Title: Looking Into DNA-Protein Interactions At The Molecular Level Using Glass Nanopores

Authors: Punitkumar Nagpure 1, Divya Shet 1, Serene R.D. 1& Gautam V. Soni. 1

1Raman Research Institute

In the cell, various molecular and cellular mechanisms involve DNA-protein interactions, such as DNA replication and transcription. Understanding the binding strength and precise location of DNA-protein complexes are essential for the proper maintenance and functioning of these biological pathways. In recent years, nanopore sensors have emerged as label-free single-molecule tools that allow us to study DNA-protein complexes in their native state while providing high throughput. In this study, we utilized a glass nanopore sensor to detect DNA-dCas9/Cas9 complexes using the resistive pulse technique. We employed CRISPR technology to target the dCas9/Cas9 protein to a specific location on DNA. Our nanopore device can sense protein binding and measure spatial positioning on DNA. It is sensitive enough to discriminate between proteins targeted to different locations on DNA, which may be used to study the off-target activity of the protein-DNA complex. In the future, we aim to use this technique to rupture DNA-protein complexes.