An Ultrastable and Dense Single-Molecule Click Platform for Sensing Protein–Deoxyribonucleic Acid Interactions

Emiel W.A. Visser (Corresponding author), Jovana Miladinovic, Joshua N. Milstein (Corresponding author)

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Abstract

An ultrastable, highly dense single-molecule assay ideal for observing protein–DNA interactions is demonstrated. Stable click tethered particle motion leverages next generation click-chemistry to achieve an ultrahigh density of surface tethered reporter particles, and has low non-specific interactions, 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 enable the authors to study previously inaccessible sequence and temperature-dependent effects on DNA binding by the bacterial protein, histone-like nucleoid-structuring protein, a global transcriptional regulator found in Escherichia coli. This greatly improved assay can directly be translated to accelerate existing tethered particle-based, single-molecule biosensing applications.

Original languageEnglish
Article number2001180
Number of pages6
JournalSmall Methods
Volume5
Issue number5
Early online date23 Feb 2021
DOIs
Publication statusPublished - 12 May 2021

Funding

The authors thank Paul Piunno for his helpful discussions on optimizing the APTES functionalization, William Navarre for providing the H‐NS proteins, and Marie Elliot and Xiafei Zhang for providing the natural DNA templates from . This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 796345, and from a National Sciences and Engineering Research Council of Canada Discovery Grant (RGPIN‐2019‐06520). Streptomyces venezuelae

FundersFunder number
European Union 's Horizon 2020 - Research and Innovation Framework Programme
Natural Sciences and Engineering Research Council of CanadaRGPIN‐2019‐06520
Horizon 2020796345

    Keywords

    • biomarker sensing platforms
    • click-chemistry
    • high-stability
    • histone-like nucleoid-structuring proteins
    • protein–deoxyribonucleic acid interactions
    • tethered particle motion

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