Scholarlink

Abstract <p>This study establishes a real-time fluorescence microscopy platform for visualizing ligand binding dynamics to His-tagged proteins bound to Ni-NTA agarose beads. By preserving solution-phase kinetics while enabling sub-minute temporal resolution in physiological buffers, the methodology overcomes critical limitations of surface-based techniques and gel electrophoretic methods. We applied this platform to investigate inhibitor action within a nucleosomal system, a more physiologically relevant context than free DNA. Through studies of PARP2-nucleosome interactions modulated by clinical inhibitors (talazoparib, veliparib) and by reaction of poly(ADP-ribosyl)ation in the presence of NAD<sup>+</sup>, we demonstrate direct spatial and temporal resolution of chromatin-protein dynamics. The virtually unlimited compatibility of the platform with buffers, real-time monitoring capabilities, and elimination of covalent immobilization artifacts provide an advanced understanding of the mechanisms of drug-chromatin interaction.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Ni-NTA Bead-Based Real-Time Monitoring of PARPs Inhibitor Binding

  • 期刊论文

  • 发表日期：

    2025年12月1日

查看全文
  • A. A. Lobanova,
  • A. A. Saulina,
  • O. V. Geraskina,
  • D. O. Koshkina,
  • N. V. Maluchenko,
  • A. V. Feofanov,
  • V. M. Studitsky

摘要

Abstract

This study establishes a real-time fluorescence microscopy platform for visualizing ligand binding dynamics to His-tagged proteins bound to Ni-NTA agarose beads. By preserving solution-phase kinetics while enabling sub-minute temporal resolution in physiological buffers, the methodology overcomes critical limitations of surface-based techniques and gel electrophoretic methods. We applied this platform to investigate inhibitor action within a nucleosomal system, a more physiologically relevant context than free DNA. Through studies of PARP2-nucleosome interactions modulated by clinical inhibitors (talazoparib, veliparib) and by reaction of poly(ADP-ribosyl)ation in the presence of NAD+, we demonstrate direct spatial and temporal resolution of chromatin-protein dynamics. The virtually unlimited compatibility of the platform with buffers, real-time monitoring capabilities, and elimination of covalent immobilization artifacts provide an advanced understanding of the mechanisms of drug-chromatin interaction.