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      Engineering a stable future for DNA-origami as a biomaterial

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          Abstract

          Reviewing the various methods and effectivity to stabilize DNA origami in biological environments.

          Abstract

          DNA as a biomaterial has evoked great interest as a potential platform for therapeutics and diagnostics and as hydrogel scaffolds due to the relative ease of programming its robust and uniform shape, site-specific functionality and controlled responsive behavior. However, for a stable self-assembled product, a relatively high cation concentration is required to prevent denaturation. Physiological and cell-culture conditions do not match these concentrations and present additional nucleases that cause a serious threat to the integrity of DNA-based materials. For the translation of this promising technology towards bioengineering challenges, stability needs to be guaranteed. Over the past years, various methods have been developed addressing the stability-related weaknesses of DNA-origami. This mini-review explains the common stability issues and compares the stabilization strategies recently developed. We present a detailed overview of each method in order to ease the selection process on which method to use for future users of DNA-origami as a biomaterial.

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          In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis.

          Dagmar Fischer, Youxin Li, Barbara Ahlemeyer … (2003)
          A comparative in vitro cytotoxicity study with different water-soluble, cationic macromolecules which have been described as gene delivery systems was performed. Cytotoxicity in L929 mouse fibroblasts was monitored using the MTT assay and the release of the cytosolic enzyme lactate dehydrogenase (LDH). Microscopic observations were carried out as indicators for cell viability. Furthermore, hemolysis of erythrocytes was quantified spectrophotometrically. To determine the nature of cell death induced by the polycations, the nuclear morphology after DAPI staining and the inhibition of the toxic effects by the caspase inhibitor zVAD.fmk were investigated. All assays yielded comparable results and allowed the following ranking of the polymers with regard to cytotoxicity: Poly(ethylenimine)=poly(L-lysine)>poly(diallyl-dimethyl-ammonium chloride)>diethylaminoethyl-dextran>poly(vinyl pyridinium bromide)>Starburst dendrimer>cationized albumin>native albumin. The magnitude of the cytotoxic effects of all polymers were found to be time- and concentration dependent. The molecular weight as well as the cationic charge density of the polycations were confirmed as key parameters for the interaction with the cell membranes and consequently, the cell damage. Evaluating the nature of cell death induced by poly(ethylenimine), we did not detect any indication for apoptosis suggesting that the polymer induced a necrotic cell reaction. Cell nuclei retained their size, chromatin was homogenously distributed and cell membranes lost their integrity very rapidly at an early stage. Furthermore, the broad spectrum caspase inhibitor zVAD.fmk did not inhibit poly(ethylenimine)-induced cell damage. Insights into the structure-toxicity relationship are necessary to optimize the cytotoxicity and biocompatibility of non-viral gene delivery systems.
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            Multiplexed 3D Cellular Super-Resolution Imaging with DNA-PAINT and Exchange-PAINT

            R A Jungmann, M.S. Avendano, J.B. Woehrstein … (2014)
            While super-resolution fluorescence microscopy is a powerful tool for biological research, obtaining multiplexed images for a large number of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed 3D super-resolution imaging inside fixed cells and achieve sub-10 nm spatial resolution in vitro using synthetic DNA structures. We also report a novel approach for multiplexing (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-“color” super-resolution imaging in vitro on synthetic DNA structures and four-“color” imaging of proteins in a fixed cell.
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              Oxidized redox state of glutathione in the endoplasmic reticulum.

              C. Hwang, A Sinskey, H Lodish (1992)
              The redox state of the endoplasmic reticulum (ER) was measured with the peptide N-Acetyl-Asn-Tyr-Thr-Cys-NH2. The peptide diffused across cellular membranes; some became glycosylated and thus trapped within the secretory pathway, and its cysteine residue underwent reversible thiol-disulfide exchanges with the surrounding redox buffer. Glycosylated peptides from cells were disulfide-linked to glutathione, indicating that glutathione is the major redox buffer in the secretory pathway. The redox state of the secretory pathway was more oxidative than that of the cytosol; the ratio of reduced glutathione to the disulfide form (GSH/GSSG) within the secretory pathway ranged from 1:1 to 3:1, whereas the overall cellular GSH/GSSG ratio ranged from 30:1 to 100:1. Cytosolic glutathione was also transported into the lumen of microsomes in a cell-free system. Although how the ER maintains an oxidative environment is not known, these results suggest that the demonstrated preferential transport of GSSG compared to GSH into the ER lumen may contribute to this redox compartmentation.
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                Author and article information

                Journal
                Journal ID (publisher-id): BSICCH
                Title: Biomaterials Science
                Abbreviated Title: Biomater. Sci.
                Publisher: Royal Society of Chemistry (RSC)
                ISSN (Print): 2047-4830
                ISSN (Electronic): 2047-4849
                Publication date Created: January 29 2019
                Publication date (Electronic): 2019
                Volume: 7
                Issue: 2
                Pages: 532-541
                Affiliations
                [1 ]Programmable Biomaterials Laboratory (PBL)
                [2 ]Institute of Materials (IMX)/Interfaculty Bioengineering Institute (IBI)
                [3 ]School of Engineering (STI)
                [4 ]École Polytechnique Federale de Lausanne (EPFL)
                [5 ]Lausanne
                Article
                DOI: 10.1039/C8BM01249K
                PubMed ID: 30534709
                SO-VID: c36b4524-9071-4f04-8291-b4e35fd2376f
                Copyright © © 2019
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                http://rsc.li/journals-terms-of-use

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