Sigurdardottir, S. | Search bioRxiv

bioRxiv2023

Novel regulatory variant in ABO intronic RUNX1 binding site inducing A3 phenotype

Background and ObjectivesMixed-field agglutination in ABO phenotyping (A3, B3) has been linked to genetically different blood cell populations like in chimerism, or to rare variants in either ABO exon 7 or regulatory regions. Clarification of such cases is challenging and would greatly benefit from sequencing technologies that allow resolving full-gene haplotypes at high resolution. Materials and MethodsWe used long-read sequencing by Oxford Nanopore Technologies to sequence the entire ABO gene, amplified in two overlapping long-range PCR fragments, in a blood donor presented with A3B phenotype. Confirmation analyses were carried out by Sanger sequencing and included samples from other family members. ResultsOur data revealed a novel heterozygous g.10924C>A variant on the ABO*A-allele located in the transcription factor binding site for RUNX1 in intron 1 (+5.8 kb site). Inheritance was shown by the results of the donors mother, who shared the novel variant and the anti-A specific mixed-field agglutination. ConclusionWe discovered a regulatory variant in the 8-bp RUNX1 motif of ABO, which extends current knowledge of three other variants affecting the same motif and also leading to A3 or B3 phenotypes. Overall, long-range PCR combined with nanopore sequencing proved powerful and showed great potential as emerging strategy for resolving cases with cryptic ABO phenotypes.

genetics↗

bioRxiv2021

A genetic trap in yeast for inhibitors of SARS-CoV-2 main protease

The ongoing COVID-19 pandemic urges searches for antiviral agents that can block infection or ameliorate its symptoms. Using dissimilar search strategies for new antivirals will improve our overall chances of finding effective treatments. Here, we have established an experimental platform for screening of small molecule inhibitors of SARS-CoV-2 main protease in Saccharomyces cerevisiae cells, genetically engineered to enhance cellular uptake of small molecules in the environment. The system consists of a fusion of the E. coli toxin MazF and its antitoxin MazE, with insertion of a protease cleavage site in the linker peptide connecting the MazE and MazF moieties. Expression of the viral protease confers cleavage of the MazEF fusion, releasing the MazF toxin from its antitoxin, resulting in growth inhibition. In the presence of a small molecule inhibiting the protease, cleavage is blocked and the MazF toxin remains inhibited, promoting growth. The system thus allows positive selection for inhibitors. The engineered yeast strain is tagged with a fluorescent marker protein, allowing precise monitoring of its growth in the presence or absence of inhibitor. We detect an established main protease inhibitor down to 10 M by a robust growth increase. The system is suitable for robotized large-scale screens. It allows in vivo evaluation of drug candidates, and is rapidly adaptable for new variants of the protease with deviant site specificities. IMPORTANCEThe COVID-19 pandemic may continue several years before vaccination campaigns can put an end it globally. Thus, the need for discovery of new antiviral drug candidates will remain. We have engineered a system in yeast cells for detection of small molecule inhibitors of one attractive drug target of SARS-CoV-2, its main protease which is required for viral replication. To detect inhibitors in live cells brings the advantage that only compounds capable of entering the cell and remain stable there, will score in the system. Moreover, by its design in yeast, the system is rapidly adaptable for tuning of detection level, eventual modification of protease cleavage site in case of future mutant variants of the SARS-CoV-2 main protease, or even for other proteases.

synthetic biology↗