Observing Single-Molecule Protein-DNA Interactions and DNA Transcription In Vitro using Transcriptomic Tethered Particle Motion

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.

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

International Journal of Molecular Sciences ◽

10.3390/ijms14023961 ◽

2013 ◽

Vol 14 (2) ◽

pp. 3961-3992 ◽

Cited By ~ 31

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

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An Ultra-Stable and Dense Single-Molecule Click Platform for Sensing Protein-DNA Interactions

10.1101/2020.11.25.397737 ◽

2020 ◽

Author(s):

Emiel W. A. Visser ◽

Jovana Miladinovic ◽

Joshua N. Milstein

Keyword(s):

Single Molecule ◽

Elevated Temperatures ◽

Regulatory Protein ◽

Bacterial Protein ◽

Dna Interactions ◽

Temperature Dependent ◽

E Coli ◽

Tethered Particle Motion ◽

Protein Dna Interactions ◽

Ultrahigh Density

AbstractWe demonstrate an ultra-stable, highly dense single-molecule assay ideal for observing protein-DNA interactions. Stable click Tethered Particle Motion (scTPM) leverages next generation click-chemistry to achieve an ultrahigh density of surface tethered reporter particles, has a high antifouling resistance, is stable at elevated temperatures to at least 45 °C, and is compatible with Mg2+, an important ionic component of many regulatory protein-DNA interactions. Prepared samples remain stable, with little degradation, for > 6 months in physiological buffers. These improvements enabled us to study previously inaccessible sequence and temperature dependent effects on DNA binding by the bacterial protein H-NS, a global transcriptional regulator found in E. Coli. This greatly improved assay can directly be translated to accelerate existing tethered particle based, single-molecule biosensing applications.

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

Essays in Biochemistry ◽

10.1042/ebc20200019 ◽

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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