RNA Molecules Research Articles

AI-integrated network for RNA complex structure and dynamic prediction.

RNA complexes are essential components in many cellular processes. The functions of these complexes are linked to their tertiary structures, which are shaped by detailed interface information, such as binding sites, interface contact, and dynamic conformational changes. Network-based approaches have been widely used to analyze RNA complex structures. With their roots in the graph theory, these methods have a long history of providing insight into the static and dynamic properties of RNA molecules. These approaches have been effective in identifying functional binding sites and analyzing the dynamic behavior of RNA complexes. Recently, the advent of artificial intelligence (AI) has brought transformative changes to the field. These technologies have been increasingly applied to studying RNA complex structures, providing new avenues for understanding the complex interactions within RNA complexes. By integrating AI with traditional network analysis methods, researchers can build more accurate models of RNA complex structures, predict their dynamic behaviors, and even design RNA-based inhibitors. In this review, we introduce the integration of network-based methodologies with AI techniques to enhance the understanding of RNA complex structures. We examine how these advanced computational tools can be used to model and analyze the detailed interface information and dynamic behaviors of RNA molecules. Additionally, we explore the potential future directions of how AI-integrated networks can aid in the modeling and analyzing RNA complex structures.

Exploring the therapeutic mechanisms of heart failure with Chinese herbal medicine: a focus on miRNA-mediated regulation

Heart failure (HF) is a clinical condition caused by abnormalities in the heart’s structure or function, primarily manifested as diminished ability of the heart to pump blood, which leads to compensatory activation of neurohormones and increased left ventricular filling pressure. HF is one of the fastest-growing cardiovascular diseases globally in terms of incidence and mortality, negatively impacting patients’ quality of life and imposing significant medical and economic burdens. Despite advancements in the treatment of HF, hospitalization and mortality remain rates high. In China, Chinese herbal medicine (CHM) has historically played a prominent role in addressing HF, with significant proven efficacy. MicroRNA (miRNA) exerts a pivotal regulatory influence on the maintenance of regular cardiac activity and the progression of HF. MiRNAs, a category of single-stranded RNA molecules, are characterized by their inability to code for proteins. They regulate gene expression by binding to the 3′-untranslated region (3′-UTR) of target mRNAs, thereby influencing the onset and progression of various diseases. Abnormal expression of specific miRNAs is closely associated with HF pathological processes, such as cardiomyocyte apoptosis, myocardial fibrosis, and cardiac hypertrophy. This abnormal expression can influence the pathological progression of HF through the regulation of miRNA expression. This article reviews the regulatory role of miRNAs in HF pathology discusses how CHM compounds and their active ingredients can ameliorate HF pathology through the regulation of miRNA expression. In conclusion, miRNAs represent promising therapeutic targets for HF, and CHM provides a novel strategy for treatment through the regulation of miRNA expression. Future studies must delve deeper into the precise mechanisms by which CHM modulates miRNAs and fully explore its potential for clinical application in HF treatment.

Expression profiles of circulating miRNAs in an endangered Piedmontese sheep breed during the estrus cycle

IntroductionThe preservation of locally endangered breeds is essential for maintaining ecosystem services that benefit both society and the environment. Reproductive fitness becomes a crucial consideration in this context. MicroRNAs (miRNAs) are small non-coding RNA molecules that play a key role in post-transcriptional regulation. Typically, they function within the tissues where they are produced. However, when they are released into extracellular fluid, they are referred to as circulating miRNAs (c-miRNAs). C-miRNAs may serve as potential biomarkers, whose profile changes under different physiological states. The purpose of this study is to establish a connection between distinctive variations in the expression of c-miRNAs and specific estrus cycle phases in Frabosana-Roaschina sheep, an endangered Piedmontese breed.MethodsTwo trials, each involving 20 ewes with different reproductive efficiencies (nulliparous in the first trial and pluriparous in the second trial), were sampled on alternate days after synchronization for blood, saliva, and feces. Ultrasound scans were performed during the induced estrus cycle. The animals’ behaviors were assessed through video recordings.ResultsIn the first trial, play behaviors were detected without sexual behaviors, whereas in the second trial, sexual behaviors were observed without play behaviors. Based on plasma trends of 17β-estradiol and progesterone and ultrasound images, two moments were identified for miRNAs analyses: the beginning of the follicular phase (day 2) and the beginning of the luteal phase (day 11). C-miRNAs of six representative animals from the second trial were sequenced. Analyses of the sequencing data have identified 12 c-miRNAs that were differentially expressed (DE) when comparing day 11 with day 2: five miRNAs were found to be upregulated, whereas seven miRNAs were downregulated. An enrichment analysis, based on predicted targets, using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases was performed. Many of these genes regulate reproductive pathways with the possible involvement of miRNAs. Finally, qRT-PCR was conducted to validate the DE miRNAs in all ewes. Differences in gene expression between the two sampling points and the two trials were observed, in line with existing literature.DiscussionInvestigating the role of these miRNAs in regulating estrus could improve the reproductive performance and welfare of Frabosana-Roaschina ewes.

Expanding the landscape of systemic sclerosis-related autoantibodies through RNA immunoprecipitation coupled with massive parallel sequencing

ObjectivesSystemic sclerosis (SSc)-related autoantibodies are widely used diagnostic and prognostic biomarkers. This study aimed to develop a new assay for detecting anti-ribonucleoprotein autoantibodies in SSc based on RNA immunoprecipitation (RNA IP) coupled with massive parallel sequencing. MethodsSerum samples and clinical data were collected from 307 SSc patients. Among these, 57 samples underwent analysis using a new protocol that combines RNA IP with massive parallel sequencing (RIP-Seq). Filtering strategies and statistical outlier detection methods were applied to select RNA molecules that could represent novel ribonucleoprotein autoantigens associated with SSc. ResultsAmong the 30,966 different RNA molecules identified by RIP-Seq in 57 SSc patients, 197 were ultimately selected. These included all RNA molecules previously identified by RNA IP, which were found to exhibit high counts almost exclusively in samples positive for the autoantibodies associated to the corresponding RNA molecule, indicating high sensitivity and specificity of the RIP-Seq technique. C/D box snoRNAs were the most abundant RNA type identified. The immunoprecipitation patterns of the detected C/D box snoRNAs varied among patients and could be associated with different clinical phenotypes. In addition, other ribonucleoproteins were identified, which could be potential targets for previously undescribed SSc-related autoantibodies. These include H/ACA box snoRNPs, vault complexes, mitochondrial tRNA synthetases, and 7SK snRNP. ConclusionA novel RIP-Seq assay has been developed to detect autoantibodies targeting ribonucleoprotein complexes in SSc patients. This method successfully identified RNA molecules associated with ribonucleoproteins known to be targeted by SSc-related autoantibodies, validating both the assay and the analysis strategy. Additionally, this approach uncovered RNA molecules associated with ribonucleoproteins that were not previously identified as targets of SSc patients’ sera, suggesting potential new autoantibody candidates in this disease.

Cationic and ionizable amphiphiles based on di-hexadecyl ester of <I>L</I>-glutamic acid for liposomal transport of RNA

Various RNAs are among the most promising and actively developed therapeutic agents for the treatment of tumors, infectious diseases and a number of other pathologies associated with the dysfunction of specific genes. Some nanocarriers are used for the effective delivery of RNAs to target cells, including liposomes based on cationic and/or ionizable amphiphiles. Cationic amphiphiles contain a protonated amino group and exist as salts in an aqueous environment. Ionizable amphiphiles are a new generation of cationic lipids that exhibit reduced toxicity and immunogenicity and undergo ionization only in the acidic environment of the cell. In this work we developed a scheme for the preparation and carried out the synthesis of new cationic and ionizable amphiphiles based on natural amino acids (L-glutamic acid, glycine, beta-alanine, and gamma-aminobutyric acid). Cationic and ionizable liposomes were formed based on the obtained compounds, mixed with natural lipids (phosphatidylcholine and cholesterol), and their physicochemical characteristics (particle size, zeta potential, and storage stability) were determined. Average diameter of particles stable for 5–7 days did not exceed 100 nm. Zeta potential of cationic and ionizable liposomes was about 30 and 1 mV, respectively. The liposomal particles were used to form complexes with RNA molecules. Such RNA complexes were characterized by atomic force microscopy and their applicability for nucleic acid transport was determined.

Directionality theory and the origin of life.

The origin of cellular life can be described in terms of the transition from inorganic matter to the emergence of cooperative assemblies of organic matter: DNA and proteins, capable of replication and metabolism. Directionality theory is a mathematical theory of the collective behaviour of networks of organic matter: activated macromolecules, cells and higher organisms. Evolutionary entropy, a generalization of the thermodynamic entropy of Boltzmann, is a statistical measure of the cooperativity of the biotic components. The cornerstone of Directionality theory is the Entropic Principle of Evolution: evolutionary entropy increases in systems driven by a stable energy source, and decreases in systems subject to a fluctuating energy source. This article invokes the Entropic Principle of Evolution-an extension to biological systems of the Second Law of Thermodynamics-to provide an adaptive rationale for the following sequence of transformations that define the emergence of cellular life: (i) the self-assembly of activated macromolecules from inorganic matter; (ii) the emergence of an RNA world, defined by RNA molecules with catalytic and replicative properties; and (iii) the origin of cellular life, the integration of the three carbon-based polymers-DNA, proteins and lipids, to generate a metabolic and replicative unit.

Measurement of circular intensity differential scattering (CIDS) from single optically trapped biological particles

The circular intensity differential scattering (CIDS), which is the normalized Mueller matrix element -S14/S11, has been measured from single biological particles as a function of scattering angle. CIDS is valuable for its potential in detecting chiral particles that may include the helical structures of DNA or RNA molecules in biological samples, and as such is a potential method for detecting biological particles. Optical trapping is employed to levitate single particles within a custom-designed elliptical reflector for CIDS measurements. The advantage of optical levitation in light-scattering measurements is that single particles can be suspended in air with sufficient working distance to prevent interference from the suspending apparatus. To measure the phase function, the reflector is used to collect the angle-dependent scattering signals. We demonstrated that we can obtain two-dimensional angular optical scattering (TAOS) patterns that cover a wide angular range from single levitated particles. These TAOS patterns are generated using 532 nm illumination of left-handed and right-handed circular polarizations and recorded from trapped single particles (silica, English Oak, Ragweed, Mulberry, Glycine, and l-Aspartic acid).

A Thorough Navigation of miRNA's Blueprint in Crafting Cardiovascular Fate.

Cardiovascular diseases contribute significantly to global morbidity and mortality. MicroRNAs are crucial in the development and progression of these diseases by regulating gene expression in various cells and tissues. Their roles in conditions like atherosclerosis, heart failure, myocardial infarction, and arrhythmias have been widely researched. The present study provides an overview of existing evidence regarding miRNAs' role in cardiovascular disease pathogenesis. Furthermore, the study examines current state-of-the-art technologies used in the study of miRNAs in cardiovascular disease. As a final point, we examine how miRNAs may serve as disease biomarkers, therapeutic targets, and prognostic indicators. In cardiology, microRNAs, small noncoding RNA molecules, are crucial to the posttranscriptional regulation of genes. Their role in regulating cardiac cell differentiation and maturation is critical during the development of the heart. They maintain the cardiac function of an adult heart by contributing to its electrical and contractile activity. By binding to messenger RNA molecules, they inhibit protein translation or degrade mRNA. Several cardiovascular diseases are associated with dysregulation of miRNAs, including arrhythmias, hypertension, atherosclerosis, and heart failure. miRNAs can be used as biomarkers to diagnose and predict diseases as well as therapeutic targets. A variety of state-of-the-art technologies have aided researchers in discovering, profiling, and analyzing miRNAs, including microarray analysis, next-generation sequencing, and others. Developing new diagnostics and therapeutic approaches is becoming more feasible as researchers refine their understanding of miRNA function. Ultimately, this will reduce the burden of cardiovascular disease around the world.

Synthetic circRNA therapeutics: innovations, strategies, and future horizons.

Small molecule drugs are increasingly emerging as innovative and effective treatments for various diseases, with mRNA therapeutics being a notable representative. The success of COVID-19 vaccines has underscored the transformative potential of mRNA in RNA therapeutics. Within the RNA family, there is another unique type known as circRNA. This single-stranded closed-loop RNA molecule offers notable advantages over mRNA, including enhanced stability and prolonged protein expression, which may significantly impact therapeutic strategies. Furthermore, circRNA plays a pivotal role in the pathogenesis of various diseases, such as cancers, autoimmune disorders, and cardiovascular diseases, making it a promising clinical intervention target. Despite these benefits, the application of circRNA in clinical settings remains underexplored. This review provides a comprehensive overview of the current state of synthetic circRNA therapeutics, focusing on its synthesis, optimization, delivery, and diverse applications. It also addresses the challenges impeding the advancement of circRNA therapeutics from bench to bedside. By summarizing these aspects, the review aims to equip researchers with insights into the ongoing developments and future directions in circRNA therapeutics. Highlighting both the progress and the existing gaps in circRNA research, this review offers valuable perspectives for advancing the field and guiding future investigations.

The Functions and Mechanisms of Long Non-coding RNA SNHGs in Gastric Cancer.

Gastric cancer (GC) is one of the most common malignancies worldwide. Despite significant advancements in surgical and adjuvant treatments, patient prognosis remains unsatisfactory. Long non-coding RNAs (lncRNAs) are a class of RNA molecules that lack protein-coding capacity but can engage in the malignant biological behaviors of tumors through various mechanisms. Among them, small nucleolar host genes (SNHGs) represent a subgroup of lncRNAs. Studies have revealed their involvement not only in gastric cancer cell proliferation, invasion, migration, epithelial- mesenchymal transition (EMT), and apoptosis but also in chemotherapy resistance and tumor stemness. This review comprehensively summarizes the biological functions, molecular mechanisms, and clinical significance of SNHGs in gastric cancer. It provides novel insights into potential biomarkers and therapeutic targets for the exploration of gastric cancer.

Circular RNA landscape in extracellular vesicles from human biofluids

BackgroundCircular RNAs (circRNAs) have emerged as a prominent class of covalently closed single-stranded RNA molecules that exhibit tissue-specific expression and potential as biomarkers in extracellular vesicles (EVs) derived from liquid biopsies. Still, their characteristics and applications in EVs remain to be unveiled.MethodsWe performed a comprehensive analysis of EV-derived circRNAs (EV-circRNAs) using transcriptomics data obtained from 1082 human body fluids, including plasma, urine, cerebrospinal fluid (CSF), and bile. Our validation strategy utilized RT-qPCR and RNA immunoprecipitation assays, complemented by computational techniques for analyzing EV-circRNA features and RNA-binding protein interactions.ResultsWe identified 136,327 EV-circRNAs from various human body fluids. Significantly, a considerable amount of circRNAs with a high back-splicing ratio are highly enriched in EVs compared to linear RNAs. Additionally, we discovered brain-specific circRNAs enriched in plasma EVs and cancer-associated EV-circRNAs linked to clinical outcomes. Moreover, we demonstrated that EV-circRNAs have the potential to serve as biomarkers for evaluating immunotherapy efficacy in non-small cell lung cancer (NSCLC). Importantly, we identified the involvement of RBPs, particularly YBX1, in the sorting mechanism of circRNAs into EVs.ConclusionsThis study unveils the extensive repertoire of EV-circRNAs across human biofluids, offering insights into their potential as disease biomarkers and their mechanistic roles within EVs. The identification of specific circRNAs and the elucidation of RBP-mediated sorting mechanisms open new avenues for the clinical application of EV-circRNAs in disease diagnostics and therapeutics.

An image-based RNAi screen identifies the EGF.R signaling pathway as a regulator of Imp/ IGF2BP RNP granules.

Biomolecular condensates have recently retained much attention since they provide a fundamental mechanism of cellular organization. Among those, cytoplasmic RNP granules selectively and reversibly concentrate RNA molecules and regulatory proteins, thus contributing to the spatio-temporal regulation of associated RNAs. Extensive in vitro work has unraveled the molecular and chemical bases of RNP granule assembly. The signaling pathways controlling this process in a cellular context are however still largely unknown. Here, we aimed at identifying regulators of cytoplasmic RNP granules characterized by the presence of the evolutionarily conserved IGF2BP/Imp/ZBP1 RNA binding protein. We performed a high-content image-based RNAi screen targeting all Drosophila genes encoding RNA binding proteins, phosphatases and kinases. This led to the identification of dozens of genes regulating the number of Imp+ RNP granules in S2R+ cells, among which components of the MAPK pathway. Combining functional approaches, phospho-mapping and generation of phospho-variants, we further showed that the EGF.R signaling inhibits Imp+ RNP granule assembly through activation of MAPK/Rolled and Imp S15 phosphosite. This work illustrates how signaling pathways can regulate cellular condensate assembly by post-translational modifications of specific components.

Viral RNA Interactome: The Ultimate Researcher’s Guide to RNA–Protein Interactions

RNA molecules in the cell are bound by a multitude of RNA-binding proteins (RBPs) with a variety of regulatory consequences. Often, interactions with these RNA-binding proteins are facilitated by the complex secondary and tertiary structures of RNA molecules. Viral RNAs especially are known to be heavily structured and interact with many RBPs, with roles including genome packaging, immune evasion, enhancing replication and transcription, and increasing translation efficiency. As such, the RNA–protein interactome represents a critical facet of the viral replication cycle. Characterization of these interactions is necessary for the development of novel therapeutics targeted at the disruption of essential replication cycle events. In this review, we aim to summarize the various roles of RNA structures in shaping the RNA–protein interactome, the regulatory roles of these interactions, as well as up-to-date methods developed for the characterization of the interactome and directions for novel, RNA-directed therapeutics.

The contribution of extracellular RNA and its derived biomaterials in disease management

AbstractThe RNA found in the circular system is known as extracellular RNA (exRNA). This kind of RNA has been found to play a biological role similar to that of a messenger. They can be used as indicators of disease status or the physiological health of an organism. A large number of RNA‐based biomaterials have been developed by simulating the biological function and structure of natural RNA molecules. The structural programmability of RNA‐based biomaterials provides the spur for scientists to pioneer new approaches in disease detection and prevention. Nevertheless, the link between exRNA function and the design of RNA‐based biomaterials has not been fully understood. Understanding the biological structure and function of exRNA will contribute to the clinical translation of this novel biotechnology. The present review discusses the research progress associated with exRNA and their derivatives to bridge the gap between natural exRNA and RNA‐based biomaterials.

Single-Cell Multiplexed Signal Amplification Strategy Based on Catalytic Hairpin Self-Assembly and CRISPR/Cas12a for Exploring the Relationship between lncRNA HOTAIR and miRNA-122 in Individual Hepatocytes.

The long noncoding RNA (lncRNA) HOTAIR has been shown to act as an oncogene in a variety of cancers, including hepatocellular carcinoma (HCC). MicroRNA-122 (miR-122) is a key liver-specific miRNA that is frequently inhibited in HCC and is associated with poor prognosis. However, a potential relationship between HOTAIR and miR-122 in individual hepatocytes has not been explored. To this end, we propose here an intracellular catalytic hairpin self-assembly-CRISPR/Cas12a tandem multiplexed signal amplification strategy for the simultaneous quantification of HOTAIR and miRNA-122 in a single hepatocyte. We applied this method to analyze both normal HL-7702 liver cells and HepG2 HCC cells, and found that HL-7702 cells contained large amounts of miRNA-122, while the content of miRNA-122 in HepG2 cells was low. However, the level of HOTAIR in HepG2 cells was much higher than that in HL-7702 cells, confirming the overexpression of HOTAIR in HCC cells. We achieved the simultaneous absolute quantification of HOTAIR and miRNA-122 in single cells, providing an important method to study the relationships between these two RNA molecules in individual cells.

Systematic analysis of the target recognition and repression by the Pumilio proteins.

RNA binding proteins orchestrate the post-transcriptional fate of RNA molecules, but the principles of their action remain poorly understood. Pumilio (PUM) proteins bind 3' UTRs of mRNAs and lead to mRNA decay. To comprehensively map the determinants of recognition of sequences by PUM proteins in cells and to study the binding outcomes, we developed a massively parallel RNA assay that profiled thousands of PUM-binding sites in cells undergoing various perturbations or RNA immunoprecipitation. By studying fragments from the NORAD long non-coding RNA, we find two features that antagonize repression by PUM proteins - G/C rich sequences, particularly those upstream of the PUM recognition element, and binding of FAM120A, which limits the repression elicited by PUM-binding sites. We also find that arrays of PUM sites separated by 8-12 bases offer particularly strong repression and use them to develop a particularly sensitive reporter for PUM repression. In contrast, PUM sites separated by shorter linkers, such as some of those found in NORAD, exhibit strong activity interdependence, likely mediated by competition between PUM binding and formation of strong secondary structures. Overall, our findings expand our understanding of the determinants of PUM protein activity in human cells.

Cross-chiral exponential amplification of an RNA enzyme.

An RNA ligase ribozyme that catalyzes the joining of RNA molecules of the opposite chiral handedness was optimized for the ability to synthesize its own enantiomer from two component fragments. The mirror-image D- and L-ligases operate in concert to provide a system for cross-chiral replication, whereby they catalyze each other's synthesis and undergo mutual amplification at constant temperature, with apparent exponential growth and a doubling time of about 1 h. Neither the D- nor the L-RNA components alone can achieve autocatalytic self-replication. Cross-chiral exponential amplification can be continued indefinitely through a serial-transfer process that provides an ongoing supply of the component D- and L-substrates. Unlike the familiar paradigm of semiconservative nucleic acid replication that relies on Watson-Crick pairing between complementary strands, cross-chiral replication relies on tertiary interactions between structured nucleic acids "across the mirror." There are few examples, outside of biology, of autocatalytic self-replication systems that undergo exponential amplification and there are no prior examples, in either biological or chemical systems, of cross-chiral replication enabling exponential amplification.

A robust deep learning approach for identification of RNA 5-methyluridine sites

RNA 5-methyluridine (m5U) sites play a significant role in understanding RNA modifications, which influence numerous biological processes such as gene expression and cellular functioning. Consequently, the identification of m5U sites can play a vital role in the integrity, structure, and function of RNA molecules. Therefore, this study introduces GRUpred-m5U, a novel deep learning-based framework based on a gated recurrent unit in mature RNA and full transcript RNA datasets. We used three descriptor groups: nucleic acid composition, pseudo nucleic acid composition, and physicochemical properties, which include five feature extraction methods ENAC, Kmer, DPCP, DPCP type 2, and PseDNC. Initially, we aggregated all the feature extraction methods and created a new merged set. Three hybrid models were developed employing deep-learning methods and evaluated through 10-fold cross-validation with seven evaluation metrics. After a comprehensive evaluation, the GRUpred-m5U model outperformed the other applied models, obtaining 98.41% and 96.70% accuracy on the two datasets, respectively. To our knowledge, the proposed model outperformed all the existing state-of-the-art technology. The proposed supervised machine learning model was evaluated using unsupervised machine learning techniques such as principal component analysis (PCA), and it was observed that the proposed method provided a valid performance for identifying m5U. Considering its multi-layered construction, the GRUpred-m5U model has tremendous potential for future applications in the biological industry. The model, which consisted of neurons processing complicated input, excelled at pattern recognition and produced reliable results. Despite its greater size, the model obtained accurate results, essential in detecting m5U.

IsRNAcirc: 3D structure prediction of circular RNAs based on coarse-grained molecular dynamics simulation.

As an emerging class of RNA molecules, circular RNAs play pivotal roles in various biological processes, thereby determining their three-dimensional (3D) structure is crucial for a deep understanding of their biological significances. Similar to linear RNAs, the development of computational methods for circular RNA 3D structure prediction is challenging, especially considering the inherent flexibility and potentially long length of circular RNAs. Here, we introduce an extension of our previous IsRNA2 model, named IsRNAcirc, to enable circular RNA 3D structure predictions through coarse-grained molecular dynamics simulations. The workflow of IsRNAcirc consists of four main steps, including input preparation, end closure, structure prediction, and model refinement. Our results demonstrate that IsRNAcirc can provide reasonable 3D structure predictions for circular RNAs, which significantly reduce the locally irrational elements contained in the initial input. Moreover, for a validation test set comprising 34 circular RNAs, our IsRNAcirc can generate 3D models with better scores than the template-based 3dRNA method. These findings demonstrate that our IsRNAcirc method is a promising tool to explore the structural details along with intricate interactions of circular RNAs.

Multiomic characterization of RNA microenvironments by oligonucleotide-mediated proximity-interactome mapping.

RNA molecules form complex networks of molecular interactions that are central to their function and to cellular architecture. But these interaction networks are difficult to probe in situ. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a method for elucidating the biomolecules near an RNA of interest, within its native context. O-MAP uses RNA-fluorescence in situ hybridization-like oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA in situ, enabling nearby molecules to be enriched by streptavidin pulldown. This induces exceptionally precise biotinylation that can be easily optimized and ported to new targets or sample types. Using the noncoding RNAs 47S, 7SK and Xist as models, we develop O-MAP workflows for discovering RNA-proximal proteins, transcripts and genomic loci, yielding a multiomic characterization of these RNAs' subcellular compartments and new regulatory interactions. O-MAP requires no genetic manipulation, uses exclusively off-the-shelf parts and requires orders of magnitude fewer cells than established methods, making it accessible to most laboratories.