Biology subjects

Fraser, A. G.

Publications and source records attributed to Fraser, A. G..

8 recordsLinked to original sources

bioRxiv2026

Deep Mutational Scanning of structure-switching aptamers identifies both base requirement and base-pairing and uncovers regions required for either sensitivity or specificity

Structure-switching aptamers (SSAs) are short oligonucleotides that undergo a large conformational change on binding their specific target. This change can be used to read out target levels and SSA-based sensors have been developed for targets including drugs, metabolites, and toxins. Despite this potential as molecular sensors, we cannot predict how an SSA folds, how it binds its target, or the final SSA:target conformation and there are very few solved SSA structures. Here we use deep mutational scanning (DMS) to probe SSA structure and function, focusing on three published SSAs that detect cortisol. We show that DMS delivers rich data: it identifies which bases are required for activity but also maps base-pairing either in the presence or absence of target. We find all three SSAs have a similar structure when bound to their target despite having different predicted structures. To broaden our understanding of how SSAs can see cortisol, we selected many new cortisol-binding SSAs. Surprisingly, we identify diverse sequences that all have similar cores to the published cortisol SSAs - many diverse sequences thus converge on the same mode of target recognition. Finally, we use DMS to show we can change target specificity with a small number of mutations and map key specificity determinants. We conclude DMS can provide rich information on how SSAs work and that generating this for many SSAs could lead to greatly improved models for SSA mechanism.

genomics↗

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↗

bioRxiv2022

Identification of a novel family of benzimidazole species-selective Complex I inhibitors as potential anthelmintics

Soil-transmitted helminths (STHs) including Ascaris, hookworm, and whipworm are major human pathogens infecting over a billion people worldwide1,2. There are few existing classes of anthelmintics and resistance is increasing3-5 -- there is thus an urgent need for new classes of these drugs. Here we focus on identifying compounds that interfere with the unusual anaerobic metabolism that STHs use to survive the highly hypoxic conditions of the host gut6-9. This requires rhodoquinone (RQ), a quinone electron carrier that is not made or used by the STH hosts10. We previously showed that C. elegans also uses this rhodoquinone-dependent metabolism (RQDM)11 and established a high throughput assay for RQDM11. We screened a collection of 480 natural products for compounds that kill worms specifically when they rely on RQDM -- these 480 are representatives of a full library of ~25,000 natural products and derivatives12,13. We identify several classes of compound including a novel family of species selective inhibitors of Complex I. These Complex I inhibitors are based on a benzimidazole core but unlike commercial benzimidazole anthelmintics they do not target microtubules14-17. We screened over 1,200 benzimidazoles and identify the key structural requirements for species selective Complex I inhibition. We suggest that these novel benzimidazole species-selective Complex I inhibitors may be potential anthelmintics.

molecular biology↗

bioRxiv2021

An intestinally secreted host factor limits bacterial colonization but promotes microsporidia invasion of C. elegans

Microsporidia are ubiquitous obligate intracellular pathogens of animals. These parasites often infect hosts through an oral route, but little is known about the function of host intestinal proteins that facilitate microsporidia invasion. To identify such factors necessary for infection by Nematocida parisii, a natural microsporidian pathogen of Caenorhabditis elegans, we performed a forward genetic screen to identify mutant animals that have a Fitness Advantage with Nematocida (Fawn). We isolated four fawn mutants that are resistant to Nematocida infection and contain mutations in T14E8.4, which we renamed aaim-1 (Antibacterial and Aids invasion by Microsporidia). Expression of AAIM-1 in the intestine of aaim-1 animals restores N. parisii infectivity and this rescue of infectivity is dependent upon AAIM-1 secretion. N. parisii spores in aaim-1 animals are improperly oriented in the intestinal lumen, leading to reduced levels of parasite invasion. Conversely, aaim-1 mutants display both increased colonization and susceptibility to the bacterial pathogen Pseudomonas aeruginosa and overexpression of AAIM-1 reduces P. aeruginosa colonization. Competitive fitness assays show that aaim-1 mutants are favoured in the presence of N. parisii but disadvantaged on P. aeruginosa compared to wild type animals. Together, this work demonstrates how microsporidia exploits a secreted protein to promote host invasion. Our results also suggest evolutionary trade-offs may exist to optimizing host defense against multiple classes of pathogens.

microbiology↗

bioRxiv2021

Identification of enzymes that are required for Rhodoquinone-dependent metabolism as targets for new species-specific inhibitors

Soil transmitted helminths (STHs) are major human pathogens that infect over a billion people. Resistance to current anthelmintics is rising and new drugs are needed. Here we combine multiple approaches to find druggable targets that are essential for RQ-dependent metabolism, an unusual form of anaerobic metabolism which STHs need to survive in their host. We identified 25 genes predicted to act in RQ-dependent metabolism from sensing hypoxia to RQ synthesis -- this includes components of the kynurenine pathway we previously showed to be essential for RQ synthesis (Del Borrello et al., 2019). We found 9 genes to be required -- since all have host orthologues, we used comparative genomics and structural modeling to identify those with helminth-specific active sites and found 4 such targets. These 4 high confidence targets open up the possibility of in silico screens to identify STH-specific inhibitors of these enzymes as new anthelmintics.

molecular biology↗

bioRxiv2018

Acute effects of drugs on C. elegans movement reveal complex responses and plasticity

Many drugs act very rapidly -- they can turn on or off their targets within minutes in a whole animal. What are the acute effects of drug treatment and how does an animal respond to these? We developed a simple assay to measure the acute effects of drugs on C.elegans movement and examined the effects of a range of compounds including neuroactive drugs, toxins, environmental stresses and novel compounds on worm movement over a time period of 3 hours. We find that many treatments show complex acute responses -- a phase of rapid paralysis is followed by one or more recovery phases. The recoveries are not the result of some generic stress response but are specific to the drug e.g. recovery from paralysis due to a neuroactive drug requires neurotransmitter pathways whereas recovery from a metabolic inhibitor requires metabolic changes. Finally, we also find that acute responses can vary greatly across development and that there is extensive and complex natural variation in acute responses. In summary, acute responses are sensitive probes of the ability of biological networks to respond to drug treatment and these responses can reveal the action of unexplored pathways.\n\nAuthor SummaryDrugs are powerful tools that let us switch on or off key pathways in whole animals and watch the effects. Here we set up a simple assay to measure how drugs affect the movement of the simple nematode C.elegans -- crucially, we look how those responses change over time. We find that worms have complex responses to many different drugs -- they go through clear phases of paralysis and recovery. The recovery from the initial effect of any drug is not due to a generic stress response but is specific to the way each drug acts. We find for example that worms can recover from paralysis driven by one neurotransmitter pathway by activating a different neurotransmitter pathway or from paralysis caused by loss of one metabolic pathway by turning on a second one. These complex responses show how the basic genetic networks that are needed for normal behaviour and function can rewire and respond to the effect of many drugs.\n\nImportantly, the responses can vary in many ways -- different aged worms or different individuals can have different responses and capturing how drug responses change over time is critical to dissect this complexity.

genetics↗

bioRxiv2017

What can an evolutionary perspective on women’s health provide to the current public health paradigm? A case study on redefining thyroid cancer as a reproductive cancer

Incidence rates of non-communicable diseases in women are increasing worldwide and now contributing more to mortality and morbidity than reproductive health. Despite this universal trend, there is no recognition by the WHO of this phenomenon in womens health. Additionally, thyroid cancer is considered a non-sex specific cancer despite female incidence rates being triple those of males. To understand why there are such conceptual limitations to womens health, biomedicine - and its prevalence in western public health authorities - will be analysed from an anthropological perspective. In mind of these conceptual limitations, insights from evolutionary perspectives on health are introduced as well as how they may alleviate the conceptual barriers in the current biomedical paradigm. Using the example of breast cancer, the difference between a reproductive cancer in women, and cancer located in the reproductive tract will be explored. From this, the possibility that a cancer outside the reproductive tract can be directly influenced by reproductive function is explored in the specific case of thyroid cancer. Thyroid cancer and current public health approaches to preventing malignancies of the thyroid are examined to show the limitations of their scope. Furthermore, evidence supporting the direct link between thyroid function and reproductive function is presented. Current academic studies into the link between thyroid cancer and womens reproductive function are analysed to show they are subject to the same conceptual limitations of womens reproductive function as found previously in biomedicine. To conclude, I will introduce a new hypothesis for exploring the impact of womens reproductive function on thyroid cancer incidence rates. This hypothesis will allow womens health to be viewed holistically, and allows reproductive function to be investigated beyond parity. Thus, the conceptual limitations of womens health in the public health paradigm will be alleviated.

evolutionary biology↗