Replicable Single-stranded DNA Origami

Dongran Han, Cameron Myhrvold, Peng Yin
Department of Systems Biology, Harvard Medical School
Wyss Institute for Biologically Inspired Engineering at Harvard
DNA has been used to create a variety of complex nanoscale shapes and devices since the birth of DNA nanotechnology in 1982. One main challenge is to create complex nanostructures from a single-strand of DNA, like proteins which typically fold from a single polymer. Here we report the design and synthesis of single-stranded DNA origami structures (ssOrigami) containing up to 4,000 nucleotides, which can fold into designed shapes. Such ssOrigami can be easily replicated by polymerases in vitro and in vivo. ssOrigami structures can also be used as a template for amplification by PCR.
In previous studies, multiple DNA strands were successfully designed to self-assemble into complex structures with or without the help of a long scaffold strand. However, biological macromolecules, such as mRNAs and proteins, typically fold from a single polymer into a well-defined structure. The ability to fold DNA nanostructures in this manner would enable robust assembly and even replication of such structures from a unimolecular template. However, folding complex nanostructures from a single molecule of single-stranded DNA (ssDNA) still remains challenging for the field of DNA nanotechnology, due to the complexity of the topology in most existing strategies and the difficulty of large-scale clonal production of suitable DNA sequences.

Figure 1. ssOrigami structures with different sizes.

In our study, ssOrigami structures with different sizes ranging from 1,000 to 4,000 nucleotides (nt) were assembled. Three diamond shape ssOrigami containing 1k, 1.6k and 2.3k nt were demonstrated (Figure. 1
A-C), and they folded with high yield.
More complicated and larger ssOrigami structures were also achieved (Figure 2). While the folding path is less obvious than simple diamond shapes, these complex ssOrigami structure were also successfully assembled with good quality. ssOrigami of up to 4,000 nt in size were demonstrated (Figure 2 C, D), which are larger than 99.99% of all proteins in the human proteome and three times the size of the largest catalytic rRNA (16S rRNA). This result will greatly broaden our vision and understanding about the self-assembly capability of macro-biomolecules, especially when such a procedure can happen during a short (2 hour)
annealing step.

Figure 2. ssOrigami structures with different shapes.

Unlike multi-stranded DNA systems such as DNA origami and DNA bricks, which contain hundreds of distinct components and assemble with undesirable defects and heterogeneity, ssOrigami is a homogenous,
â??pureâ?? system. Additionally, ssOrigami can be easily replicated and amplified by polymerases in vitro and in vivo, which can greatly decrease the cost of DNA nanotechnology, especially when large amount of DNA nanostructures are required. Finally, ssOrigami structures can be used as a template for amplification by PCR.