Revealing dynamics of helicase translocation on single-stranded DNA using high-resolution nanopore tweezers

Craig, Jonathan M.; Laszlo, Andrew H.; Brinkerhoff, Henry; Derrington, Ian M.; Noakes, Matthew T.; Nova, Ian C.; Tickman, Benjamin I.; Doering, Kenji; Leeuw, Noah F. de; Gundlach, Jens H.

doi:10.1073/pnas.1711282114

Cited by 61 publications

(93 citation statements)

References 45 publications

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“…Ideally, the DNA-controlling enzyme would move DNA unidirectionally through the pore in discrete steps of uniform length. However, the stochastic stepping of enzymes frequently diverges from this ideal behavior 26 . In addition to regular forward steps, “skips” can occur when multiple forward steps occur in quick succession, too fast to electronically resolve the intermediate step or steps.…”

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confidence: 99%

“…Improved single-read accuracy should enable fewer reads to be assembled into a high-accuracy consensus sequence, thereby reducing sequencing time and cost. Additionally, variable-voltage sequencing overcomes systematic errors, such as sequence-dependent enzyme mis-steps 26 and indistinguishable signals, that persist even when the information from many reads is combined. Variable-voltage reads can be more confidently identified with only single-read coverage.…”

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confidence: 99%

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Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage

et al. 2019

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Nanopore DNA sequencing is limited by low base calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage across an MspA nanopore between 100 to 200 mV with a frequency of 200 Hz, changing how the DNA strand moves through the nanopore. As a DNA helicase moves the DNA through the nanopore in discrete steps, the variable voltage positions the DNA continuously between these steps. The resulting electronic signal can be analysed to overcome the primary error modes in base-calling. Single-passage de novo base-calling accuracy in our device increases from 62.7 ± 0.5% with a constant driving voltage to 79.3 ± 0.3% with a variable driving voltage. Our variable-voltage sequencing mode is complementary to other advances in nanopore sequencing and is amenable to incorporation into other enzyme-actuated nanopore sequencing devices.

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Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage

et al. 2019

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“…Single-molecule studies have greatly expanded our knowledge of the mode of action and kinetics of DNA-protein interactions at the nanoscale 1 . Single-molecule Förster resonance energy transfer (smFRET) and optical/magnetic tweezers, for example, are well suited techniques to study forces, conformational changes and displacements of DNA-binding proteins such as DNA and RNA polymerases 2,3 , helicases 4,5 and CRISPR-Cas proteins 6,7 in vitro with high spatiotemporal resolution [8][9][10][11] . In vivo, however, single-particle tracking (SPT) remains the most convenient choice to study dynamic interactions 12 .…”

Section: Introductionmentioning

confidence: 99%

Extracting transition rates in single-particle tracking using analytical diffusion distribution analysis

Vink

Brouns

Hohlbein

2020

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Single-particle tracking is an important technique in the life sciences to understand the kinetics of biomolecules. Observed diffusion coefficients in vivo, for example, enable researchers to determine whether biomolecules are moving alone, as part of a larger complex or are bound to large cellular components such as the membrane or chromosomal DNA. A remaining challenge has been to retrieve quantitative kinetic models especially for molecules that rapidly interchange between different diffusional states. Here, we present analytic diffusion distribution analysis (anaDDA), a framework that allows extracting transition rates from distributions of observed diffusion coefficients. We show that theoretically predicted distributions accurately match simulated distributions and that anaDDA outperforms existing methods to retrieve kinetics especially in the fast regime of 0.1-10 transitions per imaging frame. AnaDDA does account for the effects of confinement and tracking window boundaries. Furthermore, we added the option to perform global fitting of data acquired at different frame times, to allow complex models with multiple states to be fitted confidently. Previously, we have started to develop anaDDA to investigate the target search of CRISPR-Cas complexes. In this work, we have optimized the algorithms and reanalysed experimental data of DNA polymerase I diffusing in live E. coli. We found that long-lived DNA interaction by DNA polymerase are more abundant upon DNA damage, suggesting roles in DNA repair. We further revealed and quantified fast DNA probing interactions that last shorter than 10 ms. AnaDDA pushes the boundaries of the timescale of interactions that can be probed with single-particle tracking and is a mathematically rigorous framework that can be further expanded to extract detailed information about the behaviour of biomolecules in living cells. the apparent diffusion coefficient D* is calculated from the mean squared displacement (MSD).The different mobilities of proteins switching between a DNA-bound state, in which proteins diffuse very slowly, and a DNA-free state, in which proteins diffuse through the cytoplasm, can provide kinetic information on the frequency and longevity of DNA-protein interactions.The ability to extract this information, however, is compromised by photo bleaching, which limits the length of each track to a few localisations in the case of fluorescent proteins 19 . Furthermore, the limited localization precision increases the apparent diffusion of immobile states. Therefore, measured displacements cannot be unambiguously assigned to either a bound or a diffusing state.As a consequence, histograms of D* values are often rather broad making a clear distinction between two diffusional states of a single species impossible. For the special case of noninterconverting D* distributions, the shape of distributions can be calculated for a fixed number of analysed steps 20,21 and, via fitting of the experimental data, used to extract the fractions of mobile and immobile proteins.Anothe...

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“…Even though this technology avoids synthesis of the complementary strand, and hence enables direct sequencing, it is assisted by a processing enzyme that slows down the otherwise too fast translocation and acts as a motor to move the DNA/RNA strand one nucleobase at a time. 19-21 Engineered versions of α –HL and MsPA with enhanced sensing capabilities report i-t data that vary as a function of a subsequence of, at least, 4 bases, and not as a function of a single nucleotide traversing the pore’s constriction point. 5 Assuming that each 4-base subsequence exhibits a distinct ion current level, I r (Figure 1C), there are 4 4 =256 I r levels to be discriminated within approximately 80 picoAmperes (pA) of total measurement space 22 with a standard deviation around ±2 pA, making base calling a challenging task.…”

Section: Introductionmentioning

confidence: 99%

Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

Sultan

Kanavarioti

2019

Preprint

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Nanopores, both protein and solid-state, are explored as single molecule analytical tools, but using an experimental platform is challenging. Here we show that a commercially available nanopore device, MinION from Oxford Nanopore Technologies (ONT), successfully accomplishes a task challenging for a conventional analytical tool. Specifically the MinION discriminates among 31 nucleotide (nt) long oligoriboadenylates with a single pyrimidine (Py) substitution, when this pyrimidine is tagged/labeled with a bulky group (Osmium tetroxide 2,2’-bipyridine or OsBp). This platform also discriminates between an osmylated Py (Py-OsBp) followed by a purine (Pu) and a Py-OsBp followed by a second Py-OsBp, leading to the conjecture that the bulky tag enables sensing of a two-nucleotide sequence. Two-nucleotide sensing could greatly improve base-calling accuracy in motor enzyme-assisted nanopore sequencing.We attribute the observed discrimination neither to the specific pore protein nor to OsBp, but to the tag’s bulkiness, that leads to markedly slower translocation and “touching” proximity at the pore’s constriction zone, that forces desolvation and reorganization, and enables strong interactions among the nanopore, the tagged pyrimidine, and the adjacent nucleobase. These results constitute proof-of-principle that size-suitable nanopores may be superior to traditional analytical tools, for the characterization of RNA oligos and microRNAs enhanced by selective labelling.

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Revealing dynamics of helicase translocation on single-stranded DNA using high-resolution nanopore tweezers

Cited by 61 publications

References 45 publications

Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage

Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage

Extracting transition rates in single-particle tracking using analytical diffusion distribution analysis

Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

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