Biology subjects

Kool, E. T.

Publications and source records attributed to Kool, E. T..

11 recordsLinked to original sources

bioRxiv2026

Capsular polysaccharide can sensitize bacteria to non-antibiotic drugs

Medications administered over long durations, such as phenothiazine antipsychotics, accumulate in the gut at concentrations that affect microbial growth. However, the bacterial features influencing sensitivity to these non-antibiotics remain poorly understood. Bacterial capsular polysaccharides (CPSs) typically confer protection against environmental stressors, including chemical, viral, and immunological pressures within the gut. But their roles under non-antibiotic drug pressure are unknown. Here, we show that the K5 CPS of Escherichia coli Nissle 1917 (EcN) sensitizes it to thioridazine (TDZ) and related antipsychotics. Among a panel of E. coli strains grown in minimal medium, EcN exhibited the highest sensitivity to TDZ. Experimentally evolving EcN under gut-relevant TDZ concentrations selected for resistant populations with convergent variants affecting the CPS locus, and TDZ-resistant clones correspondingly had lower CPS expression than their drug-susceptible counterparts. Genetic, transcriptomic, and phenotypic analyses confirmed that the K5 CPS enhances, rather than mitigates, TDZ sensitivity. These findings demonstrate that canonically protective surface structures can become vulnerabilities under non-antibiotic pharmaceutical pressure. Human medications may therefore inadvertently shape the expression and evolution of bacterial surface structures in the gastrointestinal tract, challenging presumptions of CPS-mediated environmental protection.

microbiology↗

bioRxiv2025

DNA Content and DNA Damage in Raw and Heat-processed Foods

DNA in foods is a source of nucleotides that are salvaged by tissues as building blocks for chromosomal and mitochondrial DNA. A recent study provided preliminary evidence that high-temperature cooking damages the DNA in foods and suggested that certain forms of DNA damage can be taken up as nucleotides via metabolic salvage in cells and animals, directly incorporating genotoxic and mutagenic species into the host DNA. To assess potential risks, we surveyed DNA in 21 food ingredients, including plant- and meat-based foods in raw and roasted form. We find large variation in extractable DNA content, implying widely variable levels of consumption. Cooking resulted in greatly elevated levels of oxidative and deaminated DNA damage in nearly all foods as indicated by 8-oxo-dG and dU nucleotides; with up to 250-fold increases. Studies of human cell lines found that incubation with these damaged nucleosides resulted in cytotoxicity and increased DNA double-strand break levels.

biochemistry↗

bioRxiv2025

RNA 2'-OH modification with stable reagents enabled bynucleophilic catalysis

RNA modification at 2-OH has typically required highly reactive acylating species that exhibit short half-lives in water, challenging purification, and limited shelf lives. Here, we investigate the use of more stable species as electrophilic reagents, employing nucleophilic catalysis to promote reactions. Results show that multiple previously unreported electrophiles can react in high stoichiometric yields with RNA under appropriate catalysis. Most notably, aryl esters can transfer acyl groups to RNA in one hour, but are stable for months even in pure water. The results expand the functional chemotypes of RNA-reactive species, and identify reagent classes with improved stability and selectivity.

biochemistry↗

bioRxiv2025

Sequence-Specific Installation of Aryl Groups in RNA via DNA-Catalyst Conjugates

Installing functional groups at specific sites in existing RNA molecules remains a challenge for modification, labeling, and therapeutic strategies. Here we describe the use of DNA oligonucleotides carrying a catalytic amine group to effect the aqueous SNAr arylation of 2'-OH groups at sequence-complementary sites in RNAs. Chloro-pyrimidine electrophiles are shown to react with amino-DNA conjugates, resulting in a proposed transient ammonium aryl intermediate that can react with RNA near the DNA binding site, delivering the heterocycle to the RNA in high yields. In a test of utility, we construct an aryl electrophile carrying an azide group, and apply this strategy to fluorescently label messenger RNAs locally at the polyA tail. We also employ the approach to direct in vitro arylation in the coding region of a messenger RNA, knocking down protein expression selectively in the presence of another coding RNA. This sequence-directed catalytic strategy enables multiple applications in RNA labeling and modification.

biochemistry↗

bioRxiv2025

Fluorogenic Covalent Probes for RNA

Sequence-generalized fluorescent labels and stains for RNA can enable imaging, tracking and analysis of the biopolymer. However, current non-covalent RNA dyes are poorly selective for RNA over DNA, interact weakly with their target, and can show limited utility in cellular RNA staining due to poor selectivity and high background signals. Here we report a fluorogenic covalent labeling approach based on acylimidazole-mediated reaction of donor-acceptor fluorophores with 2'-hydroxyl (2'-OH) groups of RNA, providing a wavelength-tunable, sequence-independent strategy for selective labeling of the biopolymer. This reactive probe design enables labeling and imaging under mild aqueous conditions, providing up to 390-fold fluorescence enhancement and 970-fold selectivity for RNA over DNA, with four emission colors documented. The covalent fluorophore platform enables improved new tools for RNA-specific analysis and imaging in gels, in solution, and in living cells.

biochemistry↗

bioRxiv2025

C-Nucleosides Stabilize RNA by Reducing Nucleophilicity at 2'-OH

Nucleotides with carbon substitution for heteroatoms are common in biological and therapeutic RNAs. Important examples include the C-nucleosides pseudouridine and N1-methyl-pseudouridine; these modifications were reported to slow degradation of large RNAs, but the mechanism is unknown. We measured kinetics of spontaneous and enzymatic cleavage at a single bond of synthetically modified RNAs, and find that carbon substitution markedly reduces strand cleavage rates in RNA by both mechanisms. Studies of nucleophilic acylation reactions of RNAs and of small alcohols of varied pKa suggest that reduced inductive effects resulting from carbon substitution for electronegative atoms results in both higher pKa and lower nucleophilicity. The results provide insight into native transcriptome modifications as well as RNA therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/666442v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@73c194org.highwire.dtl.DTLVardef@1aa0af7org.highwire.dtl.DTLVardef@b5f8a0org.highwire.dtl.DTLVardef@d601fa_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOTOC GraphicC_FLOATNO C_FIG SynopsisThis study reveals mechanisms by which modifications found in native and therapeutic RNAs enhance RNA stability. Carbon substitution for nitrogen or oxygen results in reduced 2'-OH nucleophilicity.

biophysics↗

bioRxiv2023

RNA Infrastructure Profiling Illuminates Transcriptome Structure in Crowded Spaces

RNAs can fold into compact three-dimensional structures, and most RNAs undergo protein interactions in the cell. These compact and occluded environments can block the ability of structure-probing agents to provide useful data about the folding and modification of the underlying RNA. The development of probes that can analyze structure in crowded settings, and differentiate the proximity of interactions, can shed new light on RNA biology. To this end, here we employ 2,-OH-reactive probes that are small enough to access folded RNA structure underlying many close molecular contacts within cells, providing considerably broader coverage for intracellular RNA structural analysis. We compare reverse transcriptase stops in RNA-Seq data from probes of small and standard size to assess RNA-protein proximity and evaluate solvent-exposed tunnels adjacent to RNA. The data are analyzed first with structurally characterized complexes (human 18S and 28S RNA), and then applied transcriptome-wide to polyadenylated transcripts in HEK293 cells. In our transcriptome profile, the smallest probe acetylimidazole (AcIm) yields 80% greater structural coverage than larger conventional reagent NAIN3, providing enhanced structural information in hundreds of transcripts. We further show that acetyl probes provide superior signals for identifying m6A modification sites in transcripts, and provide information regarding methylation sites that are inaccessible to a larger standard probe. RNA infrastructure profiling (RISP) enables enhanced analysis of transcriptome structure, modification, and interactions in living cells, especially in spatially crowded settings.

biochemistry↗

bioRxiv2022

Pervasive Transcriptome Interactions of Protein-Targeted Drugs

The off-target toxicity of drugs targeted to proteins imparts substantial health and economic costs. Proteome interaction studies can reveal off-target effects with unintended proteins; however, little attention has been paid to intracellular RNAs as potential off targets that may contribute to toxicity. To begin to assess this, we developed a reactivity-based RNA profiling (RBRP) methodology, and applied it to uncover transcriptome interactions of a set of FDA-approved small-molecule drugs in vivo. We show that these protein-targeted drugs pervasively interact with the human transcriptome and can exert unintended biological effects on RNA function. In addition, we show that many off-target interactions occur at RNA loci associated with protein binding and structural changes, allowing us to generate hypotheses to infer the biological consequences of RNA off-target binding. The results suggest that rigorous characterization of drugs' transcriptome interactions may help assess target specificity and potentially avoid toxicity and clinical failures.

pharmacology and toxicology↗

bioRxiv2021

Microbial byproducts determine reproductive fitness of free-living and parasitic nematodes

A distinguishing feature of Trichuris nematodes is that these parasitic worms reproduce within the digestive tracts of humans and other mammalian hosts shedding thousands of eggs daily, facilitating their sustained presence in the environment and hampering eradication efforts. Although this aspect of the lifecycle places Trichuris in a microbiota-rich environment, metabolic byproducts of bacteria that facilitate the reproductive development of parasites are unknown. Here, we employ a pipeline using the well-characterized free-living nematode C. elegans to identify microbial factors with conserved roles in the reproduction of nematodes. A screen for E. coli mutants that impair C. elegans fertility identified genes in fatty acid biosynthesis and ethanolamine utilization pathways, including fabH and eutN. Trichuris muris eggs displayed defective hatching in the presence of E. coli deficient in fabH or eutN due to reduction in arginine or elevated levels of aldehydes, respectively. Remarkably, T. muris reared in gnotobiotic mice colonized with these E. coli mutants displayed profound abnormalities including morphological defects and a failure to lay viable eggs. These findings indicate that microbial byproducts mediate evolutionarily conserved transkingdom interactions that impact the reproductive fitness of distantly-related nematodes.

microbiology↗

bioRxiv2021

Integrating transcription factor abundance with chromatin accessibility in human erythroid lineage commitment

We present InTAC-seq, a method for simultaneous quantification of genome-wide open chromatin and intracellular protein abundance in fixed cells. Using InTAC we directly observe variation in chromatin accessibility and transcription factor motif occupancy driven by differences in transcription factor protein abundance. By purifying bone marrow progenitor cells based on GATA1 protein expression, we establish its role in both functional and epigenetic restriction of erythroid cell identity in human hematopoiesis.

genomics↗

bioRxiv2020

Site-specific RNA Functionalization via DNA-induced Structure

Site-specific RNA functionalization is in high demand, but remains a challenge, particularly for RNAs produced by transcription rather than by total synthesis. Recent studies have described acylimidazole reagents that react in high yields at 2-OH groups in RNAs. To date, the reactions occur stochastically at non-base-paired regions of RNA, covering much of the RNA in scattered acyl esters. Localized reactions, if possible, could prove useful in many applications, providing functional handles at specific sites and sequences of the biopolymer. Here we describe a DNA-directed strategy for in vitro functionalization of RNA at site-localized 2-OH groups. The method, RNA Acylation at Induced Loops (RAIL), utilizes complementary helper DNA oligonucleotides that expose gaps or loops at selected positions while protecting the remainder in DNA-RNA duplexes. Reaction with acylimidazole reagents is then carried out, providing high yields of 2-OH conjugation at predetermined sites. Subsequent removal of the DNA provides the RNA functionalized as desired. Experiments reveal optimal helper oligodeoxynucleotide designs and conditions for the reaction, and tests of the approach were carried out to control ribozyme activities and to label RNAs with dual-color fluorescent dyes. The RAIL approach offers a simple new strategy for site-specific labeling and controlling RNAs of any length and origin.

biochemistry↗