Loading of DOX into a tetrahedron DNA nanostructure: The corner does matter

With the rapid development of nanotechnology, various DNA nanostructures have been synthesized and widely used in the drug delivery. However, the underlying mechanisms of drug molecules loading into the DNA...

Novel label-free electrochemiluminescence aptasensor using tetrahedral DNA nanostructure as a scaffold for ultrasensitive detection of organophosphorus pesticides in luminol-H2O2 system

In this work, a new type of Au-Tetrahedral DNA nanostructure (Au-TDN) was originally proposed and successfully applied in an electrochemiluminescence aptasensor to detect organophosphorus pesticides (Ops). The aptamers modified by...

Characterization of DNA nanostructure stability by size exclusion chromatography

DNA-based nanostructures (DNs) are advantageous for the design of functional materials for biology and medicine due to the nanoscale control provided by their predictable self-assembly. However, the use of DNs in vivo has been limited due to structural instability in biofluids. As the stability of a particular DN sets the scope of its potential biological applications, efficient methods to characterize stability are required. Here, we apply size exclusion chromatography (SEC) to study the stability of a tetrahedron DNA nanostructure (TDN) and demonstrate the analytical capabilities of our method in characterizing degradation by enzymes and a diluted human serum matrix. We show that SEC analysis can reliably assay TDN degradation by a nuclease through direct injection and peak integration. Furthermore, data analysis using a ratio chromatogram technique enables TDN peak deconvolution from the matrix of serum proteins. Using our method, we found that TDNs exhibit half-lives of 23.9 hours and 10.1 hours in 20% and 50% diluted human serum, respectively, which is consistent with reported stability studies in 10% fetal bovine serum. We anticipate that this method could be broadly applicable to characterize a variety of DNs and serve as an efficient technique toward analysis of the stability of new DN designs in complex biological matrixes.

CRISPR-Cas9 mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

DNA nanostructures are a promising tool for delivery of a variety of molecular payloads to cells. DNA origami structures, where 1000's of bases are folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent-protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein (RNP) binding sites on the DNA nanostructures to increase shuttling of structures into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA (dsDNA) templates in human primary cells. Furthermore, our study validates virus-like particles (VLPs) as an efficient method of DNA nanostructure delivery, opening the possibility of delivering DNA nanostructures in vivo to specific cell types. Together these results provide new approaches to gene delivery with DNA nanostructures and establish their use as large HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as measuring biophysical properties, into cell nuclei.