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Solid‐Phase Synthesis and Purification of Protein–DNA Origami Nanostructures
Author(s) -
Burgahn Teresa,
Garrecht Ruben,
Rabe Kersten S.,
Niemeyer Christof M.
Publication year - 2019
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201805506
Subject(s) - ligation , solid phase synthesis , recombinant dna , dna , cleavage (geology) , oligonucleotide , combinatorial chemistry , chemistry , materials science , steric effects , nanotechnology , biophysics , peptide , crystallography , biochemistry , stereochemistry , microbiology and biotechnology , biology , fracture (geology) , gene , composite material
We present a facile method for the combined synthesis and purification of protein‐decorated DNA origami nanostructures (DONs). DONs bearing reductively cleavable biotin groups in addition to ligands for ligation of recombinant proteins are bound to magnetic beads. Protein immobilization is conducted with a large protein excess to achieve high ligation yields. Subsequent to cleavage from the solid support, pure sample solutions are obtained which are suitable for direct AFM analysis of occupation patterns. We demonstrate the method's utility using three different orthogonal ligation methods, the “halo‐based oligonucleotide binder” (HOB), a variant of Halo‐tag, the “SpyTag/SpyCatcher” (ST/SC) system, and the enzymatic “ybbR tag” coupling. We find surprisingly low efficiency for ST/SC ligation, presumably due to electrostatic repulsion and steric hindrance, whereas the ybbR method, despite its ternary nature, shows good ligation yields. Our method is particularly useful for the development of novel ligation methods and the synthesis of mechanically fragile DONs that present protein patterns for surface‐based cell assays.