DiMeLo-seq: Directed Methylation with Long-read sequencing v2

2021 ◽  
Author(s):  
Nicolas Altemose ◽  
Annie Maslan ◽  
Owen Smith ◽  
Kousik Sundararajan ◽  
Rachel Brown ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Pcr Amplification ◽  
Dna Interaction ◽  
Dna Interactions ◽  
Single Molecule Level ◽  
Multiple Protein ◽  
Protein Dna Interactions ◽  
Adenine Methylation ◽  
Long Read

Directed Methylation and Long-read sequencing (DiMeLo-seq) is a powerful method to map protein-DNA interactions at a single-molecule level across the genome (including repetitive regions). It can be multiplexed to analyze multiple base modifications at once (e.g. endogenous CpG methylation and directed pA-Hia5 adenine methylation). Additionally, PCR amplification is not necessary for this protocol, which means that sequencing readout is proportional to protein-DNA interaction frequency. Finally, DiMeLo-seq can be used to map multiple protein interactions across a long single molecule.

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.

scholarly journals Stretching and immobilization of DNA for studies of protein–DNA interactions at the single-molecule level

2007 ◽  
Vol 2 (4) ◽  
pp. 185-201 ◽  
Author(s):  
Ji Hoon Kim ◽  
Venkat Ram Dukkipati ◽  
Stella W. Pang ◽  
Ronald G. Larson
Keyword(s):  
Single Molecule ◽  
Dna Interactions ◽  
Single Molecule Level ◽  
Protein Dna Interactions

Nanoscale “Curtain Rods”: High-Throughput Tools for Studying DNA-Protein Interactions

2008 ◽  
Vol 1138 ◽  
Author(s):  
Teresa Fazio ◽  
Mari-Liis Visnapuu ◽  
Shalom J. Wind ◽  
Eric Greene
Keyword(s):  
Data Acquisition ◽  
Real Time ◽  
Lipid Bilayer ◽  
High Throughput ◽  
Single Molecule ◽  
Protein Interactions ◽  
Massively Parallel ◽  
Dna Interactions ◽  
Parallel Data ◽  
Protein Dna Interactions

AbstractIn this work, we combine nanoscale engineering with single-molecule biology to probe the biochemical interactions between individual proteins and DNA. This approach, a vast improvement over previous methods, constructs a platform to observe thousands of protein-DNA interactions in real time with unprecedented detail. A key challenge in these experiments involves collecting enough statistically relevant data in order to analyze reactions which are designed to be probed individually. “DNA curtains” are formed by flowing the DNA tethered to a lipid bilayer across nanopatterned barriers, facilitating massively parallel data acquisition.

scholarly journals Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level

2013 ◽  
Vol 14 (2) ◽  
pp. 3961-3992 ◽  
Author(s):  
Carina Monico ◽  
Marco Capitanio ◽  
Gionata Belcastro ◽  
Francesco Vanzi ◽  
Francesco Pavone
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Optical Methods ◽  
Dna Interactions ◽  
Single Molecule Level ◽  
Protein Dna Interactions

scholarly journals DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome-wide

2021 ◽  
Author(s):  
Nicolas Altemose ◽  
Annie Maslan ◽  
Owen K Smith ◽  
Kousik Sundararajan ◽  
Rachel R Brown ◽  
...  
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Dna Amplification ◽  
Mapping Method ◽  
Dna Interactions ◽  
Short Reads ◽  
Interaction Sites ◽  
Genome Wide ◽  
Protein Dna Interactions ◽  
Long Read

Molecular studies of genome regulation often rely on the ability to map where specific proteins interact with genomic DNA. Existing techniques for mapping protein-DNA interactions genome-wide rely on DNA amplification methods followed by sequencing with short reads, which dissociates joint binding information at neighboring sites, removes endogenous DNA methylation information, and precludes the ability to reliably map interactions in repetitive regions of the genome. To address these limitations, we created a new protein-DNA mapping method, called Directed Methylation with Long-read sequencing (DiMeLo-seq), which methylates DNA near each target protein's DNA binding site in situ, then leverages the ability to distinguish methylated and unmethylated bases on long, native DNA molecules using long-read, single-molecule sequencing technologies. We demonstrate the optimization and utility of this method by mapping the interaction sites of a variety of different proteins and histone modifications across the human genome, achieving a single-molecule binding site resolution of less than 200 bp. Furthermore, we mapped the positions of the centromeric histone H3 variant CENP-A in repetitive regions that are unmappable with short reads, while simultaneously analyzing endogenous CpG methylation and joint binding events on single molecules. DiMeLo-seq is a versatile method that can provide multimodal and truly genome-wide information for investigating protein-DNA interactions.

scholarly journals Visualizing protein–DNA interactions at the single-molecule level

2010 ◽  
Vol 14 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Jovencio Hilario ◽  
Stephen C Kowalczykowski
Keyword(s):  
Single Molecule ◽  
Dna Interactions ◽  
Single Molecule Level ◽  
Protein Dna Interactions

scholarly journals Low biological fluctuation of mitochondrial CpG and non-CpG methylation at the single-molecule level

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chloe Goldsmith ◽  
Jesús Rafael Rodríguez-Aguilera ◽  
Ines El-Rifai ◽  
Adrien Jarretier-Yuste ◽  
Valérie Hervieu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Single Molecule ◽  
Nuclear Dna ◽  
Pcr Amplification ◽  
Nanopore Sequencing ◽  
High Coverage ◽  
Single Molecule Level ◽  
Modified Dna ◽  
Long Read ◽  
Low Levels

AbstractMammalian cytosine DNA methylation (5mC) is associated with the integrity of the genome and the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mC has a regulatory role in this context. Here, we used bisulfite-independent single-molecule sequencing of native human and mouse DNA to study mitochondrial 5mC across different biological conditions. We first validated the ability of long-read nanopore sequencing to detect 5mC in CpG (5mCpG) and non-CpG (5mCpH) context in nuclear DNA at expected genomic locations (i.e. promoters, gene bodies, enhancers, and cell type-specific transcription factor binding sites). Next, using high coverage nanopore sequencing we found low levels of mtDNA CpG and CpH methylation (with several exceptions) and little variation across biological processes: differentiation, oxidative stress, and cancer. 5mCpG and 5mCpH were overall higher in tissues compared to cell lines, with small additional variation between cell lines of different origin. Despite general low levels, global and single-base differences were found in cancer tissues compared to their adjacent counterparts, in particular for 5mCpG. In conclusion, nanopore sequencing is a useful tool for the detection of modified DNA bases on mitochondria that avoid the biases introduced by bisulfite and PCR amplification. Enhanced nanopore basecalling models will provide further resolution on the small size effects detected here, as well as rule out the presence of other DNA modifications such as oxidized forms of 5mC.

scholarly journals Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexandra Stützer ◽  
Luisa M. Welp ◽  
Monika Raabe ◽  
Timo Sachsenberg ◽  
Christin Kappert ◽  
...  
Keyword(s):  
Uv Light ◽  
Amino Acid Level ◽  
Dna Interaction ◽  
Cross Linking ◽  
Dna Interactions ◽  
Single Experiment ◽  
Protein Dna Interactions ◽  
Nucleosome Binding ◽  
Binding Interfaces

Abstract Protein–DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein–DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein–RNA and DNA interactions in one single experiment, we project that it will be possible to obtain insights into chromatin and its regulation in the future.

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