scholarly journals Quantifying epigenetic modulation of nucleosome breathing by high-throughput AFM imaging

2021 ◽  
Author(s):  
Sebastian Ferdinand Konrad ◽  
Willem Vanderlinden ◽  
Jan Lipfert
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Mode Of Action ◽  
Chromatin Accessibility ◽  
Wild Type ◽  
Analysis Pipeline ◽  
Data Set ◽  
Single Molecule Level ◽  
Afm Imaging ◽  
Conformational Landscape

Nucleosomes are the basic units of chromatin and critical to the storage and expression of eukaryotic genomes. Chromatin accessibility and gene readout are heavily regulated by epigenetic marks of which post-translational modifications of histones play a key role. However, the mode of action and the structural implications on the single-molecule level of nucleosomes is often still poorly understood. Here, we apply a high-throughput AFM imaging and analysis pipeline to investigate the conformational landscape of the nucleosome variants H3K36me3, H3S10phos and H4K5/8/12/16ac. Our data set of >25,000 nucleosomes reveals nucleosomal unwrapping steps corresponding to 5 bp DNA. We find that H3K36me3 nucleosomes unwrap significantly more than wild type nucleosomes and additionally unwrap stochastically from both sides similar to CENP-A nucleosomes and in contrast to the highly anti-cooperative unwrapping of wild type nucleosomes. Nucleosomes with H3S10phos or H4K5/8/12/16ac modifications show unwrapping populations similar to wild type nucleosomes and also retain the same level of anti-cooperativity. Our findings help putting the mode of action of these modifications into context: While H3K36me3 likely partially acts by directly affecting nucleosome structure on the single-molecule level, H3S10phos and H4K5/8/12/16ac must predominantly act through higher-order processes. Our analysis pipeline is readily applicable to other nucleosome variants and will facilitate future high-resolution studies of the conformational landscape of nucleoprotein complexes.

scholarly journals Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

2018 ◽  
Vol 115 (33) ◽  
pp. 8346-8351 ◽  
Author(s):  
Xiang Li ◽  
Yu Jiang ◽  
Shaorong Chong ◽  
David R. Walt
Keyword(s):  
Single Molecule ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Subunit Function ◽  
Enzyme Molecules ◽  
Individual Enzyme ◽  
Kinetics Of

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10−4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.

scholarly journals Simple nanofluidic devices for high-throughput, non-equilibrium studies at the single-molecule level

2017 ◽  
Author(s):  
Carel Fijen ◽  
Mattia Fontana ◽  
Serge G. Lemay ◽  
Klaus Mathwig ◽  
Johannes Hohlbein
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Single Particle Tracking ◽  
Biological Interactions ◽  
Dynamic Heterogeneity ◽  
High Throughput Analysis ◽  
Equilibrium Studies ◽  
Single Molecule Level ◽  
Nanofluidic Devices ◽  
And Performance

ABSTRACTSingle-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. Probing chemical and biological interactions and reactions with high throughput and time resolution, however, remains challenging and often requires surface-immobilized entities. Here, utilizing camera-based fluorescence microscopy, we present glass-made nanofluidic devices in which fluorescently labelled molecules flow through nanochannels that confine their diffusional movement. The first design features an array of parallel nanochannels for high-throughput analysis of molecular species under equilibrium conditions allowing us to record 200.000 individual localization events in just 10 minutes. Using these localizations for single particle tracking, we were able to obtain accurate flow profiles including flow speeds and diffusion coefficients inside the channels.A second design featuring a T-shaped nanochannel enables precise mixing of two different species as well as the continuous observation of chemical reactions. We utilized the design to visualize enzymatically driven DNA synthesis in real time and at the single-molecule level. Based on our results, we are convinced that the versatility and performance of the nanofluidic devices will enable numerous applications in the life sciences.

scholarly journals DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

Applied Nano ◽  
2022 ◽  
Vol 3 (1) ◽  
pp. 16-41
Author(s):  
Aurimas Kopūstas ◽  
Mindaugas Zaremba ◽  
Marijonas Tutkus
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Dna Interaction ◽  
Dna Interactions ◽  
Ensemble Averaging ◽  
Target Search ◽  
Single Molecule Level ◽  
Protein Dna Interactions ◽  
Long Time

Protein-DNA interactions are the core of the cell’s molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surface-immobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms.

SNARE-seq2 v2

2021 ◽  
Author(s):  
Nongluk Plongthongkum ◽  
Dinh H Diep ◽  
Song Chen ◽  
Blue Lake ◽  
Kun Zhang
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Chromatin Accessibility ◽  
Single Experiment ◽  
Data Set ◽  
Whole Cells ◽  
High Throughput Method ◽  
Single Nucleus ◽  
Accessible Chromatin ◽  
Entire Procedure

To study the heterogeneity of complex tissues by joint profiling of gene expression and its regulation, we require an accurate and high-throughput method. Here we described improved high-throughput combinatorial indexing-based single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-Seq2) co-assay. This protocol involves fixing and permeabilizing the nucleus followed by tagmentation, chromatin barcode ligation, reverse transcription, pooling and splitting for the next rounds of cell barcode ligation into cDNA and accessible chromatin (AC) on the same nucleus. The captured cDNA and AC are co-amplified before splitting and enrichment into single-nucleus RNA and single-nucleus AC sequencing libraries. The protocol can also be applied to both nuclei and whole cells to capture mRNA in the cytoplasm. This improvement allows us to generate hundreds of thousands of data set of each assay and can be scaled up to half a million cells from a single experiment. The entire procedure can be complete in 3.5 d for generating joint single-nucleus RNA and single-nucleus ATAC sequencing libraries.

scholarly journals Mobility of nucleosomes is regulated by their binding partners

2022 ◽  
Author(s):  
Daniel P Melters ◽  
Keir C Neuman ◽  
Tatini Rakshit ◽  
Yamini Dalal
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
High Speed ◽  
Diffusion Constant ◽  
Chromatin Accessibility ◽  
Chromatin Dynamics ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force

Chromatin accessibility is modulated in a variety of ways, both to create open and closed chromatin states which are critical for eukaryotic gene regulation. At the mechanistic single molecule level, how accessibility is regulated remains a fundamental question in the field. Here, we use single molecule tracking by high-speed atomic force microscopy to investigate this question using chromatin arrays and extend our findings into the nucleus. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and observed that the essential kinetochore protein CENP-C reduces the diffusion constant of CENP-A nucleosomes and the linker H1.5 protein restricts H3 nucleosome mobility. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. These data suggest a model in which inner kinetochore proteins are critically involved in modulating chromatin accessibility and consequently, noncoding transcription at human centromeres.

SNARE-seq2 v2

2021 ◽  
Author(s):  
Nongluk Plongthongkum ◽  
Dinh H Diep ◽  
Song Chen ◽  
Blue Lake ◽  
Kun Zhang
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Chromatin Accessibility ◽  
Single Experiment ◽  
Data Set ◽  
Whole Cells ◽  
High Throughput Method ◽  
Single Nucleus ◽  
Accessible Chromatin ◽  
Entire Procedure

To study the heterogeneity of complex tissues by joint profiling of gene expression and its regulation, we require an accurate and high-throughput method. Here we described improved high-throughput combinatorial indexing-based single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-Seq2) co-assay. This protocol involves fixing and permeabilizing the nucleus followed by tagmentation, chromatin barcode ligation, reverse transcription, pooling and splitting for the next rounds of cell barcode ligation into cDNA and accessible chromatin (AC) on the same nucleus. The captured cDNA and AC are co-amplified before splitting and enrichment into single-nucleus RNA and single-nucleus AC sequencing libraries. The protocol can also be applied to both nuclei and whole cells to capture mRNA in the cytoplasm. This improvement allows us to generate hundreds of thousands of data set of each assay and can be scaled up to half a million cells from a single experiment. The entire procedure can be complete in 3.5 d for generating joint single-nucleus RNA and single-nucleus ATAC sequencing libraries.

VISUALIZATION EX SITU OF SINGLE DNA MOLECULES INCUBATION: A FIRST STEP FOR QUANTITATIVE ANALYSIS ON MULTI-SITE DEGRADATION AND ENZYMATIC KINETICS

2009 ◽  
Vol 16 (01) ◽  
pp. 79-85
Author(s):  
XINYAN WANG ◽  
HAIJUN YANG ◽  
HUABIN WANG ◽  
PENG WANG ◽  
HAI LI
Keyword(s):  
Single Molecule ◽  
Ex Situ ◽  
In Vitro Tests ◽  
Dna Molecules ◽  
Single Molecule Level ◽  
Single Dna Molecules ◽  
Enzymatic Kinetics ◽  
Afm Imaging ◽  
Molecular Combing

Herein, we showed a different approach to directly single-molecule level visualization of the degradation of DNA in vitro tests using DNase I incubation based on high-resolution AFM imaging ex situ with fine relocation nanotechnology. A method of nanomanipulation termed as "modified dynamic molecular combing" (MDMC) was used to pattern DNA samples for further degradation and enzymatic kinetics. This strategy is potentially able to quantitatively address the mechanical-induced kinetic profiles of multi-site degradation of individual DNA molecules with very stable tension and strong immobilization on a surface and discover the mechanochemistry.

scholarly journals In Silico Model for Chemical-Induced Chromosomal Damages Elucidates Mode of Action and Irrelevant Positives

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1181
Author(s):  
Yurika Fujita ◽  
Osamu Morita ◽  
Hiroshi Honda
Keyword(s):  
Functional Groups ◽  
High Throughput ◽  
Mode Of Action ◽  
In Silico ◽  
High Throughput Screening ◽  
Data Set ◽  
Chemical Structures ◽  
In Silico Model ◽  
Organic Functional Groups ◽  
In Silico Tools

In silico tools to predict genotoxicity have become important for high-throughput screening of chemical substances. However, current in silico tools to evaluate chromosomal damage do not discriminate in vitro-specific positives that can be followed by in vivo tests. Herein, we establish an in silico model for chromosomal damages with the following approaches: (1) re-categorizing a previous data set into three groups (positives, negatives, and misleading positives) according to current reports that use weight-of-evidence approaches and expert judgments; (2) utilizing a generalized linear model (Elastic Net) that uses partial structures of chemicals (organic functional groups) as explanatory variables of the statistical model; and (3) interpreting mode of action in terms of chemical structures identified. The accuracy of our model was 85.6%, 80.3%, and 87.9% for positive, negative, and misleading positive predictions, respectively. Selected organic functional groups in the models for positive prediction were reported to induce genotoxicity via various modes of actions (e.g., DNA adduct formation), whereas those for misleading positives were not clearly related to genotoxicity (e.g., low pH, cytotoxicity induction). Therefore, the present model may contribute to high-throughput screening in material design or drug discovery to verify the relevance of estimated positives considering their mechanisms of action.

scholarly journals Cellular Lensing and Near Infrared Fluorescent Nanosensor Arrays to Enable Chemical Efflux Cytometry

2020 ◽  
Author(s):  
Soo-Yeon Cho ◽  
Xun Gong ◽  
Volodymyr Koman ◽  
Matthias Kuehne ◽  
Sun Jin Moon ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Near Infrared ◽  
Microfluidic Channel ◽  
Human Monocyte ◽  
Cellular Heterogeneity ◽  
Label Free ◽  
Temporal Precision ◽  
Single Molecule Level ◽  
Chemical Cytometry

Abstract Nanosensor have proven to be powerful tools to monitor single biological cells and organisms, achieving spatial and temporal precision even at the single molecule level. However, there has not been a way of extending this approach to statistically relevant numbers of living cells and organisms. Herein, we design and fabricate a high throughput nanosensor array in a microfluidic channel that addresses this limitation, creating a Nanosensor Chemical Cytometry (NCC). An array of nIR fluorescent single walled carbon nanotube (SWNT) nanosensors is integrated along a microfluidic channel through which a population of flowing cells is guided. We show that one can utilize the flowing cell itself as highly informative Gaussian lenses projecting nIR emission profiles and extract rich information on a per cell basis at high throughput. This unique biophotonic waveguide allows for quantified cross-correlation of the biomolecular information with physical properties such as cellular diameter, refractive index (RI), and eccentricity and creates a label-free chemical cytometer for the measurement of cellular heterogeneity with unprecedented precision. As an example, the NCC can profile the immune response heterogeneities of distinct human monocyte populations at attomolar (10-18 moles) sensitivity in a completely non-destructive and real-time manner with a rate of ~100 cells/frame, highest range demonstrated to date for state of the art chemical cytometry. We demonstrate distinct H2O2 efflux heterogeneities between 330 and 624 attomole/cell·min with cell projected areas between 271 and 263 µm2, eccentricity values between 0.405 and 0.363 and RI values between 1.383 and 1.377 for non-activated and activated human monocytes, respectively. Hence, we show that our nanotechnology based biophotonic cytometer has significant potential and versatility to answer important questions and provide new insight in immunology, cell manufacturing and biopharmaceutical research.

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