title: Protocol for sortase-mediated construction of DNA-protein hybrids and functional nanostructures.
abstract: Recent methods in DNA nanotechnology are enabling the creation of intricate nanostructures through the use of programmable, bottom-up self-assembly. However, structures consisting only of DNA are limited in their ability to act on other biomolecules. Proteins, on the other hand, perform a variety of functions on biological materials, but directed control of the self-assembly process remains a challenge. While DNA-protein hybrids have the potential to provide the best-of-both-worlds, they can be difficult to create as many of the conventional techniques for linking proteins to DNA render proteins dysfunctional. We present here a sortase-based protocol for covalently coupling proteins to DNA with minimal disturbance to protein function. To accomplish this we have developed a two-step process. First, a small synthetic peptide is bioorthogonally and covalently coupled to a DNA oligo using click chemistry. Next, the DNA-peptide chimera is covalently linked to a protein of interest under protein-compatible conditions using the enzyme sortase. Our protocol allows for the simple coupling and purification of a functional DNA-protein hybrid. We use this technique to form oligos bearing cadherin-23 and protocadherin-15 protein fragments. Upon incorporation into a linear M13 scaffold, these protein-DNA hybrids serve as the gate to a binary nanoswitch. The outlined protocol is reliable and modular, facilitating the construction of libraries of oligos and proteins that can be combined to form functional DNA-protein nanostructures. These structures will enable a new class of functional nanostructures, which could be used for therapeutic and industrial processes.
authors.affiliation: Program in Neuroscience, Department of Neurobiology, Harvard Medical School, Boston, MA, United States.
authors.forename: Mounir A
authors.initials: MA
authors.lastname: Koussa
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, United States.
authors.forename: Marcos
authors.initials: M
authors.lastname: Sotomayor
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, United States; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, United States; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, United States. Electronic address: wong@idi.harvard.edu.
authors.forename: Wesley P
authors.initials: WP
authors.lastname: Wong
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D055162:Biocatalysis; D057930:Click Chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D008969:Molecular Sequence Data; D058726:Nanoconjugates / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis* / Q000302:isolation & purification; D055550:Protein Stability
chemical_list: D001426:Bacterial Proteins; D058726:Nanoconjugates; D009841:Oligonucleotides; D010455:Peptides; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
keywords: Bioconjugation; DNA origami; DNA–protein hybrids/chimeras; Molecular self-assembly; Site-directed; Sortase coupling