Orthogonal Protein Decoration of DNA Origami

Abstract

Structural DNA nanotechnology [1,2] and the technique of DNA origami [3] enable the rapid generation of a plethora of complex self-assembled nanostructures. [4][5][6] Since DNA molecules themselves display limited chemical, optical, and electronic functionality, it is of utmost importance to devise methods to decorate DNA scaffolds with functional moieties to realize applications in sensing, catalysis, and device fabrication. Protein functionalization is particulary desirable because it allows exploitation of an almost unlimited variety of functional elements which nature has evolved over billions of years.[7] The delicate architecture of proteins has resulted in no generally applicable method being currently available to selectively couple these components on DNA scaffolds, and thus approaches used so far are based on reversible antibodyantigen interactions, [8,9] aptamer binding, [10,11] nucleic acid hybridization of DNA-tagged proteins, [12,13] or predominantly biotin-streptavidin (STV) interactions. [14][15][16][17][18][19] We demonstrate here that DNA nanostructures can be site-specifically decorated with several different proteins by using coupling systems orthogonal to the biotin-STV system. In particular, benzylguanine (BG) and chlorohexane (CH) groups incorporated in DNA origami have been used as suicide ligands for the site-specific coupling of fusion proteins containing the self-labeling protein tags O 6 -alkylguanine-DNA-alkyltransferase (hAGT), which is often referred to as "Snap-tag", [20] or haloalkane dehalogenase, which is also known as "HaloTag".[21] By using various model proteins we demonstrate the general applicability of this approach for the generation of DNA superstructures that are selectively decorated with multiple different proteins.To realize orthogonal protein immobilization on DNA origami using self-ligating protein tags, we chose the Snap-tag, developed by Johnsson and co-workers, [20] and the commercially available HaloTag [21] system. The respective smallmolecule suicide tags (O 6 -benzylguanine (BG) and 5-chlorohexane (CH)) for both self-labeling protein tags are readily available as amino-reactive N-hydroxysuccinimide (NHS) derivatives (BG-NHS and CH-NHS; Figure 1 a). Complete derivatization of alkylamino-modified oligonucleotides was achieved by coupling with 30 molar equivalents of BG-NHS or CH-NHS, as indicated by electrophoretic analysis (Figure 1 b). To gain access to fusion proteins bearing the complementary Snap-and Halo-protein tags, we constructed expression plasmids by genetic fusion of the genes encoding the protein of interest (POI) and Snap-tag or HaloTag (see the Supporting Information). As model POIs we chose the fluorescent proteins enhanced yellow fluorescent protein (EYFP) [22] and mKate, [23] the enzymes cytochrome C peroxidase (CCP) [24] and esterase 2 from Alicyclobacillus acidocaldarius thermos (EST2), [25] to which the self-labeling tags were fused at the C terminus (POI-Snap or POI-Halo, respectively). In addition, the bispecific Halo-Snap fusion protein...

Spatially-Interactive Biomolecular Networks Organized by Nucleic Acid Nanostructures

“…(B) Patterning macromolecules on DNA origami: streptavidin (top), 29 virus capsid (middle) 32 and orthogonal protein decoration (bottom). 36 Reproduce from ref (C) Site-specific protein-oligo conjugation using His-tag, ybbR-tag, STV-tag, HALO-tag, SNAP-tag and Intein-tag (from top to bottom). Figures are reproduced with permissions from IOP Publishing, ACS, and Wiley.…”

Challenges and opportunities for structural DNA nanotechnology

“…Taking advantage of the sequence specificity and the resulting spatial addressability of DNA nanostructures, many of the DNA nanoarchitectures listed above have been used for the organization of heteroelements such as proteins 22,29,51–57 , peptides 58 , virus capsids 59 , nanoparticles 60–74 and carbon nanotubes 75 . And in turn, several of these DNA-directed assemblies have led to unique and improved functional properties, such as increased enzyme-cascade activities 76,77 due to spatially positioned enzyme pairs, and shifts of surface plasmon resonance controlled by custom arrangement of nanoparticles 78–80 through DNA-mediated self-assembly.…”

Programmable motion of DNA origami mechanisms

“…Scaffolded DNA origami, the process of folding a long singlestranded DNA (ssDNA) strand into a custom structure (16)(17)(18), has enabled the fabrication of nanoscale objects with unprecedented geometric complexity that have recently been implemented in applications such as containers for drug delivery (19,20), nanopores for single-molecule sensing (21)(22)(23), and templates for nanoparticles (24,25) or proteins (26)(27)(28). The majority of these and other applications of DNA origami have largely focused on static structures.…”