Sensing of protein and DNA complexes using solid-state nanopores.

Abstract

The field of nanopores has garnered increased interest for nucleic acid and peptide sensing and sequencing to develop targeted therapies for various diseases, including cancer, due to versatile pore sizes, measurement conditions, signal-to-noise ratio, and integration into electronics. We utilize a SiNx solid-state nanopore platform for studying the interaction and specificity of DNA-binding proteins with DNA. These DNA-binding proteins include various transcription factors, nuclear receptors, polymerases, and nucleases, which are responsible for several essential cellular processes and intra- and intercellular signaling cascades. Some of these DNA-binding proteins are known to be diagnostic and therapeutic targets for various diseases, specifically breast, ovarian, and prostate cancers. We seek to use these DNA-binding proteins as a clinically relevant model to detect their binding sites on the DNA and identify the protein mutations that would have a detrimental effect on its functionality, as they are highly dependent on accurate post-translational modifications. Using our nanopores, we successfully observe the binding of DNA-binding proteins onto the conventional binding sites on the target DNA and correlate these with the gold-standard Electrophoretic Mobility Shift Assay (EMSA) carried out parallelly. These proteins are observed as a pronounced local spike over and above the DNA strand translocating through the nanopore, signifying their binding to the DNA. The protein binding is stable and unaffected at a relatively high salt concentration (1 M LiCl, pH 8). Furthermore, the isoelectronic point of the protein is higher than this pH, imparting a positive charge on the protein and slowing down the translocation of such nuclear receptor-DNA complex, leading to a better signal resolution. We believe studying DNA binding proteins, including nuclear receptors, using nanopores could improve understanding of these proteins on a single molecule level, furthering the development of novel targeted treatment and diagnostic strategies.