Programming and preparing long single-stranded DNA with highly integrated sequence information for the self-assembly of DNA nanostructures

Abstract

As a popular assembly technique, DNA origami offered a simple way to fold long, single-stranded (ss) DNA molecules into arbitrary two-dimensional (2D) shapes. To design a desired shape, a 7-kilobase M13mp18 genomic DNA was raster filled into the shape as a scaffold, and over hundreds short oligonucleotide staple strands were carefully chosen and added in excessive molar ratios to hold the scaffold in place. The assembly of a desired 2D shape with a diameter of ~100 nm can be achieved in a simple annealing step in hours. Due to its convenience during preparation and the large address-able surface area of the assembled shapes, DNA origami has been rapidly explored as templates for many types of bio-material and biomedical applications. However, a few limitations hinder the further development of this assembly technique. One is about the relatively high cost: When it comes to large scale preparation (e.g., gram-level) for potential drug evaluation in animals, the requirement of large scale of hundreds of synthetic DNA strands usually brings about bills near to 0.1-1 Million Chinese Yuan. Another issue is the leftover of the excess of the staple strands: They are hardly to be completely removed from the desired DNA structures, which may cause unwanted side-effects during biomedical applications. If a desired shape could be assembled with only one ssDNA in a high yield, the second issue would not exist anymore. As early as in 2007, the Yan's group reported that the DNA tile with odd number of the paranemic crossover could be assembled by one ssDNA. Later on, several groups found that the tetrahedron and prism-like scaffold objects could also be assembled with an ssDNA. However these DNA nanostructures created by a ssDNA (~hundred bases in length) were relatively small, with diameters <10 nm. The challenge to create large shapes with only one ssDNA is that in the one polymer folding pathway, the crossovers that bundle many helical domains to form the shapes would inevitably encounter the topological knotting issue, which set the shape formation into many kinetic traps. About ten years later, recently the Yan and Yin's group developed the ssDNA origami technique as a derivative of the traditional DNA origami by integrating all sequences required by a desired shape into one long single strand. The key innovation is to use partially complemented double-stranded DNA and parallel crossover cohesion to construct a knot-free structure that can be folded smoothly from a single strand. The removal of the excess of staple strands yet still possessing a high yield (>90%) of the desired shapes and the adaptiveness to single-stranded RNA origami hint a promising future of this new technique in biomedical and biomaterial applications. Moreover, the recent development of single-stranded DNA preparation, for example, extraction of single-stranded genomic DNA with customized sequences up to 30000 bases from the helper phage/phagemid system, enabled the acquisition of gram-level of long single-stranded DNA in a cost-effective manner (~10 Thousand Chinese Yuan) and offered a solid foundation for large scale assembly of single-stranded DNA origami. The settlement of the two major issues in the traditional DNA origami should pave the way for applying DNA origami-based nanostructures in clinical tests. In this paper, we summarized the research progress of DNA self-assembly with one long single strand and the current ways to prepare long single-stranded DNAs, and finished with our thoughts on the future of the new single-stranded DNA origami technique.