title: Click Addition of a DNA Thread to the N-Termini of Peptides for Their Translocation through Solid-State Nanopores.
abstract: Foremost among the challenges facing single molecule sequencing of proteins by nanopores is the lack of a universal method for driving proteins or peptides into nanopores. In contrast to nucleic acids, the backbones of which are uniformly negatively charged nucleotides, proteins carry positive, negative and neutral side chains that are randomly distributed. Recombinant proteins carrying a negatively charged oligonucleotide or polypeptide at the C-termini can be translocated through a α-hemolysin (α-HL) nanopore, but the required genetic engineering limits the generality of these approaches. In this present study, we have developed a chemical approach for addition of a charged oligomer to peptides so that they can be translocated through nanopores. As an example, an oligonucleotide PolyT20 was tethered to peptides through first selectively functionalizing their N-termini with azide followed by a click reaction. The data show that the peptide-PolyT20 conjugates translocated through nanopores, whereas the unmodified peptides did not. Surprisingly, the conjugates with their peptides tethered at the 5'-end of PolyT20 passed the nanopores more rapidly than the PolyT20 alone. The PolyT20 also yielded a wider distribution of blockade currents. The same broad distribution was found for a conjugate with its peptide tethered at the 3'-end of PolyT20, suggesting that the larger blockades (and longer translocation times) are associated with events in which the 5'-end of the PolyT20 enters the pore first.
authors.affiliation: Biodesign Institute, ‡Department of Chemistry and Biochemistry, §Department of Physics, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Sudipta
authors.initials: S
authors.lastname: Biswas
authors.affiliation: Biodesign Institute, ‡Department of Chemistry and Biochemistry, §Department of Physics, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Weisi
authors.initials: W
authors.lastname: Song
authors.affiliation: Biodesign Institute, ‡Department of Chemistry and Biochemistry, §Department of Physics, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Chad
authors.initials: C
authors.lastname: Borges
authors.affiliation: Biodesign Institute, ‡Department of Chemistry and Biochemistry, §Department of Physics, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Stuart
authors.initials: S
authors.lastname: Lindsay
authors.affiliation: Biodesign Institute, ‡Department of Chemistry and Biochemistry, §Department of Physics, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Peiming
authors.initials: P
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D057930:Click Chemistry; D004247:DNA / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000737:chemistry; D008958:Models, Molecular; D058608:Nanopores / Q000648:ultrastructure; D036103:Nanotechnology / Q000379:methods; D009841:Oligonucleotides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*
chemical_list: D006460:Hemolysin Proteins; D009841:Oligonucleotides; D010455:Peptides; D004247:DNA
keywords: DNA thread; click addition; nanopore; peptide translocation; peptide-PolyT20 conjugate; protein sequencing