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

Keywords

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

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