Biology subjects

Tan, J. H.

Publications and source records attributed to Tan, J. H..

7 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

Fasting Induces a Highly Resilient Deep Quiescent State in Muscle Stem Cells via Ketone Body Signaling

Short-term fasting is beneficial for the regeneration of multiple tissue types. However, the effects of fasting on muscle regeneration are largely unknown. Here we report that fasting slows muscle repair both immediately after the conclusion of fasting as well as after multiple days of refeeding. We show that ketosis, either endogenously produced during fasting or a ketogenic diet, or exogenously administered, promotes a deep quiescent state in muscle stem cells (MuSCs). Although deep quiescent MuSCs are less poised to activate, slowing muscle regeneration, they have markedly improved survival when facing sources of cellular stress. Further, we show that ketone bodies, specifically {beta}-hydroxybutyrate, directly promote MuSC deep quiescence via a non-metabolic mechanism. We show that {beta}-hydroxybutyrate functions as an HDAC inhibitor within MuSCs leading to acetylation and activation of an HDAC1 target protein p53. Finally, we demonstrate that p53 activation contributes to the deep quiescence and enhanced resilience observed during fasting.

cell biology↗

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↗

bioRxiv2020

Alternative splicing of COQ-2 determines the choice between ubiquinone and rhodoquinone biosynthesis in helminths

Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In aerobic environments they use ubiquinone (UQ) but in anaerobic environments inside the host, they require rhodoquinone (RQ) and greatly increase RQ levels. The switch to RQ synthesis is driven by substrate selection by the polyprenyltransferase COQ-2 but the mechanisms underlying this substrate choice are unknown. We found that helminths make two coq-2 isoforms, coq-2a and coq-2e, by alternative splicing. COQ-2a is homologous to COQ2 from other eukaryotes but the COQ-2e-specific exon is only found in species that make RQ and its inclusion changes the enzyme core. We show COQ-2e is required for RQ synthesis and for survival in cyanide in C. elegans. Crucially, we see a switch from COQ-2a to COQ-2e as parasites transition into anaerobic environments. We conclude that under anaerobic conditions helminths switch from UQ to RQ synthesis via alternative splicing of coq-2.

molecular biology↗

bioRxiv2019

Identification of the pathway of Rhodoquinone biosynthesis in C. elegans

Parasitic helminths infect over a billion humans. To survive in the low oxygen environment of their hosts, these parasites use unusual anaerobic metabolism. This requires Rhodoquinone (RQ), an electron carrier that is made by very few animal species -- crucially it is not present in any parasitic hosts. RQ synthesis is thus an ideal target for anthelmintics but little is known about how RQ is made and no drugs are known to block RQ synthesis. C.elegans makes RQ and can use RQ-dependent metabolic pathways -- here, we use C.elegans genetics to identify the pathway for RQ synthesis and show that C.elegans requires RQ for survival in hypoxic conditions. Finally, we establish a robust assay for drugs that block RQ-dependent metabolism. This study identifies for the first time how RQ is made in any animal and establishes a novel assay that can drive the development of a new class of anthelmintic drugs.

biochemistry↗