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Self-assembled endogenous DNA nanoparticles for auto-release and expression of the eGFP gene in Bacillus subtilis.

ABSTRACT: The development of DNA delivery techniques is critical to promote the wider use of deoxyribonucleic acids as cellular transporters. The present study aimed to develop a type of DNA nanoparticle (citZ-box) to automatically load and release cargo. The restriction enzyme can cleave citZ-boxes at pro-designed sites, and the enhanced green fluorescent protein gene (eGFP) can be delivered into the B. subtilis protoplasts by them. The process of eGFP expression is recorded using a confocal microscope over 4 h. Here, multiscaffold and multimodular designs are used for citZ-box assembly with a DAEDALUS module, DX_cage_design and rem (edge_length, 21), to ensure the structure was predicted as B-type DNA. Finally the citZ-box is estimated to be a 50.7 nm cube. The 3D structure of the citZ-box particle is detected to be approximately 50.3 ± 0.3 nm. DNA nanoparticles prepared as citZ-boxes have great potential as drug carriers with automatic loading and releasing abilities.

SUBMITTER: Cao L

PROVIDER: S-EPMC9751278 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Self-assembled endogenous DNA nanoparticles for auto-release and expression of the eGFP gene in Bacillus subtilis.

Cao Linfeng L Meng Ziwen Z Tan Junjie J Ying Ming M Ying Ming M Bi Meiying M Liu Yanjun Y Tong Xinrui X Wei Jiaxun J Huang Lei L

Communications biology 20221214 1

The development of DNA delivery techniques is critical to promote the wider use of deoxyribonucleic acids as cellular transporters. The present study aimed to develop a type of DNA nanoparticle (citZ-box) to automatically load and release cargo. The restriction enzyme can cleave citZ-boxes at pro-designed sites, and the enhanced green fluorescent protein gene (eGFP) can be delivered into the B. subtilis protoplasts by them. The process of eGFP expression is recorded using a confocal microscope o ...[more]

PMID: 36517556

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Project description:Nonviral siRNA vectors prepared by the direct mixing of siRNA and mixtures of an asymmetric N(4),N(9)-diacyl spermine conjugate, N(4)-linoleoyl-N(9)-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), with either cholesterol or DOPE, at various molar ratios of the neutral lipids, are reported. The effects of varying the lipid formulation and changing the N/P charge ratio on the intracellular delivery of siRNA to HeLa cells and on the siRNA-mediated gene silencing of a stably expressed reporter gene (EGFP) were evaluated. The presence of either cholesterol or DOPE in the mixture resulted in a marked increase in the delivery of the siRNA as well as enhanced EGFP silencing as evaluated by FACS. A LinOS/Chol 1:2 mixture resulted in the highest siRNA delivery and the most efficient EGFP silencing (reduced to 20%) at N/P = 3.0. Lowering the amount of siRNA from 15 pmol to 3.75 pmol, thus increasing the N/P charge ratio to 11.9, resulted in decreasing the amount of delivered siRNA, while the efficiency of gene silencing was comparable to that obtained with 15 pmol (N/P = 3.0) of siRNA. Mixtures of symmetrical N(4),N(9)-dioleoyl spermine (DOS) with cholesterol at 1:2 molar ratio showed less siRNA delivery than with LinOS/Chol at N/P = 3.0 (15 pmol of siRNA), and comparable delivery at N/P = 11.9 (3.75 pmol of siRNA). The EGFP silencing was comparable with LinOS and with DOS when mixed with cholesterol 1:2 (lipoplexes prepared with 15 pmol of siRNA), but LinOS mixtures showed better EGFP silencing when the siRNA was reduced to 3.75 pmol. Lipoplex particle size determination by DLS of cholesterol mixtures was 106-118 nm, compared to 194-356 nm for lipoplexes prepared with the spermine conjugates only, and to 685 nm for the LinOS/DOPE 1:1 mixture. Confocal microscopy showed successful siRNA delivery of red tagged siRNA and quantitative EGFP knockdown in HeLa EGFP cells; Z-stack photomicrographs showed that the delivered siRNA is distributed intracellularly. Cryo-TEM of siRNA LinOS/Chol 1:2 lipoplexes shows the formation of multilamellar spheres with a size of ∼100 nm, in good agreement with the particle size measured by DLS. The constant distance between lamellar repeats is ∼6 nm, with the electron-dense layers fitting a monolayer of siRNA. AlamarBlue cell viability assay showed that the lipoplexes resulted in cell viability ≥81%, with LinOS/Chol 1:2 mixtures resulting in cell viabilities of 89% and 94% at siRNA 15 nM and 3.75 nM respectively. These results show that lipoplexes of siRNA and LinOS/Chol mixtures prepared by the direct mixing of the lipid mixture and siRNA, without any preceding preformulation steps, result in enhanced siRNA delivery and EGFP knockdown, with excellent cell viability. Thus, LinOS/Chol 1:2 mixture is a promising candidate as a nontoxic nonviral siRNA vector.

Dataset Information

Self-assembled endogenous DNA nanoparticles for auto-release and expression of the eGFP gene in Bacillus subtilis.

Publications

Self-assembled endogenous DNA nanoparticles for auto-release and expression of the eGFP gene in Bacillus subtilis.

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