A Rationally Designed Connector for Assembly of Protein-Functionalized DNA Nanostructures.

Chembiochem : a European journal of chemical biology

PubMedID: 26972311

Koßmann KJ, Ziegler C, Angelin A, Meyer R, Skoupi M, Rabe KS, Niemeyer CM. A Rationally Designed Connector for Assembly of Protein-Functionalized DNA Nanostructures. Chembiochem. 2016;.
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.