Purification of DNA Nanoparticles Using Photocleavable Biotin Tethers

Abstract

The number of applications of self-assembled deoxyribonucleic acid (DNA) origami nanoparticles (DNA NPs) has increased drastically, following the development of a variety of single-stranded template DNA (ssDNA) that can serve as the scaffold strand. In addition to viral genomes, such as M13 bacteriophage and lambda DNAs, enzymatically produced ssDNA from various template sources is rapidly gaining traction and being applied as the scaffold for DNA NP preparation. However, separating fully formed DNA NPs that have custom scaffolds from crude assembly mixes is often a multistep process of first separating the ssDNA scaffold from its enzymatic amplification process and then isolating the assembled DNA NPs from excess precursor strands. Only then is the DNA NP sample ready for downstream characterization and application. In this work, we highlight a single-step purification of custom sequence-or M13derived scaffold-based DNA NPs using photocleavable biotin tethers. The process only requires an inexpensive ultraviolet (UV) lamp, and DNA NPs with up to 90% yield and high purity are obtained. We show the versatility of the process in separating two multihelix bundle structures and a wireframe polyhedral architecture.

Design and Characterization of a Gene-Encoding DNA Nanoparticle in a Cell-Free Transcription–Translation System

“…Naturally, such application of gene-encoded DNA NPs would require concomitant ways to produce the ss custom scaffold strand in desirable quantities. While producing custom plasmids in large quantities is an established process, scaling up of the synthesis of gene-encoded DNA NPs is rapidly advancing as well due to leading efforts to formulate enzymatic and phage-based techniques of producing custom scaffold strands. , Traditionally gel extraction is used to acquire purified ssDNA for custom scaffold strands, but new analytical techniques are emerging that reduce material loss and improve DNA NP production efficiency . And the assembly process of DNA NPs lends itself directly to scale up in that the reactions can be done in a massively parallel format.…”