Abstract
Nanopores have unlocked an accurate long-read sequencing technology for DNA and RNA molecules based on their translocation through the nanopore and subsequent ionic current blockade by nucleotides. It is tempting to expand this success from nucleic acids to proteins. However, proteins have a net charge various in polarity and amount. This hinders the transfer of nanopore nucleic acid sequencing technology to proteins because the capturing of nucleic acid molecules by the nanopore depends on their negative net charge. Furthermore, native proteins exist with stable higher order structures that are not readily unwind like those in nucleic acids, which could obstruct the translocation of proteins through nanopores. Our recent study demonstrated steady translocation of full-length proteins with genetically engineered poly-aspartate tails through biological nanopores, where guanidinium chloride was used to denature and linearize the protein. The charged poly-aspartate tail at the terminal of the protein facilitates protein threading, an essential prerequisite for the translocation process. Here, we use bioconjugation chemistry to end-tag native full-length proteins with charged polymers. We investigated the specificity and efficiency of various tagging strategies, and the performance of tags on the capturing of peptides and full-length proteins, and their characteristic current blockade signal. Our work should facilitate the application of nanopore technology for analysis of full-length proteins in native samples like cell cultures and clinical samples, which features label-free, low cost, and high throughput detection of concentration and modification of protein molecules of biological importance.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.