Mercado, M. P. | bioRxiv Science

bioRxiv2023

A novel platform for metabolomics using barcoded structure-switching aptamers

Small organic molecules like metabolites and drugs are critical for diagnostics, treatment, and synthetic biology. Measuring them presents two key challenges however: they are biochemically highly diverse and there is no method to amplify them. Mass spectrometry has been the workhorse of metabolomics for decades but is costly and slow and single-cell metabolomics remains very challenging. Here we describe an alternative platform for metabolomics based on structure-switching aptamers (SSAs). SSAs are short nucleic acid molecules that each recognise a specific target ligand and undergo a major conformational change on ligand binding. This conformational change can drive detection such as fluorescence allowing SSAs to be used as sensors. We adapted conventional SSAs to a novel readout: barcode release. Each SSA recognises a unique ligand and each SSA releases a unique barcode allowing many ligands to be detected in parallel. We show that these barcode SSAs (bSSAs) can be multiplexed and act as independent sensors and that barcode release can be massively amplified to allow high sensitivity. Finally, we establish methods for the generation of large collections of bSSAs where barcode-SSA matching is completely directed. We believe that this novel platform which converts metabolite detection into barcode sequencing will allow the deep multiplexed detection of metabolites and drugs down to the scale of single cells.

genomics↗

bioRxiv2023

Natural variation in expression of the mitochondrial flavoprotein WAH-1 alters response to cyanide in C. elegans

C. elegans is a free-living nematode that must adapt to a wide range of environments including both aerobic and anaerobic conditions. To survive in low oxygen, C. elegans can use an unusual form of anaerobic respiration that relies on rhodoquinone (RQ) as an alternative electron carrier. Parasitic nematodes like hookworm and whipworm also require rhodoquinone-dependent metabolism (RQDM) to survive in the highly anaerobic conditions in the human gut. Understanding how RQDM is regulated in C. elegans may thus identify new ways to combat these closely-related major human pathogens. We previously established a simple movement-based assay for RQDM in C. elegans. In this study, we tested a panel of wild-type isolates of C. elegans in our RQDM assay and find substantial variation in their ability to use RQDM. We carried out a genome-wide association study (GWAS) to identify loci that affect RQDM -- this identified a single major QTL on the right arm of Chromosome III. We used RNAi to test almost all genes within the QTL region for involvement in RQDM and found one gene, wah-1, that strongly modulates RQDM-dependent recovery in C. elegans. WAH-1 is a mitochondrial flavoprotein that affects the electron transport chain, consistent with a role in RQDM. We show that wah-1 expression varies between isolates due to major changes in wah-1 transcript structures and this correlates tightly with variation in RQDM. Finally, we show that there is similar complexity to wah-1 transcription in parasitic nematodes and that wah-1 transcript structures change as parasites shift from aerobic to anaerobic, RQ- requiring metabolism. We thus conclude that reduced wah-1 expression correlates with increased ability to survive in conditions where RQDM is essential.

molecular biology↗