Anna M. Kietrys

We developed a series of nitro reduction-reversible acylating reagents. Following optimization of the acylation conditions, these reagents were tested for deacylation with sodium dithionite in vitro . We applied this reversible acylation to modulate RNAzyme-mediated pre-tRNA maturation, demonstrating its ability to regulate RNA-RNA interactions. Furthermore, the in vitro reversible acylation of EGFP mRNA indicated effective control of its translational activity. To explore cellular applications, we validated NQO1-mediated deacylation in vitro and then induced hypoxia in HepG2 cells using cobalt chloride, thereby reactivating the function of acylated EGFP mRNA via endogenous NQO1. Overall, this study highlights the potential for developing nitro reduction-reversible acylation as a new strategy for RNA functional control and RNA-based drug modification.

RNase P was one of the first enzymes discovered to have an RNA-based catalytic component. Since its identification, it has been extensively studied, particularly in E. coli , due to the ability of its M1 RNA to exhibit in vitro catalytic activity even in the absence of associated protein. In this study, we report G-quadruplex formation as a potential regulatory mechanism that modulates the catalytic activity of this RNA. We observed a significantly higher propensity for G-quadruplex formation in the linear isoform (linM1) compared to its circular counterpart (circM1). G-quadruplex formation was confirmed through circular dichroism spectroscopy and a fluorescence-based assay using a G-quadruplex-binding small molecule. We compared the catalytic activity of linM1 and circM1 in lithium and potassium environments and found that G-quadruplex formation specifically reduced linM1 activity. Furthermore, we observed distinct condensate properties of linM1 in the presence or absence of G-quadruplex structures. Overall, our findings suggest that G-quadruplex formation serves as a regulatory switch for RNA activity in linM1, whereas circM1 resists G-quadruplex formation and remains catalytically active even under conditions that favor G-quadruplex assembly.

RNase P, one of the earliest enzymes identified with RNA as its catalytic component, has been extensively studied across all three domains of life. In this research, we unveil circular isoforms of RNase P RNA within bacterial, fungal, and human cell lines. Comparing the bacterial variant, circM1, with its linear counterpart under diverse conditions revealed its enhanced temperature resistance and superior tolerance to Mn ²⁺ . Moreover, our findings suggest distinct protein associations for both isoforms in the presence of FBS. The human counterpart, circH1, was proved to be active in cellulo .

Reversible 2′-OH acylation is a powerful strategy for switching RNA function, but existing systems often rely on nonphysiological or cytotoxic triggers for deacylation. Here we present EST1A , a hydrolysis-responsive 2′-OH acylating reagent whose RNA adducts are efficiently removed by endogenous esterases in vitro and in cellulo . EST1A acylates model oligonucleotides, an EGFP-targeting antisense strand, and reporter mRNAs, thereby modulating their activity; notably, the acylated antisense strand shows enhanced EGFP knockdown in HepG2 cells. By tuning carboxylesterase and cholinesterase activity and comparing EST1A- acylated mCherry mRNA across noncancerous and cancer-derived cell lines, we reveal a positive correlation between intracellular esterase activity and functional recovery of acylated RNA. These results establish EST1A -mediated, hydrolysis-responsive 2′-OH acylation as a simple platform for enzyme-guided, cell-selective activation of RNA function and point toward esterase-activated RNA therapeutics. Entry for the Table of Contents: Liu et al. introduce EST1A , a hydrolysis-responsive 2′-OH acylating reagent whose RNA adducts are removed by endogenous esterases or histidine, enabling reversible control of RNA function. By exploiting differences in esterase activity between noncancerous and cancer-derived cell lines, EST1A -treated mRNA exhibits enzyme-guided and cell-selective translational reactivation.

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 avoid toxicity and clinical failures.

We report the development of post-transcriptional chemical methods that enable control over CRISPR–Cas9 gene editing activity both in in vitro assays and in living cells. We show that an azide-substituted acyl imidazole reagent (NAI-N₃) efficiently acylates CRISPR single guide RNAs (sgRNAs) in 20 minutes in buffer. Poly-acylated (“cloaked”) sgRNA was completely inactive in DNA cleavage with Cas9 in vitro, and activity was quantitatively restored after phosphine treatment. Delivery of cloaked sgRNA and Cas9 mRNA into HeLa cells was enabled by the use of charge-altering releasable transporters (CARTs), which outperformed commercial transfection reagents in transfecting sgRNA co-complexed with Cas9 encoding functional mRNA. Genomic DNA cleavage in the cells by CRISPR–Cas9 was efficiently restored after treatment with phosphine to remove the blocking acyl groups. Our results highlight the utility of reversible RNA acylation as a novel method for temporal control of genome-editing function.

Oxidative damage in DNA is one of the primary sources of mutations in the cell. The activities of repair enzymes 8-oxoguanine DNA glycosylase (OGG1) and human MutT Homologue 1 (NUDT1 or MTH1), which work together to ameliorate this damage, are closely linked to mutagenesis, genotoxicity, cancer, and inflammation. Here we have undertaken the development of small-molecule dual inhibitors of the two enzymes as tools to test the relationships between these pathways and disease. The compounds preserve key structural elements of known inhibitors of the two enzymes, and they were synthesized and assayed with recently developed luminescence assays of the enzymes. Further structural refinement of initial lead molecules yielded compound 5 (SU0383) with IC₅₀(NUDT1) = 0.034 μM and IC₅₀(OGG1) = 0.49 μM. The compound SU0383 displayed low toxicity in two human cell lines at 10 μM. Experiments confirm the ability of SU0383 to increase sensitivity of tumor cells to oxidative stress. Dual inhibitors of these two enzymes are expected to be useful in testing multiple hypotheses regarding the roles of 8-oxo-dG in multiple disease states.

Acylation of RNA at 2′-OH groups is widely applied in mapping RNA structure and recently for controlling RNA function. Reactions are described that install the smallest 2-carbon acyl groups on RNA—namely, 2′-O-acetyl and 2′-O-carbonate groups. Hybridization and thermal melting experiments are performed to assess the effects of the acyl groups on duplex formation. Both reagents can be employed at lower concentrations to map RNA secondary structure by reverse transcriptase primer extension (SHAPE) methods.