Eric T. Kool

Bio-reversible 2′-OH polyacylation (cloaking) has been described recently as a post-transcriptional modification strategy for stabilizing RNAs while enabling recovery of biological function after delivery into cells. Here, we probe the chemical space and utility of this method by evaluating a series of new acyl adducts for their ability to maintain protein expression levels after being substituted at high levels (ca. 25% of nucleotides) on transcripts. Reagent designs focused on the inclusion of electron-withdrawing groups near the acyl carbonyl to enhance electrophilicity. Multiple new reagents demonstrated efficient messenger RNA acylation and stabilization of the RNA against thermal cleavage for two days at 37 °C. Cellular transfection experiments with these acylated transcripts showed that certain modifications maintained native or near-native levels of translation of a protein-coding RNA, confirming the robustness of the approach across a broad set of chemotypes. We further report that protein-coding RNAs polyacylated with selected reversible acyl groups show transiently reduced proinflammatory cytokine responses relative to unmodified RNA. Together, these findings expand the functional boundaries of bio-reversible 2′-OH acylation and provide guidance for future development of RNA protection and modification strategies.

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.

The nucleophilic reactivity of sidechains of biopolymers is broadly enabling for bioscience and biomedicine, providing selective means for modifying them and analyzing their structures, interactions, and biological roles. Recent studies of RNA have shown that the 2′‐OH group is unusually reactive as a nucleophile to acyl and aryl electrophiles, reacting in water at rates far higher than simple alcohols, but the origins of this high reactivity are not well understood. Here we investigate the chemical origins of this nucleophilicity at the basic monomer level, measuring reaction kinetics of a broadly varied set of nucleosides, nucleotides, and sugars with four distinct electrophiles documented to react with RNA. We find that the nucleobases play unexpectedly influential roles in enhancing reactivity, by their presence, their size, and possibly their dipoles. We also find that the 2′‐OH of ribose (pentose) framework is unusually reactive as compared with that in structurally related hexose sugars. The results demonstrate that specific ribonucleoside structure contributes essentially to this nucleophilic reactivity, and likely influences RNA biology as a result.

Elevated activity of DNA repair enzymes that surveil pyrimidine damage has been associated with resistance to antitumor therapies. The DNA base excision repair (BER) enzyme SMUG1 recognizes and excises a number of modified and damaged pyrimidines that arise in cellular DNA, including deoxyuridine and 5-hydroxymethyl-dU, as well as the cytotoxic antitumor pyrimidine 5-fluorodeoxyuridine (5-FdU). Here we report the discovery and development of the first SMUG1 inhibitor scaffolds, derived from a small molecule sphingosine kinase inhibitor identified as a hit in assays against the DNA repair enzyme. Partial structural optimization resulted in compound SU0617, which inhibits SMUG1 with IC₅₀ = 1.5 ± 0.1 μM and has ablated kinase inhibitory activity. We expect that the new inhibitor can be useful as a tool in studies of the overlapping substrate preferences of base excision repair enzymes and in the study of the pathways involved in responses to therapeutic nucleoside drugs.

Sequence-generalized fluorescent labels and stains for RNA can enable imaging, tracking, and analysis of the biopolymer. However, current noncovalent 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.

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 forms. We found a 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.

Nucleotides with a carbon substitution for heteroatoms are common in biological and therapeutic RNAs. Important examples include the C-nucleosides pseudouridine and N1-methylpseudouridine; these modifications were reported to slow the 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 found that carbon substitution markedly reduces strand cleavage rates in RNA by both mechanisms. Studies of nucleophilic acylation reactions of RNAs and small alcohols of varied pK _a suggest that reduced inductive effects resulting from carbon substitution for electronegative atoms results in both higher pK _a and lower nucleophilicity. The results provide insight into native transcriptome modifications as well as RNA therapies.