A Rationally Designed Connector for Assembly of Protein‐Functionalized DNA Nanostructures

Abstract

We report on the rational engineering of the binding interface of the self-ligating HaloTag protein to generate an optimized linker for DNA nanostructures. Five amino acids positioned around the active-site entry channel for the chlorohexyl ligand (CH) of the HaloTag protein were exchanged for positively charged lysine amino acids to produce the HOB (halo-based oligonucleotide binder) protein. HOB was genetically fused with the enzyme cytochrome P450 BM3, as well as with BMR, the separated reductase domain of BM3. The resulting HOB-fusion proteins revealed significantly improved rates in ligation with CH-modified oligonucleotides and DNA origami nanostructures. These results suggest that the efficient self-assembly of protein-decorated DNA structures can be greatly improved by fine-tuning of the electrostatic interactions between proteins and the negatively charged nucleic acid nanostructures.

Solid‐Phase Synthesis and Purification of Protein–DNA Origami Nanostructures

“…This is remarkable because even small amounts of unbound protein usually make AFM analyses very difficult if not impossible (Figure E, Figure S12). In agreement with an earlier study, a small excess of 1.3 equivalents of protein allows for AFM analysis of crude coupling products but only affords low occupancy rates (about 49 %) (Figure F). In contrast, on‐bead coupling with a large excess of protein followed by subsequent washing and cleavage yields pure protein‐DON samples with high occupancies of about 73 % (Figure G).…”

“…To establish the system, as a positive control, we firstly used the “halo‐based oligonucleotide binder” (HOB), a self‐labeling protein tag (293 amino acids) that forms a covalent bond with small‐molecule chlorohexane (CH)‐ligands in a similar fashion as the regular Halo‐tag protein, which is commonly used for imaging in cell biology . HOB was genetically engineered to bind to CH‐ligands attached to DNA oligonucleotides and DNA nanostructures with a significantly higher efficiency than Halo . Then we used two coupling systems, which had not yet been tested for the direct modification of DONs with proteins.…”

“…For an initial assessment of the MB‐based purification and as a positive control, we tested the HOB coupling system, which has previously been demonstrated to deliver high ligation yields of about 75 % for enzymes of comparable and even larger size as the enzyme Gre2 . We used DON‐1 that contained three CH‐ligands to enable the direct ligation with the enzyme Gre2 that was genetically fused with the HOB domain (Figure A).…”

Orthogonal Surface Tags for Whole‐Cell Biocatalysis

“…[25] Thes elf-labeling Halo-based oligonucleotide binder (HOB) tag protein (293 amino acids) forms acovalent bond with small-molecule chlorohexane (CH) ligands in as imilar fashion as the regular HaloTag protein, which is commonly used for imaging in cell biology. [27] [*] T. Peschke Plasmids encoding for afusion of the bacterial membrane protein Lpp-ompA that is connected to the SBP,S T, SC,o r HOB polypeptide tags via af lexible GGGGS linker were cloned into the pTF16 backbone of aplasmid that carries an chloramphenicol resistance and ap15A-ori and for which the protein expression is tightly controlled with an arabinosedependent promotor.Full details are given in the Supporting Information. [27] [*] T. Peschke Plasmids encoding for afusion of the bacterial membrane protein Lpp-ompA that is connected to the SBP,S T, SC,o r HOB polypeptide tags via af lexible GGGGS linker were cloned into the pTF16 backbone of aplasmid that carries an chloramphenicol resistance and ap15A-ori and for which the protein expression is tightly controlled with an arabinosedependent promotor.Full details are given in the Supporting Information.…”

“…[26] HOB was genetically engineered to bind to CH ligands attached to DNAo ligonucleotides and DNAn anostructures with as ignificantly higher efficiency than Halo. [27] [*] T. Peschke Plasmids encoding for afusion of the bacterial membrane protein Lpp-ompA that is connected to the SBP,S T, SC,o r HOB polypeptide tags via af lexible GGGGS linker were cloned into the pTF16 backbone of aplasmid that carries an chloramphenicol resistance and ap15A-ori and for which the protein expression is tightly controlled with an arabinosedependent promotor.Full details are given in the Supporting Information. Tr ansformation of E. coli BL21(DE3) with the engineered plasmids led to the formation of recombinant bacteria displaying either the SBP,S T, SC,o rH OB tag on their surface,w hich are denoted as E.coli-SBP, E.coli-ST, E.coli-SC,orE.coli-HOB,r espectively,int he following.…”