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1.
ACS Nano ; 16(6): 8954-8966, 2022 06 28.
Article in English | MEDLINE | ID: mdl-35640255

ABSTRACT

Viruslike particles (VLPs) fabricated using wireframe DNA origami are emerging as promising vaccine and gene therapeutic delivery platforms due to their programmable nature that offers independent control over their size and shape, as well as their site-specific functionalization. As materials that biodegrade in the presence of endonucleases, specifically DNase I and II, their utility for the targeting of cells, tissues, and organs depends on their stability in vivo. Here, we explore minor groove binders (MGBs) as specific endonuclease inhibitors to control the degradation half-life of wireframe DNA origami. Bare, unprotected DNA-VLPs composed of two-helix edges were found to be stable in fetal bovine serum under typical cell culture conditions and in human serum for 24 h but degraded within 3 h in mouse serum, suggesting species-specific endonuclease activity. Inhibiting endonucleases by incubating DNA-VLPs with diamidine-class MGBs increased their half-lives in mouse serum by more than 12 h, corroborated by protection against isolated DNase I and II. Our stabilization strategy was compatible with the functionalization of DNA-VLPs with HIV antigens, did not interfere with B-cell signaling activity of DNA-VLPs in vitro, and was nontoxic to B-cell lines. It was further found to be compatible with multiple wireframe DNA origami geometries and edge architectures. MGB protection is complementary to existing methods such as PEGylation and chemical cross-linking, offering a facile protocol to control DNase-mediated degradation rates for in vitro and possibly in vivo therapeutic and vaccine applications.


Subject(s)
Nanostructures , Mice , Humans , Animals , Nucleic Acid Conformation , DNA , Endonucleases , Deoxyribonuclease I
2.
Nat Mater ; 20(9): 1272-1280, 2021 09.
Article in English | MEDLINE | ID: mdl-34112975

ABSTRACT

DNA is an ultrahigh-density storage medium that could meet exponentially growing worldwide demand for archival data storage if DNA synthesis costs declined sufficiently and if random access of files within exabyte-to-yottabyte-scale DNA data pools were feasible. Here, we demonstrate a path to overcome the second barrier by encapsulating data-encoding DNA file sequences within impervious silica capsules that are surface labelled with single-stranded DNA barcodes. Barcodes are chosen to represent file metadata, enabling selection of sets of files with Boolean logic directly, without use of amplification. We demonstrate random access of image files from a prototypical 2-kilobyte image database using fluorescence sorting with selection sensitivity of one in 106 files, which thereby enables one in 106N selection capability using N optical channels. Our strategy thereby offers a scalable concept for random access of archival files in large-scale molecular datasets.


Subject(s)
DNA/chemistry , Information Storage and Retrieval , Archives , Fluorescence , Plasmids , Polymerase Chain Reaction , Silicon Dioxide/chemistry , Synthetic Biology
3.
Sci Rep ; 9(1): 6121, 2019 04 16.
Article in English | MEDLINE | ID: mdl-30992517

ABSTRACT

Scalable production of kilobase single-stranded DNA (ssDNA) with sequence control has applications in therapeutics, gene synthesis and sequencing, scaffolded DNA origami, and archival DNA memory storage. Biological production of circular ssDNA (cssDNA) using M13 addresses these needs at low cost. However, one unmet goal is to minimize the essential protein coding regions of the exported DNA while maintaining its infectivity and production purity to produce sequences less than 3,000 nt in length, relevant to therapeutic and materials science applications. Toward this end, synthetic miniphage with inserts of custom sequence and size offers scalable, low-cost synthesis of cssDNA at milligram and higher scales. Here, we optimize growth conditions using an E. coli helper strain combined with a miniphage genome carrying only an f1 origin and a ß-lactamase-encoding (bla) antibiotic resistance gene, enabling isolation of pure cssDNA with a minimum sequence genomic length of 1,676 nt, without requiring additional purification from contaminating DNA. Low-cost scalability of isogenic, custom-length cssDNA is demonstrated for a sequence of 2,520 nt using a bioreactor, purified with low endotoxin levels (<5 E.U./ml). We apply these exonuclease-resistant cssDNAs to the self-assembly of wireframe DNA origami objects and to encode digital information on the miniphage genome for biological amplification.


Subject(s)
Bioreactors/virology , DNA, Single-Stranded/biosynthesis , Escherichia coli/metabolism , Industrial Microbiology/methods , Bacteriophage M13/genetics , Bioreactors/economics , DNA, Single-Stranded/isolation & purification , Escherichia coli/genetics , Escherichia coli/virology , Industrial Microbiology/economics , Nanotechnology/economics , Nanotechnology/methods , Plasmids/genetics
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