ABSTRACT: In this study, the modified DAEDALUS module, DX_cage_design, was used to design a 50.7 nm nanocube by reading in the sequence of citZ gene, encoding citrate synthase of B. subtilis 168, and circulating it nine times with a remainder function, rem (edge_length, 21), ensuring to the predicted structure as B-type DNA29. The sequence of citZ, used as scaffold strand, was folded nine times to form deoxynucleotide nanoparticles by multimodular self-assembly, named a citZ-box. The scaffold strands were elongated to 9984 nt, which involved seven 1116 nt scaffolds and two short main scaffold strands of 812 nt and 1034 nt, respectively. Atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) revealed a nanocube with an average edge of 50.3 ± 0.3 nm, which was only 0.4 ± 0.3 nm different to the predicted size, which verified the feasibility of the modified software. To implement the auto loading and release function, BamHI restriction enzyme sites were inserted into the two short scaffolds and used to construct a “can-lip” domain that is expected to load and encapsulate cargoes outside the cells, depending on sequence-specific cleavage. Sequence-specific bacterial endonucleases are suitable for developing DNA delivery platform, so long as the loaded DNA nanoparticles can be opened in cells. Fluorescence resonance energy transfer (FRET) demonstrated the automatic on-off performance of the “can-lip”. The whole process of “can-lip” opening was tracked using Cy3, recorded under a confocal microscope. Surprisingly, the expression of eGFP in B. subtilis protoplasts was observed within four hours from delivery to release and expression. The results demonstrated that the DNA nanoparticles, designed and prepared as citZ-boxes using endogenous DNA with restriction sites as a lip, have good potential as a good drug delivering platform.
