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 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

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.

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 Single-Molecule Kinetics in Living Cells

2019 ◽  
Vol 88 (1) ◽  
pp. 635-659 ◽  
Author(s):  
Johan Elf ◽  
Irmeli Barkefors
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
The Past

In the past decades, advances in microscopy have made it possible to study the dynamics of individual biomolecules in vitro and resolve intramolecular kinetics that would otherwise be hidden in ensemble averages. More recently, single-molecule methods have been used to image, localize, and track individually labeled macromolecules in the cytoplasm of living cells, allowing investigations of intermolecular kinetics under physiologically relevant conditions. In this review, we illuminate the particular advantages of single-molecule techniques when studying kinetics in living cells and discuss solutions to specific challenges associated with these methods.

scholarly journals An Optical Setup for the Study of Mechanotransduction in Living Cells at the Single Molecule Level

2017 ◽  
Vol 112 (3) ◽  
pp. 588a
Author(s):  
Marios Sergides ◽  
Tommaso Galgani ◽  
Claudia Arbore ◽  
Francesco S. Pavone ◽  
Marco Capitanio
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Single Molecule Level ◽  
Optical Setup

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.

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