scholarly journals NAP1-Assisted Nucleosome Assembly on DNA Measured in Real Time by Single-Molecule Magnetic Tweezers

PLoS ONE ◽  
2012 ◽  
Vol 7 (9) ◽  
pp. e46306 ◽  
Author(s):  
Rifka Vlijm ◽  
Jeremy S. J. Smitshuijzen ◽  
Alexandra Lusser ◽  
Cees Dekker
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Magnetic Tweezers ◽  
Nucleosome Assembly

scholarly journals NAP1-Assisted Nucleosome Assembly on DNA Measured in Real Time by Single-Molecule Magnetic Tweezers

2013 ◽  
Vol 104 (2) ◽  
pp. 38a-39a
Author(s):  
Rifka Vlijm ◽  
Jeremy S.J. Smitshuijzen ◽  
Alexandra Lusser ◽  
Cees Dekker
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Magnetic Tweezers ◽  
Nucleosome Assembly

scholarly journals Real-time detection of condensin-driven DNA compaction reveals a multistep binding mechanism

2017 ◽  
Author(s):  
Jorine M. Eeftens ◽  
Shveta Bisht ◽  
Jacob Kerssemakers ◽  
Christian H. Haering ◽  
Cees Dekker
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Electrostatic Interactions ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Magnetic Tweezers ◽  
Condensed State ◽  
Binding Modes ◽  
Dna Molecules ◽  
Dna Compaction

ABSTRACTCondensin, a conserved member of the SMC protein family of ring-shaped multi-subunit protein complexes, is essential for structuring and compacting chromosomes. Despite its key role, its molecular mechanism has remained largely unknown. Here, we employ single-molecule magnetic tweezers to measure, in real-time, the compaction of individual DNA molecules by the budding yeast condensin complex. We show that compaction proceeds in large (~200nm) steps, driving DNA molecules into a fully condensed state against forces of up to 2pN. Compaction can be reversed by applying high forces or adding buffer of high ionic strength. While condensin can stably bind DNA in the absence of ATP, ATP hydrolysis by the SMC subunits is required for rendering the association salt-insensitive and for subsequent compaction. Our results indicate that the condensin reaction cycle involves two distinct steps, where condensin first binds DNA through electrostatic interactions before using ATP hydrolysis to encircle the DNA topologically within its ring structure, which initiates DNA compaction. The finding that both binding modes are essential for its DNA compaction activity has important implications for understanding the mechanism of chromosome compaction.

scholarly journals Rescuing Replication from Barriers: Mechanistic Insights from Single-Molecule Studies

2019 ◽  
Vol 39 (10) ◽  
Author(s):  
Bo Sun
Keyword(s):  
Real Time ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Replication Fork ◽  
Magnetic Tweezers ◽  
Origin Of Replication ◽  
Replication Fork Progression ◽  
Nucleotide Incorporation ◽  
Single Molecule Studies ◽  
Replication Failure

ABSTRACT To prevent replication failure due to fork barriers, several mechanisms have evolved to restart arrested forks independent of the origin of replication. Our understanding of these mechanisms that underlie replication reactivation has been aided through unique dynamic perspectives offered by single-molecule techniques. These techniques, such as optical tweezers, magnetic tweezers, and fluorescence-based methods, allow researchers to monitor the unwinding of DNA by helicase, nucleotide incorporation during polymerase synthesis, and replication fork progression in real time. In addition, they offer the ability to distinguish DNA intermediates after obstacles to replication at high spatial and temporal resolutions, providing new insights into the replication reactivation mechanisms. These and other highlights of single-molecule techniques and remarkable studies on the recovery of the replication fork from barriers will be discussed in this review.

scholarly journals Multiplexed Nanometric 3D Tracking of Microbeads using a FFT-Phasor Algorithm

2019 ◽  
Author(s):  
T. B. Brouwer ◽  
N. Hermans ◽  
J. van Noort
Keyword(s):  
Diffraction Pattern ◽  
Real Time ◽  
Single Molecule ◽  
Fourier Transforms ◽  
Magnetic Tweezers ◽  
Fast Fourier Transforms ◽  
Tracking Algorithm ◽  
Wide Field ◽  
3D Tracking ◽  
Chromatin Fibers

AbstractMany single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorentz Mie scattering theory yields the bead position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method. Here, we introduce 3D phasor tracking, a fast and robust bead tracking algorithm with nanometric localization accuracy in a z-range of over 10 µm. The algorithm is based on a 2D cross-correlation using Fast Fourier Transforms with computer-generated reference images, yielding a processing rate of up to 10.000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real-time on a generic CPU. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead tracking applications, especially where high throughput is required.SignificanceMicrobeads are often used in biophysical single-molecule manipulation experiments and accurately tracking their position in 3 dimensions is key for quantitative analysis. Holographic imaging of these beads allows for multiplexing bead tracking but image analysis can be a limiting factor. Here we present a 3D tracking algorithm based on Fast Fourier Transforms that is fast, has nanometric precision, is robust against common artifacts and is accurate over 10’s of micrometers. We show its real-time application for magnetic tweezers based force spectroscopy on more than 100 chromatin fibers in parallel, but we anticipate that many other bead-based biophysical essays can benefit from this simple and robust 3 phasor algorithm.

scholarly journals Combined Transcriptome Analysis Reveals the Ovule Abortion Regulatory Mechanisms in the Female Sterile Line of Pinus tabuliformis Carr.

2021 ◽  
Vol 22 (6) ◽  
pp. 3138
Author(s):  
Zaixin Gong ◽  
Rui Han ◽  
Li Xu ◽  
Hailin Hu ◽  
Min Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Regulatory Mechanisms ◽  
Cdna Libraries ◽  
Ovule Development ◽  
Ovule Abortion ◽  
Pinus Tabuliformis ◽  
Wild Type Female ◽  
Fertile Line ◽  
Next Generation Sequencing Ngs

Ovule abortion is a common phenomenon in plants that has an impact on seed production. Previous studies of ovule and female gametophyte (FG) development have mainly focused on angiosperms, especially in Arabidopsis thaliana. However, because it is difficult to acquire information about ovule development in gymnosperms, this remains unclear. Here, we investigated the transcriptomic data of natural ovule abortion mutants (female sterile line, STE) and the wild type (female fertile line, FER) of Pinus tabuliformis Carr. to evaluate the mechanism of ovule abortion during the process of free nuclear mitosis (FNM). Using single-molecule real-time (SMRT) sequencing and next-generation sequencing (NGS), 18 cDNA libraries via Illumina and two normalized libraries via PacBio, with a total of almost 400,000 reads, were obtained. Our analysis showed that the numbers of isoforms and alternative splicing (AS) patterns were significantly variable between FER and STE. The functional annotation results demonstrate that genes involved in the auxin response, energy metabolism, signal transduction, cell division, and stress response were differentially expressed in different lines. In particular, AUX/IAA, ARF2, SUS, and CYCB had significantly lower expression in STE, showing that auxin might be insufficient in STE, thus hindering nuclear division and influencing metabolism. Apoptosis in STE might also have affected the expression levels of these genes. To confirm the transcriptomic analysis results, nine pairs were confirmed by quantitative real-time PCR. Taken together, these results provide new insights into ovule abortion in gymnosperms and further reveal the regulatory mechanisms of ovule development.

scholarly journals A machine learning approach for accurate and real-time DNA sequence identification

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yiren Wang ◽  
Mashari Alangari ◽  
Joshua Hihath ◽  
Arindam K. Das ◽  
M. P. Anantram
Keyword(s):  
Real Time ◽  
Dna Sequence ◽  
Single Molecule ◽  
Identification Accuracy ◽  
Measuring System ◽  
Identification System ◽  
Genetic Sequencing ◽  
Sequence Identification ◽  
Tree Classifier ◽  
Single Mismatch

Abstract Background The all-electronic Single Molecule Break Junction (SMBJ) method is an emerging alternative to traditional polymerase chain reaction (PCR) techniques for genetic sequencing and identification. Existing work indicates that the current spectra recorded from SMBJ experimentations contain unique signatures to identify known sequences from a dataset. However, the spectra are typically extremely noisy due to the stochastic and complex interactions between the substrate, sample, environment, and the measuring system, necessitating hundreds or thousands of experimentations to obtain reliable and accurate results. Results This article presents a DNA sequence identification system based on the current spectra of ten short strand sequences, including a pair that differs by a single mismatch. By employing a gradient boosted tree classifier model trained on conductance histograms, we demonstrate that extremely high accuracy, ranging from approximately 96 % for molecules differing by a single mismatch to 99.5 % otherwise, is possible. Further, such accuracy metrics are achievable in near real-time with just twenty or thirty SMBJ measurements instead of hundreds or thousands. We also demonstrate that a tandem classifier architecture, where the first stage is a multiclass classifier and the second stage is a binary classifier, can be employed to boost the single mismatched pair’s identification accuracy to 99.5 %. Conclusions A monolithic classifier, or more generally, a multistage classifier with model specific parameters that depend on experimental current spectra can be used to successfully identify DNA strands.

Highly Sensitive Detection of Paraquat with Pillar[5]arene as an aptamer in α-Hemolysin Nanopore

2021 ◽  
Author(s):  
Xiaojia Jiang ◽  
Mingsong Zang ◽  
Fei Li ◽  
Chunxi Hou ◽  
Quan Luo ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Single Molecule Detection ◽  
Sensitive Detection ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
The Real ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

Biological nanopore-based techniques have attracted more and more attention recently in the field of single-molecule detection, because they allow the real-time, sensitive, high-throughput analysis. Herein, we report an engineered biological...

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