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 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

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 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.

Biology on track: single-molecule visualisation of protein dynamics on linear DNA substrates

2020 ◽  
Author(s):  
Gurleen Kaur ◽  
Lisanne M. Spenkelink
Keyword(s):  
Single Molecule ◽  
Molecular Motor ◽  
Imaging Techniques ◽  
Biological Research ◽  
Dna Interactions ◽  
Single Molecule Fluorescence ◽  
Linear Dna ◽  
Protein Dna Interactions ◽  
Dna Substrates

Abstract Single-molecule fluorescence imaging techniques have become important tools in biological research to gain mechanistic insights into cellular processes. These tools provide unique access to the dynamic and stochastic behaviour of biomolecules. Single-molecule tools are ideally suited to study protein–DNA interactions in reactions reconstituted from purified proteins. The use of linear DNA substrates allows for the study of protein–DNA interactions with observation of the movement and behaviour of DNA-translocating proteins over long distances. Single-molecule studies using long linear DNA substrates have revealed unanticipated insights on the dynamics of multi-protein systems. In this review, we provide an overview of recent methodological advances, including the construction of linear DNA substrates. We highlight the versatility of these substrates by describing their application in different single-molecule fluorescence techniques, with a focus on in vitro reconstituted systems. We discuss insights from key experiments on DNA curtains, DNA-based molecular motor proteins, and multi-protein systems acting on DNA that relied on the use of long linear substrates and single-molecule visualisation. The quality and customisability of linear DNA substrates now allows the insertion of modifications, such as nucleosomes, to create conditions mimicking physiologically relevant crowding and complexity. Furthermore, the current technologies will allow future studies on the real-time visualisation of the interfaces between DNA maintenance processes such as replication and transcription.

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