Synthesis of DNA Origami Scaffolds: Current and Emerging Strategies

Abstract

DNA origami nanocarriers have emerged as a promising tool for many biomedical applications, such as biosensing, targeted drug delivery, and cancer immunotherapy. These highly programmable nanoarchitectures are assembled into any shape or size with nanoscale precision by folding a single-stranded DNA scaffold with short complementary oligonucleotides. The standard scaffold strand used to fold DNA origami nanocarriers is usually the M13mp18 bacteriophage’s circular single-stranded DNA genome with limited design flexibility in terms of the sequence and size of the final objects. However, with the recent progress in automated DNA origami design—allowing for increasing structural complexity—and the growing number of applications, the need for scalable methods to produce custom scaffolds has become crucial to overcome the limitations of traditional methods for scaffold production. Improved scaffold synthesis strategies will help to broaden the use of DNA origami for more biomedical applications. To this end, several techniques have been developed in recent years for the scalable synthesis of single stranded DNA scaffolds with custom lengths and sequences. This review focuses on these methods and the progress that has been made to address the challenges confronting custom scaffold production for large-scale DNA origami assembly.

Mechanical Properties of DNA Hydrogels: Towards Highly Programmable Biomaterials

“…The source of DNA material is an important factor as synthetically synthesized DNA can cost a few hundred to a few thousand dollars for a few milligrams required for milliliter scale DNA hydrogel synthesis [56]; typically requiring 2-4 milligrams (mg) of DNA per 100 µL, or 2-4 percent weight/volume (%w/v) of hydrogel. However, RCA is able to produce a few hundred micrograms of DNA per milliliter of overnight reaction [57][58][59], though it is currently limited to short tandem repeats (~100 nucleotides [nt]) without control over the repeat number. Multiprimed chain amplification (MCA) is the subsequent priming of the concatemeric strand produced by RCA (Figure 1a); this approach allows for a significant amount of DNA to be produced with a multitude of complementary domains enabling partial hybridization between multiple strands ranging from a few hundred to thousands of nucleotides in length.…”

One-Pot Synthesis of Defined-Length ssDNA for Multiscaffold DNA Origami

“…The classical, multistep method to make custom-length DNA origami is by synthesizing ssDNA scaffolds followed by one or more purification steps. 3 Next, the DNA origami structure is commonly formed by thermal annealing and subjected to a further round of purification to remove excess staples. 16 After PCR and T7 exonuclease digestion to form ssDNA, described above, we reasoned that the subsequent addition of Proteinase K would inactivate T7 exonuclease.…”

“…Creating scaffolds with tailor-made lengths opens up the possibility of making DNA origami of any size. To this end, custom-sized ssDNA scaffolds have been made using a wide variety of techniques, 3 including the use of phages and helper phages, 4 asymmetric polymerase chain reaction (PCR), 5 restriction endonucleases (RE) digestion, 6 selective digestion of plasmids and PCR products, 7 , 8 rolling circle amplification, 9 and even using RNA and dsDNA as a scaffold. 10 , 11 Despite this multitude of available techniques, few have found widespread use, primarily because they often require specialized knowledge and equipment in order to be implemented.…”

Organizing Multi-Enzyme Systems into Programmable Materials for Biocatalysis

“…The Watson-Crick base pairing of DNA makes it naturally a highly engineerable material for the predictable design of nanoscale structures [56][57][58][59]. Over a decade ago, a technique dubbed DNA origami was used for the design of patterned DNA nanomaterials [60] and has since then been applied for the design of a multitude of higher order structures [61,62]. Many of these materials were designed for biomedical applications [63][64][65][66][67] but it was soon realized that DNA scaffolds could also be used for the spatial organization of proteins to create programmable, functional materials [68,69].…”