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

Visser, Emiel W. A.; Miladinovic, Jovana; Milstein, Joshua N.

doi:10.1002/smtd.202001180

Cited by 3 publications

(3 citation statements)

References 33 publications

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“…Traditional immobilization approaches, such as thiol/maleimide or NH 2 /NHS reactions, , often involve a functional group common to proteins, potentially compromising the target protein and the overall system. Advances in bioorthogonal click chemistry have improved immobilization methods for DNA and proteins, , enhancing their study at the single-molecule level. − Inspired by these advancements, we combined click-chemistry and enzymatic ligation to immobilize DNA and protein within an atomic force microscopy (AFM) system. As a result, a high-precision AFM-SMFS system optimized for analyzing protein–DNA interactions is achieved.…”

Section: Introductionmentioning

confidence: 99%

Click Chemistry-Based Force Spectroscopy Revealed Enhanced Binding Dynamics of Phosphorylated HMGB1 to Cisplatin-DNA

Liu,

Song,

et al. 2024

J. Am. Chem. Soc.

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Cisplatin, a cornerstone in cancer chemotherapy, is known for its DNAbinding capacity and forms lesions that lead to cancer cell death. However, the repair of these lesions compromises cisplatin's effectiveness. This study investigates how phosphorylation of HMGB1, a nuclear protein, modifies its binding to cisplatin-modified DNA (CP-DNA) and thus protects it from repair. Despite numerous methods for detecting protein−DNA interactions, quantitative approaches for understanding their molecular mechanism remain limited. Here, we applied click chemistry-based singlemolecule force spectroscopy, achieving high-precision quantification of the interaction between phosphorylated HMGB1 and CP-DNA. This method utilizes a synergy of click chemistry and enzymatic ligation for precise DNA−protein immobilization and interaction in the system. Our results revealed that HMGB1 binds to CP-DNA with a significantly high rupture force of ∼130 pN, stronger than most natural DNA−protein interactions and varying across different DNA sequences. Moreover, Ser14 is identified as the key phosphorylation site, enhancing the interaction's kinetic stability by 35-fold. This increase in stability is attributed to additional hydrogen bonding suggested by molecular dynamics (MD) simulations. Our findings not only reveal the important role of phosphorylated HMGB1 in potentially improving cisplatin's therapeutic efficacy but also provide a precise method for quantifying protein−DNA interactions.

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Section: Introductionmentioning

confidence: 99%

Click Chemistry-Based Force Spectroscopy Revealed Enhanced Binding Dynamics of Phosphorylated HMGB1 to Cisplatin-DNA

Liu,

Song,

et al. 2024

J. Am. Chem. Soc.

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“…The cleaning of cover glasses for single-molecule experiments creates terminal silanol groups on the surface, which deprotonate in aqueous environments, leaving a negative interfacial charge density. There are a number of different methods designed to minimize adsorption of biomolecules to negatively charged glass surfaces. − The most common is the passivation of surfaces by either unlabeled proteins or synthetic polymers. In protein blocking methods, the binding sites on a glass surface are populated by an inert protein and become unavailable to make interactions with other molecules. − The most common protein used for this purpose is bovine serum albumin (BSA), but the method suffers from the weakness of the noncovalent interaction of BSA with the surface-binding sites, leading to a lack of uniformity of the inert protein layer .…”

Section: Introductionmentioning

confidence: 99%

“…There are a number of different methods designed to minimize adsorption of biomolecules to negatively charged glass surfaces. 14 − 16 The most common is the passivation of surfaces by either unlabeled proteins or synthetic polymers. In protein blocking methods, the binding sites on a glass surface are populated by an inert protein and become unavailable to make interactions with other molecules.…”

Section: Introductionmentioning

confidence: 99%

Surface Passivation with a Perfluoroalkane Brush Improves the Precision of Single-Molecule Measurements

Bueno-Alejo

Vega

Chaplin

et al. 2022

ACS Appl. Mater. Interfaces

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Single-molecule imaging is invaluable for investigating the heterogeneous behavior and interactions of biological molecules. However, an impediment to precise sampling of single molecules is the irreversible adsorption of components onto the surfaces of cover glasses. This causes continuous changes in the concentrations of different molecules dissolved or suspended in the aqueous phase from the moment a sample is dispensed, which will shift, over time, the position of chemical equilibria between monomeric and multimeric components. Interferometric scattering microscopy (iSCAT) is a technique in the single-molecule toolkit that has the capability to detect unlabeled proteins and protein complexes both as they adsorb onto and desorb from a glass surface. Here, we examine the reversible and irreversible interactions between a number of different proteins and glass via analysis of the adsorption and desorption of protein at the single-molecule level. Furthermore, we present a method for surface passivation that virtually eliminates irreversible adsorption while still ensuring the residence time of molecules on surfaces is sufficient for detection of adsorption by iSCAT. By grafting high-density perfluoroalkane brushes on cover-glass surfaces, we observe approximately equal numbers of adsorption and desorption events for proteins at the measurement surface (±1%). The fluorous–aqueous interface also prevents the kinetic trapping of protein complexes and assists in establishing a thermodynamic equilibrium between monomeric and multimeric components. This surface passivation approach is valuable for in vitro single-molecule experiments using iSCAT microscopy because it allows for continuous monitoring of adsorption and desorption of protein without either a decline in detection events or a change in sample composition due to the irreversible binding of protein to surfaces.

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Cortisol Monitoring Devices toward Implementation for Clinically Relevant Biosensing In Vivo

2023

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Cortisol is a steroid hormone that regulates energy metabolism, stress reactions, and immune response. Cortisol is produced in the kidneys’ adrenal cortex. Its levels in the circulatory system are regulated by the neuroendocrine system with a negative feedback loop of the hypothalamic–pituitary–adrenal axis (HPA-axis) following circadian rhythm. Conditions associated with HPA-axis disruption cause deteriorative effects on human life quality in numerous ways. Psychiatric, cardiovascular, and metabolic disorders as well as a variety of inflammatory processes accompanying age-related, orphan, and many other conditions are associated with altered cortisol secretion rates and inadequate responses. Laboratory measurements of cortisol are well-developed and based mainly on the enzyme linked immunosorbent assay (ELISA). There is a great demand for a continuous real-time cortisol sensor that is yet to be developed. Recent advances in approaches that will eventually culminate in such sensors have been summarized in several reviews. This review compares different platforms for direct cortisol measurements in biological fluids. The ways to achieve continuous cortisol measurements are discussed. A cortisol monitoring device will be essential for personified pharmacological correction of the HPA-axis toward normal cortisol levels through a 24-h cycle.

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

Cited by 3 publications

References 33 publications

Click Chemistry-Based Force Spectroscopy Revealed Enhanced Binding Dynamics of Phosphorylated HMGB1 to Cisplatin-DNA

Click Chemistry-Based Force Spectroscopy Revealed Enhanced Binding Dynamics of Phosphorylated HMGB1 to Cisplatin-DNA

Surface Passivation with a Perfluoroalkane Brush Improves the Precision of Single-Molecule Measurements

Cortisol Monitoring Devices toward Implementation for Clinically Relevant Biosensing In Vivo

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