Functional Patterning of DNA Origami by Parallel Enzymatic Modification

Abstract

We demonstrate here a rapid and cost-effective technique for nanoscale patterning of functional molecules on the surface of a DNA origami. The pattern is created enzymatically by transferring a functionalized dideoxynucleotide to the 3'-end of an arbitrary selected set of synthetic DNA oligonucleotides positioned approximately 6 nm apart in a 70 × 100 nm(2) rectangular DNA origami. The modifications, which are performed in a single-tube reaction, provide an origami surface modified with a variety of functional groups including chemical handles, fluorescent dyes, or ligands for subsequent binding of proteins. Efficient labeling and patterning was demonstrated by gel electrophoresis shift assays, reverse-phase HPLC, mass spectrometry, atomic force microscopy (AFM) analysis, and fluorescence measurements. The results show a very high yield of oligonucleotide labeling and incorporation in the DNA origami. This method expands the toolbox for constructing several different modified DNA origami from the same set of staple strands.

Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

“…A combination of ddN*TPs and the TdT has also been used to label staple strands for the construction of functionalized origamis . Indeed, Jahn et al.…”

“…The staple strands are enzymatically modified with a single biotin, digoxigenin, or Texas Red dye by using the corresponding ddN*TPs and are then combined with the other 205 staple strands as well as the 7‐kb M13mp18 phage ssDNA to form the desired rectangular origamis. The authors note that the presence of the modifications on the origamis can be ascertained by AFM imaging …”

Enzymatic production of single-molecule FISH and RNA capture probes

“…This enzyme could elongate 3 ′ ends indefinitely; however, the use of dideoxy nucleotides ensures that only a single labeled nucleotide is incorporated into each and almost every oligonucleotide molecule added to the reaction. Although the feasibility of such a direct labeling strategy was demonstrated previously for individual oligonucleotides (Jahn et al 2011;Winz et al 2015), here we show that it is possible to label sets of conventional PCR oligos using custom-made dye/ biotin conjugated ddUTPs synthesized in a standard biology laboratory (Fig. 1A).…”

DNA Origami Seesaws as Comparative Binding Assay

“…The simple, computer‐aided design process has enabled the self‐assembly of a variety of complex 2D and 3D geometries with nanometer addressability. DNA origami structures have been frequently employed to arrange molecules, binding moieties and proteins in designed patterns . Furthermore, conformational changes of these structures have been demonstrated, for example by endonuclease activity, aptamer‐ligand interactions, toehold‐mediated branch migration and with hydrophobic moieties such as cholesterol …”