Construction of a novel phagemid to produce custom DNA origami scaffolds

Abstract

DNA origami, a method for constructing nanoscale objects, relies on a long single strand of DNA to act as the 'scaffold' to template assembly of numerous short DNA oligonucleotide 'staples'. The ability to generate custom scaffold sequences can greatly benefit DNA origami design processes. Custom scaffold sequences can provide better control of the overall size of the final object and better control of low-level structural details, such as locations of specific base pairs within an object. Filamentous bacteriophages and related phagemids can work well as sources of custom scaffold DNA. However, scaffolds derived from phages require inclusion of multi-kilobase DNA sequences in order to grow in host bacteria, and those sequences cannot be altered or removed. These fixed-sequence regions constrain the design possibilities of DNA origami. Here, we report the construction of a novel phagemid, pScaf, to produce scaffolds that have a custom sequence with a much smaller fixed region of 393 bases. We used pScaf to generate new scaffolds ranging in size from 1512 to 10 080 bases and demonstrated their use in various DNA origami shapes and assemblies. We anticipate our pScaf phagemid will enhance development of the DNA origami method and its future applications.

Keywords: DNA nanotechnology; DNA origami; phagemid.

Figure 1.

DNA origami design would benefit from custom scaffolds. (A) Many DNA origami shapes can be folded from a single scaffold. (B) New scaffolds will expand the space of possible designs. (C) Phagemids are excellent sources of scaffolds, but have multi-kilobase sequence constraints. Previous studies offer hints of how these constraints could be circumvented. (D) Dotto et al. (2) used phagemids with modified origins to show that f1-ori ssDNA initiation and termination functions overlap, but can be inactivated separately by modifying distinct sequences. (E) Specthrie et al. (3) produced phage-like particles with ssDNA as short as 292 bases using a truncated f1-ori that acts as a terminator (f1-oriΔ29).

Figure 2.

Construction and optimization of the pScaf phagemid. (A) The pScaf phagemid is derived from the pUC18 plasmid and M13mp18 phage vectors. The phagemid includes an M13 origin of replication (M13ori) that contains ssDNA start site S1 and ssDNA termination site T2, a KpnI and BamHI cloning site, a packaging sequence (PS), and a terminator of ssDNA synthesis (M13term) containing ssDNA termination site T1 and ssDNA start site S2. The M13term sequence was adapted from the M13ori sequence by selectively deactivating the S2 ssDNA initiation function. (B) The ssDNA synthesis initiation and termination functions of the origin overlap near the γ and δ regions that form an inverted repeat (highlighted in gray). Therefore, we used a mutational screen of the δ region to optimize the M13term performance. (C) Three primary species were observed: S1T2, representing failed termination at terminator T1, S2T2, representing spurious initiation at initiator S2, and S1T1, the desired product. (D) We analyzed the variants by agarose gel. Substituting 0–2 thymines yielded the S2T2 species (*). Substituting 4–6 bases yielded the S1T2 species (**). Substituting three thymines yielded the best balance between spurious S2 initiation and failed T1 termination, producing the most pure S1T1 species (***).

Figure 3.

Cloning scheme and gel analysis of new scaffolds. (A) Custom sequence inserts are PCR-amplified with a forward primer containing KpnI and BglII sites and reverse primer containing a BamHI site. Inserts up to 3 kb in length were directly transformed into E. coli bearing helper plasmid. Larger scaffolds can be assembled by iterative PvuI+BamHI digestion of the vector containing the 5′ fragment, and PvuI+BglII digestion of the vector containing the 3′ fragment, followed by ligation, transformation and miniprep. (B) Twelve inserts (A–L) were cloned into pScaf vector at the KpnI-BamHI site. Inserts A, B and C were used to produce scaffolds with lengths of 1512, 2268 and 3024 bases. Larger scaffolds were assembled in multiple rounds as shown. (C) All scaffolds were grown in XL1-Blue cells containing helper plasmid M13cp, recovered and analyzed by agarose gel electrophoresis to determine purities ranging from 46% to 83%.

Figure 4.

Folding custom scaffolds into DNA origami shapes and assemblies. Six scaffolds of varying sizes were folded into DNA origami shapes and assemblies. Folding reactions were analyzed by gel electrophoresis, and origami analyzed negative stain transmission electron microscopy. (A) Brick-like DNA origami shapes were folded from scaffolds of lengths 1512, 2268, 3024, 5544, 8064 and 10080 bases. Each brick was PEG-purified and imaged by negative stain TEM. Class averages for each brick are shown, along with a 20-nm scale bar. (B) Scaffolds of lengths 1512, 2268 and 3024 were folded separately into six-helix-bundle (6hb) nanotubes. All three scaffolds and 6hb-staple sets were combined into one-pot reaction mixtures. When connector staples are included, the three scaffolds assemble into a 6hb trimer. TEM micrographs of the individual 6hb nanotubes and combined trimer are shown with 100-nm scale bars.