells. Thus, an accurate picture of the cytosine epigenetic status in target cells should also include these C5-cytosine variants. In the present study, we used a patch clamp amplifier to acquire ionic current traces caused by phi29 DNA polymerase-controlled translocation of DNA templates through the M2MspA pore. Decision boundaries based on three consecutive ionic current states were implemented to call mC, hmC, caC, fC, or cytosine at CG dinucleotides in ∼4400 individual DNA molecules. We found that the percentage of correct base calls for single pass reads ranged from 91.6% to 98.3%. This accuracy depended upon the identity of nearest neighbor bases surrounding the CG dinucleotide.

Cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine were identified during translocation of single DNA template strands through a modified Mycobacterium smegmatis porin A (M2MspA) nanopore under control of phi29 DNA polymerase. This identification was based on three consecutive ionic current states that correspond to passage of modified or unmodified CG dinucleotides and their immediate neighbors through the nanopore limiting aperture. To establish quality scores for these calls, we examined ~3,300 translocation events for 48 distinct DNA constructs. Each experiment analyzed a mixture of cytosine-, 5-methylcytosine-, and 5-hydroxymethylcytosine-bearing DNA strands that contained a marker that independently established the correct cytosine methylation status at the target CG of each molecule tested. To calculate error rates for these calls, we established decision boundaries using a variety of machine-learning methods. These error rates depended upon the identity of the bases immediately 5' and 3' of the targeted CG dinucleotide, and ranged from 1.7% to 12.2% for a single-pass read. We estimate that Q40 values (0.01% error rates) for methylation status calls could be achieved by reading single molecules 5-19 times depending upon sequence context.

Complexes formed between phi29 DNA polymerase (DNAP) and DNA fluctuate discretely between the pre-translocation and post-translocation states on the millisecond time scale. The translocation fluctuations can be observed in ionic current traces when individual complexes are captured atop the α-hemolysin nanopore in an electric field. The presence of complementary 2'-deoxynucleoside triphosphate (dNTP) shifts the equilibrium across the translocation step toward the post-translocation state. Here we have determined quantitatively the kinetic relationship between the phi29 DNAP translocation step and dNTP binding. We demonstrate that dNTP binds to phi29 DNAP-DNA complexes only after the transition from the pre-translocation state to the post-translocation state; dNTP binding rectifies the translocation but it does not directly drive the translocation. Based on the measured time traces of current amplitude, we developed a method for determining the forward and reverse translocation rates and the dNTP association and dissociation rates, individually at each dNTP concentration and each voltage. The translocation rates, and their response to force, match those determined for phi29 DNAP-DNA binary complexes and are unaffected by dNTP. The dNTP association and dissociation rates do not vary as a function of voltage, indicating that force does not distort the polymerase active site and that dNTP binding does not directly involve a displacement in the translocation direction. This combined experimental and theoretical approach and the results obtained provide a framework for separately evaluating the effects of biological variables on the translocation transitions and their effects on dNTP binding.

Using nanopores to sequence biopolymers was proposed more than a decade ago. Recent advances in enzyme-based control of DNA translocation and in DNA nucleotide resolution using modified biological pores have satisfied two technical requirements of a functional nanopore DNA sequencing device. Nanopore sequencing of proteins was also envisioned. Although proteins have been shown to move through nanopores, a technique to unfold proteins for processive translocation has yet to be demonstrated. Here we describe controlled unfolding and translocation of proteins through the α-hemolysin (α-HL) pore using the AAA+ unfoldase ClpX. Sequence-dependent features of individual engineered proteins were detected during translocation. These results demonstrate that molecular motors can reproducibly drive proteins through a model nanopore--a feature required for protein sequence analysis using this single-molecule technology.

Complexes formed between the bacteriophage phi29 DNA polymerase (DNAP) and DNA fluctuate between the pre-translocation and post-translocation states on the millisecond time scale. These fluctuations can be directly observed with single-nucleotide precision in real-time ionic current traces when individual complexes are captured atop the α-hemolysin nanopore in an applied electric field. We recently quantified the equilibrium across the translocation step as a function of applied force (voltage), active-site proximal DNA sequences, and the binding of complementary dNTP. To gain insight into the mechanism of this step in the DNAP catalytic cycle, in this study, we have examined the stochastic dynamics of the translocation step. The survival probability of complexes in each of the two states decayed at a single exponential rate, indicating that the observed fluctuations are between two discrete states. We used a robust mathematical formulation based on the autocorrelation function to extract the forward and reverse rates of the transitions between the pre-translocation state and the post-translocation state from ionic current traces of captured phi29 DNAP-DNA binary complexes. We evaluated each transition rate as a function of applied voltage to examine the energy landscape of the phi29 DNAP translocation step. The analysis reveals that active-site proximal DNA sequences influence the depth of the pre-translocation and post-translocation state energy wells and affect the location of the transition state along the direction of the translocation.

Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.

An emerging DNA sequencing technique uses protein or solid-state pores to analyze individual strands as they are driven in single-file order past a nanoscale sensor. However, uncontrolled electrophoresis of DNA through these nanopores is too fast for accurate base reads. Here, we describe forward and reverse ratcheting of DNA templates through the α-hemolysin nanopore controlled by phi29 DNA polymerase without the need for active voltage control. DNA strands were ratcheted through the pore at median rates of 2.5-40 nucleotides per second and were examined at one nucleotide spatial precision in real time. Up to 500 molecules were processed at ∼130 molecules per hour through one pore. The probability of a registry error (an insertion or deletion) at individual positions during one pass along the template strand ranged from 10% to 24.5% without optimization. This strategy facilitates multiple reads of individual strands and is transferable to other nanopore devices for implementation of DNA sequence analysis.

During each catalytic cycle, DNA polymerases select deoxyribonucleoside triphosphate (dNTP) substrates complementary to a templating base with high fidelity from a pool that includes noncomplementary dNTPs and both complementary and noncomplementary ribonucleoside triphosphates (rNTPs). The Klenow fragment of Escherichia coli DNA polymerase I (KF) achieves this through a series of conformational transitions that precede the chemical step of phosphodiester bond formation. Kinetic evidence from fluorescence and FRET experiments indicates that discrimination of the base and sugar moieties of the incoming nucleotide occurs in distinct, sequential steps during the selection pathway. Here we show that KF-DNA complexes formed with complementary rNTPs or with noncomplementary nucleotides can be distinguished on the basis of their properties when captured in an electric field atop the α-hemolysin nanopore. The average nanopore dwell time of KF-DNA complexes increased as a function of complementary rNTP concentration. The increase was less than that promoted by complementary dNTP, indicating that the rNTP complexes are more stable than KF-DNA binary complexes but less stable than KF-DNA-dNTP ternary complexes. KF-DNA-rNTP complexes could also be distinguished from KF-DNA-dNTP complexes on the basis of ionic current amplitude. In contrast to complementary rNTPs, noncomplementary dNTPs and rNTPs diminished the average nanopore dwell time of KF-DNA complexes in a concentration-dependent manner, suggesting that binding of a noncomplementary nucleotide keeps the KF-DNA complex in a less stable state. These results imply that nucleotide selection proceeds through a series of complexes of increasing stability in which substrates with the correct moiety promote the forward transitions.

Coupling nucleic acid processing enzymes to nanoscale pores allows controlled movement of individual DNA or RNA strands that is reported as an ionic current/time series. Hundreds of individual enzyme complexes can be examined in single-file order at high bandwidth and spatial resolution. The bacteriophage phi29 DNA polymerase (phi29 DNAP) is an attractive candidate for this technology, due to its remarkable processivity and high affinity for DNA substrates. Here we show that phi29 DNAP-DNA complexes are stable when captured in an electric field across the α-hemolysin nanopore. DNA substrates were activated for replication at the nanopore orifice by exploiting the 3'-5' exonuclease activity of wild-type phi29 DNAP to excise a 3'-H terminal residue, yielding a primer strand 3'-OH. In the presence of deoxynucleoside triphosphates, DNA synthesis was initiated, allowing real-time detection of numerous sequential nucleotide additions that was limited only by DNA template length. Translocation of phi29 DNAP along DNA substrates was observed in real time at Ångstrom-scale precision as the template strand was drawn through the nanopore lumen during replication.

Nanopores can be used to analyse DNA by monitoring ion currents as individual strands are captured and driven through the pore in single file by an applied voltage. Here, we show that serial replication of individual DNA templates can be achieved by DNA polymerases held at the α-haemolysin nanopore orifice. Replication is blocked in the bulk phase, and is initiated only after the DNA is captured by the nanopore. We used this method, in concert with active voltage control, to observe DNA replication catalysed by bacteriophage T7 DNA polymerase (T7DNAP) and by the Klenow fragment of DNA polymerase I (KF). T7DNAP advanced on a DNA template against an 80-mV load applied across the nanopore, and single nucleotide additions were measured on the millisecond timescale for hundreds of individual DNA molecules in series. Replication by KF was not observed when this enzyme was held on top of the nanopore orifice at an applied potential of 80 mV. Sequential nucleotide additions by KF were observed upon applying controlled voltage reversals.

By controlling the microarchitecture of bioengineered scaffolds for artificial tissues, their material and cell-interaction properties can be designed to mimic native correspondents. Current understanding of this relationship is sparse and based on microscopy requiring harsh sample preparation and labeling, leaving it open to which extent the natural morphology is studied. This work introduces multimodal nonlinear microscopy for label-free imaging of tissue scaffolds, exemplified by bacterial cellulose. Unique three-dimensional images visualizing the formation of nanofiber networks throughout the biosynthesis, revealing that supra-structures (layered structures, cavities) are formed. Cell integration in compact scaffolds was visualized and compared with porous scaffolds. While the former showed distinct boundaries to the native tissue, gradual cell integration was observed for the porous material. Thus, the degree of cell integration can be controlled through scaffold supra-structures. This illustrates the potential of nonlinear microscopy for noninvasive imaging of the intriguing interaction mechanisms between scaffolds and cells.

DNA polymerases are molecular motors that catalyze template-dependent DNA replication, advancing along template DNA by one nucleotide with each catalytic cycle. Nanopore-based measurements have emerged as a single molecule technique for the study of these enzymes. Using the alpha-hemolysin nanopore, we determined the position of DNA templates bearing inserts of abasic (1',2'-dideoxy) residues, bound to the Klenow fragment of Escherichia coli DNA polymerase I (KF) or to bacteriophage T7 DNA polymerase. Hundreds of individual polymerase complexes were analyzed at 5 A precision within minutes. We generated a map of current amplitudes for DNA-KF-deoxynucleoside triphosphate (dNTP) ternary complexes, using a series of templates bearing blocks of three abasic residues that were displaced by approximately 5 A in the nanopore lumen. Plotted as a function of the distance of the abasic insert from n = 0 in the active site of the enzyme held atop the pore, this map has a single peak. The map is similar when the primer length, the DNA sequences flanking the abasic insert, and the DNA sequences in the vicinity of the KF active site are varied. Primer extension catalyzed by KF using a three abasic template in the presence of a mixture of dNTPs and 2',3'-dideoxynucleoside triphosphates resulted in a ladder of ternary complexes with discrete amplitudes that closely corresponded to this map. An ionic current map measured in the presence of 0.15 M KCl mirrored the map obtained with 0.3 M KCl, permitting experiments with a broader range of mesophilic DNA and RNA processing enzymes. We used the abasic templates to show that capture of complexes with the KF homologue, T7 DNA polymerase, yields an amplitude map nearly indistinguishable from the KF map.

DNA polymerases catalyze template-dependent genome replication. The assembly of a high affinity ternary complex between these enzymes, the double strand-single strand junction of their DNA substrate, and the deoxynucleoside triphosphate (dNTP) complementary to the first template base in the polymerase active site is essential to this process. We present a single molecule method for iterative measurements of DNA-polymerase complex assembly with high temporal resolution, using active voltage control of individual DNA substrate molecules tethered noncovalently in an alpha-hemolysin nanopore. DNA binding states of the Klenow fragment of Escherichia coli DNA polymerase I (KF) were diagnosed based upon their ionic current signature, and reacted to with submillisecond precision to execute voltage changes that controlled exposure of the DNA substrate to KF and dNTP. Precise control of exposure times allowed measurements of DNA-KF complex assembly on a time scale that superimposed with the rate of KF binding. Hundreds of measurements were made with a single tethered DNA molecule within seconds, and dozens of molecules can be tethered within a single experiment. This approach allows statistically robust analysis of the assembly of complexes between DNA and RNA processing enzymes and their substrates at the single molecule level.

Nanoscale pores are a tool for single molecule analysis of DNA or RNA processing enzymes. Monitoring catalytic activity in real time using this technique requires that these enzymes retain function while held atop a nanopore in an applied electric field. Using an alpha-hemolysin nanopore, we measured the dwell time for complexes of DNA with the Klenow fragment of Escherichia coli DNA polymerase I (KF) as a function of the concentration of deoxynucleoside triphosphate (dNTP) substrate. We analyzed these dwell time measurements in the framework of a two-state model for captured complexes (DNA-KF binary and DNA-KF-dNTP ternary states). Average nanopore dwell time increased without saturating as a function of correct dNTP concentration across 4 orders of magnitude. This arises from two factors that are proportional to dNTP concentration: (1) The fraction of complexes that are in the ternary state when initially captured predominantly affects dwell time at low dNTP concentrations. (2) The rate of binding and rebinding of dNTP to captured complexes affects dwell time at higher dNTP concentrations. Thus there are two regimes that display a linear relationship between average dwell time and dNTP concentration. The transition from one linear regime to the other occurs near the equilibrium dissociation constant (K(d)) for dNTP binding to KF-DNA complexes in solution. We conclude from the combination of titration experiments and modeling that DNA-KF complexes captured atop the nanopore retain iterative, sequence-specific dNTP binding, as required for catalysis and fidelity in DNA synthesis.