Abstract
We demonstrate an approach that allows attachment of single-stranded DNA (ssDNA) to a defined residue in a protein of interest (POI) so as to provide optimal and well-defined multicomponent assemblies. Using an expanded genetic code system, azido-phenylalanine (azF) was incorporated at defined residue positions in each POI; copper-free click chemistry was used to attach exactly one ssDNA at precisely defined residues. By choosing an appropriate residue, ssDNA conjugation had minimal impact on protein function, even when attached close to active sites. The protein-ssDNA conjugates were used to (i) assemble double-stranded DNA systems with optimal communication (energy transfer) between normally separate groups and (ii) generate multicomponent systems on DNA origami tiles, including those with enhanced enzyme activity when bound to the tile. Our approach allows any potential protein to be simply engineered to attach ssDNA or related biomolecules, creating conjugates for designed and highly precise multiprotein nanoscale assembly with tailored functionality.
Highlights
The use of DNA origami tiles as a well-defined and addressable template has emerged as a versatile tool for assembling proteins
By attaching glucose oxidase (GOx) and horseradish peroxidase (HRP) onto DNA origami tiles at specific positions, the substrate transfer from GOx to HRP was monitored as a function of distance, showing that optimal rates were achieved when the enzymes are in close proximity.[3]
Superfolder green fluorescent protein[29] and TEM β-lactamase (BL)[30] were selected as model energy capture and catalytic proteins, respectively. Both sfGFP and BL have recently been shown to be amenable to strained ring promoted alkyne−azide cycloaddition (SPAAC), through the introduction of an azide handle into the protein via the noncanonical amino acid p-azido-L-phenylalanine in conjunction with a reprogrammed genetic code
Summary
The use of DNA origami tiles as a well-defined and addressable template has emerged as a versatile tool for assembling proteins. While assembly on DNA origami tiles is precise, attachment of the addressing ssDNA strands to proteins is still limited, mostly due to lack of a single well-defined designed anchoring point. CuAAC can adversely affect protein structure (both at the primary and tertiary levels) and function.[18−20] Proteins have been modified with azides for CuAAC modification with alkyneDNA; multiple azides were introduced by global replacement of methionine to azido-homoalanine, leaving only nonburied azides to react with DNA.[21,22] This methodology is, limiting with respect to site selectivity and the requirement for Cu-catalyst
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