The potential of coding RNAs as a general therapeutic modality is limited by their short intracellular lifetime. Here, we investigate the effects of localized post-transcriptional RNA modification on protein expression over time. While 2 '-OH acylation of GFP RNA with stable adducts in the protein-coding region strongly suppressed protein expression, acylation at the poly(A) tail extended translation duration, with protein output increased by up to 8-fold at 36 h. Aryl amino acid derivatives proved to be most effective, while alkyl variants showed little effect. Preliminary mechanistic experiments point to disruption of the poly(A) helical structure as a contributing factor. Our study demonstrates the potential of post-transcriptional localized 2 '-acylation as a simple molecular solution to enhance protein-expression capabilities of RNAs.

A potentially promising approach to targeted cancer prevention in genetically at-risk populations is the pharmacological upregulation of DNA repair pathways. SMUG1 is a base excision repair enzyme that ameliorates adverse genotoxic and mutagenic effects of hydrolytic and oxidative damage to pyrimidines. Here we describe the discovery and initial cellular activity of a small-molecule activator of SMUG1. Screening of a kinase inhibitor library and iterative rounds of structure-activity relationship studies produced compound 40 (SU0547), which activates SMUG1 by as much as 350 +/- 60% in vitro at 100 nM, with an AC50 of 4.3 +/- 1.1 mu M. To investigate the effect of compound 40 on endogenous SMUG1, we performed in vitro cell-based experiments with 5-hydroxymethyl-2'-deoxyuridine (5-hmdU), a pyrimidine oxidation product that is selectively removed by SMUG1. In several human cell lines, compound 40 at 3-5 mu M significantly reduces the cytotoxicity of 5-hmdU and decreases levels of double-strand breaks induced by the damaged nucleoside. We conclude that the SMUG1 activator compound 40 is a useful tool to study the mechanisms of 5-hmdU toxicity and the potentially beneficial effects of suppressing damage to pyrimidines in cellular DNA.

The efficacy of strategies targeting oncogenic RAS, prevalent in lung adenocarcinoma (LUAD), is limited by rapid adaptive resistance mechanisms. These include loss of RAS addiction and hyperactivation of downstream signaling pathways, such as PI3K/AKT. We previously reported that oncogenic RAS-driven LUAD cells possess an enhanced reliance on MTH1, the mammalian 8-oxodGTPase, to prevent genomic incorporation of oxidized nucleotides, and that MTH1 depletion compromises tumorigenesis and oncogenic signaling. Here, we show that elevated MTH1 correlates with poor prognosis in LUAD and that its redox-protective 8-oxodGTPase activity is variably regulated in KRAS-addicted vs. non-addicted states. Multiple oncogenic KRAS mutants or overexpression of wildtype (wt) KRAS increased MTH1 expression. Conversely, KRAS depletion or its inhibition by AMG-510 (sotorasib) decreased MTH1 in KRASG12C-addicted LUAD cells. Separation-of-function MEK/ERK1/2-activating mutants recapitulated the elevated MTH1 expression induced by oncogenic RAS in wt KRAS LUAD cells. However, upon inhibition of the MEK/ERK1/2 pathway, compensatory AKT activation maintained MTH1 expression. Indeed, elevated AKT signaling maintained high MTH1 expression even when KRAS oncoprotein was low. We previously reported that cancer cells possess variable MTH1-specific and MTH1-independent 8-oxodGTPase activity levels. Whereas both ERK1/2 and AKT could regulate MTH1 protein levels in KRAS-addicted cells, only AKT signaling was associated with elevated MTH1-specific 8-oxodGTPase activity under KRAS-low or KRAS non-addicted states. Our studies suggest that despite loss of KRAS dependency, LUAD cells retain the requirement for high MTH1 8-oxodGTPase activity due to redox vulnerabilities associated with AKT signaling. Thus, MTH1 may serve as a novel orthogonal vulnerability in LUAD that has lost KRAS addiction.

DNA methylation at cytosine bases (5-methylcytosine, 5mC) is a heritable epigenetic mark regulating gene expression. While enzymes that metabolize 5mC are well-characterized, endogenous signaling molecules that regulate DNA methylation machinery have not been described. We report that physiological nitric oxide (NO) concentrations reversibly inhibit the DNA demethylases TET and ALKBH2 by binding to the mononuclear non-heme iron atom forming a dinitrosyliron complex (DNIC) and preventing cosubstrates from binding. In cancer cells treated with exogenous NO, or endogenously synthesizing NO, 5mC and 5-hydroxymethylcytosine (5hmC) increase, with no changes in DNA methyltransferase activity. 5mC is also significantly increased in NO-producing patient-derived xenograft tumors from mice. Genome-wide methylome analysis of cells chronically treated with NO (10 days) shows enrichment of 5mC and 5hmC at gene-regulatory loci, correlating with altered expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a unique epigenetic role for NO.

RNA modification at 2'-OH has typically required highly reactive acylating species that exhibit short half-lives in water, challenging purification, and limiting shelf lives. Here, we investigate the use of more stable species as electrophilic reagents, employing nucleophilic catalysis to promote reactions. The 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.

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.

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.

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.

Abstract Background: Pharmacological upregulation of DNA repair pathways presents a potentially promising approach for cancer prevention in populations that are genetically high-risk. SMUG1, an enzyme involved in base excision repair, removes uracil and certain oxidized bases from DNA to mitigate adverse genotoxic and mutagenic effects and represents a promising target for cancer therapy. A growing body of evidence suggests that SMUG1 is involved in tumorigenesis, and deficiency of SMUG1 correlates with poor prognosis in several cancers. We hypothesize that upregulation of SMUG1 by a small molecule activator may be a viable strategy for reducing tumorigenesis in at-risk populations. We report on the properties of SU0547 - a SMUG1 activator that was derived from the EGFR inhibitor drug gefitinib - which shows reduced kinase inhibition and potent activation of SMUG1 both in vitro and in human cell lines. Methods: MTT proliferation assays were used to screen for toxicity and to determine the effect of SU0547 on 5-hydroxymethyl-2’-deoxyuridine (5-hmdU)-treated cell lines. The effect of the compound on SMUG1 expression was determined by Western blot and RT-qPCR while its effect on DSBs via γH2AX expression in 5-hmdU-treated cells was determined by immunofluorescence and Western blot. Results: SU0547 activates SMUG1 by 347 ± 50% in vitro at 100 nM, with an AC50 of 2.55 ± 0.62 µM. We examined whether it can modulate SMUG1 activity in a panel of human cell lines by artificially inducing DNA damage with the native SMUG1 substrate 5-hmdU and treating cells with the activator. In all five human cancer cell lines tested, SU0547 caused the cells to become significantly more resistant to 5-hmdU by factors of 2.1 to 5.3-fold. A series of CRISPR-Cas9 generated SMUG1 KO cell lines showed resistance to 5-hmdU and SU0547 had no effect on these treated cells, confirming that this activator acts on endogenous SMUG1. To determine the effect of the activator on double-strand breaks (DSBs), cells were treated with 5-hmdU with or without the compound and DSBs were visualized and quantitated by examining γH2AX foci. 5-hmdU treatment induced γH2AX foci while SU0547 treatment significantly reduced these foci to near or below baseline levels. These results were confirmed using Western blot quantitation of γH2AX. Conclusions: We envision that this activator will serve as a useful tool for further studying the role of SMUG1 in mediating 5-hmdU toxicity and will enable the testing of our hypothesis that enhancing DNA repair processes may serve to inhibit mutagenesis and slow progression of pre-neoplastic lesions to invasive cancers associated with genomic instability. Citation Format: Lisa A. McPherson, Yixuan Gao, Shanthi Adimoolam, Samyuktha Suresh, David L. Wilson, Ishani Das, Elizabeth R. Park, Christine S. Ng, Yong Woong Jun, James M. Ford, Eric T. Kool. A small-molecule SMUG1 activator enhances repair of 5-hydroxymethyl-2’-deoxyuridine-mediated pyrimidine lesions in DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4777.

Activated acyl species have proven versatile in the esterification of 2 '-OH groups in RNA, enabling structure mapping, caging, profiling, and labeling of the biopolymer. Nearly all reagents developed for this reaction have been achiral; however, a recent study reported that simple chiral amino acid acylimidazole derivatives could yield diastereoselective reactions at RNA 2 '-OH in water, enabling up to 4:1 selectivity in screening. Here, we investigated the effect of steric bulk on the stereoselectivity of RNA reaction and on the stability of adducts with a library of 36 chiral acylimidazole scaffolds with increasing steric demand. The results document the highest stereoselectivity yet achieved in RNA acylation reactions, with as high as >99:1 diastereoselectivity at >70% conversion. Also notably, the bulky adducts were found to have markedly improved stability on RNA.

Hydrolytic and oxidative damage to pyrimidine nucleobases in DNA represents a significant source of mutations in the human genome. To better understand how these lesions are incorporated and repaired in human cells, it is desirable to have ready access to isotopically enriched nucleosides for use in isotope tracing and mass spectrometry-based quantification experiments. Here we report on improved syntheses of deoxyuridine, deoxycytidine, 5-hydroxydeoxyuridine, and 5-hydroxydeoxycytidine nucleosides labeled with 13C and 15N. Deoxyuridine was synthesized from uracil in a direct glycosylation reaction with excellent stereoselectivity without the need to reduce a ribonucleoside intermediate. Deoxyuridine was further converted to deoxycytidine using mild O4 activation conditions with high efficiency. Finally, we document the synthetic details of preparative oxidation of deoxyuridine and deoxycytidine to their 5-hydroxy counterparts. Overall, our protocols avoid hazardous reagents and tedious conditions found in previous methods.

DNA methylation at cytosine bases of eukaryotic DNA (5-methylcytosine, 5mC) is a heritable epigenetic mark that can regulate gene expression in health and disease. Enzymes that metabolize 5mC have been well-characterized, yet the discovery of endogenously produced signaling molecules that regulate DNA methyl-modifying machinery have not been described. Herein, we report that the free radical signaling molecule nitric oxide (NO) can directly inhibit the Fe(II)/2-OG-dependent DNA demethylases ten-eleven translocation (TET) and human AlkB homolog 2 (ALKBH2). Physiologic NO concentrations reversibly inhibited TET and ALKBH2 demethylase activity by binding to the mononuclear non-heme iron atom which formed a dinitrosyliron complex (DNIC) preventing cosubstrates (2-OG and O2) from binding. In cancer cells treated with exogenous NO, or cells endogenously synthesizing NO, there was a global increase in 5mC and 5-hydroxymethylcytosine (5hmC) in DNA, the substrates for TET, that could not be attributed to increased DNA methyltransferase activity. 5mC was also elevated in NO-producing cell-line-derived mouse xenograft and patient-derived xenograft tumors. Genome-wide DNA methylome analysis of cells chronically treated with NO (10 days) demonstrated enrichment of 5mC and 5hmC at gene-regulatory loci which correlated to changes in the expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a novel epigenetic role for NO.

A key intermediate in the inflammatory pathway is 7,8-dihydro-8-oxoguanine (8-oxoG), the most frequent oxidation DNA product of reactive oxygen species (ROS). In the process of repair and release of 8-oxoG from the genome, 8-Oxoguanine-DNA glycosylase-1 (OGG1) has been reported to increase expression of pro-inflammatory cytokines. In our previous study, we showed that 8-oxoG and OGG1 were highly expressed in peripheral CD14+ monocytes from patients with sickle cell disease (SCD) and peritoneal macrophages from Townes HbSS mice. Macrophages incubated with plasma from SCD-HbSS patients showed increased expression of pro-inflammatory cytokines IL-1ꞵ, IL-6, TNF-α, which was largely blocked by pretreatment with SU0268, a selective OGG1 inhibitor, further confirmed using OGG1-unique siRNAs. Objective: To investigate the mechanism of how OGG1 regulates expression of the pro-inflammatory cytokines utilizing mass-spectrometry analysis. Methods: Blood samples were obtained from ethnic-matched healthy controls (HbAA) and patients with SCD-HbSS enrolled under protocols NCT03049475 and NCT00047996). CD14+ monocytes were isolated from PBMCs of HbAA and HbSS subjects using EasySep™ Human Monocyte Isolation Kit (STEMCELL Technologies). Human THP-1 monocytic cells were grown in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. To acquire macrophages from monocytes, THP1 cells were incubated with 50 ng/ml PMA (Sigma-Aldrich, MO USA) for 24h. One transfection reagent and two siRNAs from an evaluation of four different transfection reagents and four OGG1-unique siRNAs were chosen for the study. After differentiation, macrophages derived from THP1 cells were transfected with OGG1 siRNA for 48h and then incubated with HbAA or HbSS steady state or crisis plasma, followed by qRT-PCR quantitation of IL-1ꞵ, IL-6, and TNF-α mRNA. To identify the binding partner of OGG1, a mass-spectrometry method was employed to analyze the product immunoprecipitated by OGG1. The identified proteins were further filtered based on the Human Transcription Factor database. Differential expressed proteins were selected based on t-test and fold change along with volcano plot. Null expression PSM was adjusted by adding negligible value. All analyses were conducted with R (v4.3.2). The interaction between OGG1 and STAT1 was confirmed by co-immunoprecipitation, and the level of tyrosine phosphorylation of STAT1 (Tyr-p-STAT1) measured by Western blotting. To confirm the effect of STAT1 on immune response, THP1 derived macrophages were treated with fludarabine (STAT1 inhibitor) or transfected with two STAT1-unique siRNAs, and then incubated with plasma from HbSS patients, followed by qRT-PCR quantitation of IL-1ꞵ, IL-6, TNF-α, and OGG1 mRNA. The putative binding sites of STAT1 on IL-1ꞵ, IL-6, and TNF-α promoters were predicted by JASPAR database. Results: We first confirmed that siRNA knockdown of OGG1 blocked the increased expression of IL-1ꞵ, IL-6, and TNF-α in macrophages treated with plasma from HbSS patients (steady and crisis status). We next sought to identify the binding partners of OGG1 in the initiation of expression of these inflammatory cytokines. Mass-spectrometry analysis of OGG1 immunoprecipitation product identified more than one thousand proteins, of which 81 were transcription factors. Signal transducer and activator of transcription 1 (STAT1) ranked as the second highest occurrence. We confirmed the interaction between OGG1 and STAT1 via co-immunoprecipitation. Interestingly, the activity of STAT1 (Tyr-p-STAT1) in HbSS CD14+ monocytes is higher than that in HbAA CD14+ monocytes, suggesting STAT1 as a potential therapeutic target of inflammation in SCD. To investigate the role of STAT1 in regulating cytokine expression, we treated THP1 derived macrophages with fludarabine or STAT1 siRNA. Plasma from HbSS patients increased the expression of OGG1, IL-1ꞵ, IL-6, and TNF-α, and the increase was largely blocked by fludarabine or STAT1 siRNA. A search of JASPAR database revealed three, three, and one putative binding sites of STAT1 on the promoters of IL-1ꞵ, TNF-α, and IL-6, respectively, which suggests a direct regulatory role of STAT1 on these pro-inflammatory cytokines. Conclusion: OGG1 recruits STAT1 to regulate the expression of pro-inflammatory cytokines in SCD. OGG1 inhibition could be a potential therapeutic approach to control sickle cell inflammation.

Incorporating stimuli-responsive components into RNA constructs provides precise spatiotemporal control over RNA structures and functions. Despite considerable advancements, the utilization of redox-responsive stimuli for the activation of caged RNAs remains scarce. In this context, we present a novel strategy that leverages post-synthetic acylation coupled with redox-responsive chemistry to exert control over RNA. To achieve this, we design and synthesize a series of acylating reagents specifically tailored for introducing disulfide-containing acyl adducts into the 2'-OH groups of RNA ("cloaking"). Our data reveal that these acyl moieties can be readily appended, effectively blocking RNA catalytic activity and folding. We also demonstrate the traceless release and reactivation of caged RNAs ("uncloaking") through reducing stimuli. By employing this strategy, RNA exhibits rapid cellular uptake, effective distribution and activation in the cytosol without lysosomal entrapment. We anticipate that our methodology will be accessible to laboratories engaged in RNA biology and holds promise as a versatile platform for RNA-based applications.

RNA plays pivotal roles in most cellular processes, serving as both the traditional carrier of genetic information and as a key regulator of cellular functions. The advent of chemical technologies has contributed critically to the analysis of cellular RNA structures, functions, and interactions. Many of these methods and molecules involve the utilization of chemically reactive handles in RNAs, either introduced externally or inherent within the polymer itself. Among these handles, the 2'-hydroxyl (2'-OH) group has emerged as an exceptionally well-suited and general chemical moiety for the modification and profiling of RNAs in intracellular studies. In this review, we provide an overview of the recent advancements in intracellular applications of acylation at the 2'-OH group of RNA. We outline progress made in probing RNA structure and interactomes, controlling RNA function, RNA imaging, and analyzing RNA-small molecule interactions, all achieved in living cells through this simple chemical handle on the biopolymer.

Electrophilic water-soluble compounds have proven versatile in reacting selectively with 2'-OH groups in RNA, enabling structure mapping, probing, caging, labeling, crosslinking, and conjugation of RNAs in vitro and in living cells. While early work focused on one or two types of reagents with limited properties, recent studies have greatly diversified the structure, properties, and applications of these reagents. Here we review the scope of documented RNA hydroxyl-reactive species reported to date, with an eye to the effects of chemical structure on reactivity with RNA and other useful properties. Multiple forms of carbonyl electrophiles are now known to react at the 2'-OH, and recently, sulfonyl and aryl electrophiles have also been documented to form bonds there in high yields as well. In addition to electrophilicity, data also point to significant effects of reagent stability, steric bulk, and chirality on reaction yields and selectivity. Finally, we outline reagent properties and principles that define utility in applications with RNA, with an eye to the design of future reagents.

The CRISPR-Cas9 system is a widely popular tool for genome engineering. There is strong interest in developing tools for temporal control of CRISPR-Cas9 activity to address some of the challenges and to broaden the scope of potential applications. In this work, we describe a bio-orthogonal chemistry-based approach to control nuclease activity with temporal precision. We report a trans-cyclooctene (TCO)-acylimidazole reagent that acylates 2'-OH groups of RNA. Poly acylation ("cloaking") of RNA was optimized in vitro using a model 18-nt oligonucleotide, as well as CRISPR single guide RNA (sgRNA). Two hours of treatment completely inactivated sgRNA for Cas9-assisted DNA cleavage. Nuclease activity was restored upon addition of tetrazine, which removes the TCO moieties via a two-step process ("uncloaking"). The approach was applied to target the GFP gene in live HEK293 cells. GFP expression was analyzed by flow cytometry. In the future, we anticipate that our approach will be useful in the field of developmental biology, by enabling investigation of genes of interest at different stages of an organism's development.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive KRAS-driven cancer that remains one of the most lethal of all human malignancies. Oncogenic KRAS relies on elevated reactive oxygen species (ROS) to support pro-tumorigenic signaling. However, these elevated ROS levels can also evoke tumor suppressive oxidative stress; thus KRAS-driven cancers must evolve protective redox adaptations to mitigate ROS-induced anti-tumor consequences. Here we report a unique adaptation centered on redox maintenance of nucleotide pool integrity, that supports aggressive PDAC tumorigenesis. MTH1 is the main mammalian 8-oxodGTPase that is known to be elevated in various RAS-driven cancers including PDAC. We have previously shown MTH1 is critical for ROS-mediated oncogenic signaling, and promotes escape from oncogene-induced senescence. We therefore hypothesized that MTH1 provides important redox support for aggressive PDAC tumor growth. Consistent with this, we found that elevated MTH1 expression was strongly correlated with poor disease-free survival in PDAC patients. These findings extended to KRAS-driven PDAC cell lines: highly proliferative human PDAC cells strongly expressed MTH1 (MTH1high), and MTH1 deletion was selectively anti-tumorigenic against MTH1high cells. Furthermore, introduction of MTH1-null backgrounds into gold standard genetically engineered PDAC mouse models selectively decreased 8-oxodGTPase activity levels and slowed PDAC growth in aggressive, short latency Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) mice, but with no effect in longer latency Pdx1Cre; LSL-KrasG12D/+; LSL-Trp53R172H/+ (KPC) mice. These selective anti-tumorigenic effects were accompanied by a reduction in circulating immunosuppressive cytokines in PKT mice, further suggesting that MTH1 potentially supports aggressive tumorigenesis through induction of systemic immunosuppression. Consistent with this, pancreatic-restricted MTH1 conditional deletion fully rescued PDAC growth in PKT mice. Our findings here thus suggest that MTH1-dependent redox protection of the nucleotide pool represents a unique TME adaptation that supports aggressive PDAC tumorigenesis. Further studies will investigate whether this adaptation is maintained through symbiotic tumor/stroma or tumor/immune cell crosstalk, as observed for other KRAS-driven traits. Ultimately, interventions designed to target MTH1-dependent protection of the nucleotide pool could hold promise as novel therapeutic strategies for this untreatable disease. Citation Format: Beatriz Mateo-Victoriano, Ling Zhang, Govindi J. Samaranayake, Vanessa Mcgaughey, Cassie Due, Clara Troccoli, Michael G. Mohsen, Nagaraj S. Nagathihalli, Oliver G. McDonald, Michael VanSaun, Eric T. Kool, Priyamvada Rai. The mammalian 8-oxodGTPase, MTH1, as a novel targetable vulnerability in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B091.

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.