03 Fakultät Chemie
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Item Open Access The effect of pooling on the detection of the nucleocapsid protein of SARS-CoV-2 with rapid antigen tests(2021) Berking, Tim; Lorenz, Sabrina; Ulrich, Alexander; Greiner, Joachim; Kervio, Eric; Bremer, Jennifer; Wege, Christina; Kleinow, Tatjana; Richert, ClemensThe COVID-19 pandemic puts significant stress on the viral testing capabilities of many countries. Rapid point-of-care (PoC) antigen tests are valuable tools but implementing frequent large scale testing is costly. We have developed an inexpensive device for pooling swabs, extracting specimens, and detecting viral antigens with a commercial lateral flow test for the nucleocapsid protein of SARS-CoV-2 as antigen. The holder of the device can be produced locally through 3D printing. The extraction and the elution can be performed with the entire set-up encapsulated in a transparent bag, minimizing the risk of infection for the operator. With 0.35 mL extraction buffer and six swabs, including a positive control swab, 43 ± 6% (n = 8) of the signal for an individual extraction of a positive control standard was obtained. Image analysis still showed a signal-to-noise ratio of approximately 2:1 at 32-fold dilution of the extract from a single positive control swab. The relative signal from the test line versus the control line was found to scale linearly upon dilution (R2 = 0.98), indicating that other pooling regimes are conceivable. A pilot project involving 14 participants and 18 pooled tests in a laboratory course at our university did not give any false positives, and an individual case study confirmed the ability to detect a SARS-CoV-2 infection with five-fold or six-fold pooling, including one swab from a PCR-confirmed COVID patient. These findings suggest that pooling can make frequent testing more affordable for schools, universities, and similar institutions, without decreasing sensitivity to an unacceptable level.Item Open Access An AZT analog with strongly pairing ethynylpyridone nucleobase and its antiviral activity against HSV1(2020) Han, Jianyang; Funk, Christina; Eyberg, Juri; Bailer, Susanne; Richert, ClemensChallenges resulting from novel viruses or new strains of known viruses call for new antiviral agents. Nucleoside analogs that act as inhibitors of viral polymerases are an attractive class of antivirals. For nucleosides containing thymine, base pairing is weak, making it desirable to identify nucleobase analogs that pair more strongly with adenine, in order to compete successfully with the natural substrate. We have recently described a new class of strongly binding thymidine analogs that contain an ethynylmethylpyridone as base and a C‐nucleosidic linkage to the deoxyribose. Here we report the synthesis of the 3′‐azido‐2′,3′‐deoxyribose derivative of this compound, dubbed AZW, both as free nucleoside and as ProTide phosphoramidate. As a proof of principle, we studied the activity against Herpes simplex virus type 1 (HSV1). Whereas the ProTide phosphoramidate suffered from low solubility, the free nucleoside showed a stronger inhibitory effect than that of AZT in a plaque reduction assay. This suggests that strongly pairing C‐nucleoside analogs of pyrimidines have the potential to become active pharmaceutical ingredients with antiviral activity.Item Open Access 2′/3′ regioselectivity of enzyme‐free copying of RNA detected by NMR(2020) Motsch, Sebastian; Pfeffer, Daniel; Richert, ClemensThe RNA‐templated extension of oligoribonucleotides by nucleotides produces either a 3′,5′ or a 2′,5′‐phosphodiester. Nature controls the regioselectivity during RNA chain growth with polymerases, but enzyme‐free versions of genetic copying have modest specificity. Thus far, enzymatic degradation of products, combined with chromatography or electrophoresis, has been the preferred mode of detecting 2′,5′‐diesters produced in enzyme‐free reactions. This approach hinges on the substrate specificity of nucleases, and is not suitable for in situ monitoring. Here we report how 1H NMR spectroscopy can be used to detect the extension of self‐templating RNA hairpins and that this reveals the regioisomeric nature of the newly formed phosphodiesters. We studied several modes of activating nucleotides, including imidazolides, a pyridinium phosphate, an active ester, and in situ activation with carbodiimide and organocatalyst. Conversion into the desired extension product ranged from 20 to 90 %, depending on the leaving group. Integration of the resonances of H1′ protons of riboses and H5 protons of pyrimidines gave regioselectivities ranging from 40:60 to 85:15 (3′,5′ to 2′,5′ diester), but no simple correlation between 3′,5′ selectivity and yield. Our results show how monitoring with a high‐resolution technique sheds a new light on a process that may have played an important role during the emergence of life.Item Open Access Hybridization networks of mRNA and branched RNA hybrids(2020) Damakoudi, Vassiliki; Feldner, Tobias; Dilji, Edina; Belkin, Andrey; Richert, ClemensMessenger RNA (mRNA) is emerging as an attractive biopolymer for therapy and vaccination. To become suitable for vaccination, mRNA is usually converted to a biomaterial, using cationic peptides, polymers or lipids. An alternative form of converting mRNA into a material is demonstrated that uses branched oligoribonucleotide hybrids with the ability to hybridize with one or more regions of the mRNA sequence. Two such hybrids with hexamer arms and adamantane tetraol as branching element were prepared by solution‐phase synthesis. When a rabies mRNA was treated with the branched hybrids at 1 M NaCl concentration, biomaterials formed that contained both of the nucleic acids. These results show that branched oligoribonucleotides are an alternative to the often toxic reagents commonly used to formulate mRNA for medical applications.