A groundbreaking advancement in cellular biology has emerged from a team of researchers who have developed an innovative method to profile mitochondrial RNA within living cells with unprecedented resolution and specificity. This new approach circumvents many of the limitations faced by traditional techniques, such as genetic manipulation dependency, contamination, and inadequate spatial resolution. The study introduces a cutting-edge bioorthogonal photocatalytic labelling and sequencing technology, termed CAT-seq, that enables researchers to dissect the mitochondrial transcriptome’s spatiotemporal dynamics in situ, ushering in a new era of RNA molecular mapping within subcellular compartments.
The mitochondrion, often referred to as the powerhouse of the cell, holds a distinct genome and transcriptional profile crucial for cellular function, energy metabolism, and signaling. Understanding how mitochondrial RNAs differ, move, and dynamically interact within the mitochondrial environment holds immense importance for elucidating fundamental biological mechanisms and disease pathogenesis, including metabolic disorders, neurodegenerative diseases, and cancer. However, existing mitochondrial RNA profiling tools frequently encounter cellular complexity, resulting in signal contamination from cytoplasmic or nuclear RNAs, and require the introduction of exogenous genetic constructs, which complicates studies especially in primary cells or delicate biological samples.
The newly developed CAT-seq method deftly eliminates these barriers by leveraging a photocatalytic quinone methide (QM) probe designed explicitly for selective RNA labeling within mitochondria of living cells. Quinone methides, known for their reactive electrophilic character, have long been recognized for their capacity to form covalent bonds with nucleophiles, making them ideal for targeted biomolecular tagging. The research team’s novel application of QM chemistry, integrated with a bioorthogonal framework, ensures high efficiency and specificity in reacting with mitochondrial RNA while preserving the native physiological milieu of the cells.
Integral to the success of CAT-seq is the meticulous optimization and validation process performed by the researchers, who fine-tuned probe concentration, illumination parameters, and reaction conditions to maximize labeling efficiency and minimize off-target modification. The approach employs a mild photoactivation step that triggers the quinone methide warhead, enabling spatiotemporally controllable covalent attachment to RNA molecules within the mitochondrial matrix. This light-driven bioorthogonal chemistry confines labeling exclusively to molecules present at the precise location and time of illumination, enhancing spatial resolution and reducing background noise typical of diffusion-based labeling techniques.
Demonstrating the robustness of CAT-seq, the authors successfully applied the method to HeLa cells, a widely used human cell line. The experiments highlighted CAT-seq’s ability to map the mitochondrial transcriptome with subcellular precision, revealing nuanced patterns of RNA distribution and turnover. The technique also facilitated the real-time tracking of RNA dynamics, capturing changes in mitochondrial RNA profiles in response to cellular stimuli and environmental perturbations. These findings underscore the method’s potential to decipher mitochondrial RNA life cycles and their adaptive mechanisms under various physiological and pathological conditions.
Beyond conventional cancer cell models, CAT-seq was deployed to investigate RAW 264.7 macrophages, representing a more challenging and physiologically relevant immune cell type. Macrophages play pivotal roles in immune defense and inflammation, with mitochondrial function intricately linked to their activation states and metabolic rewiring. Using CAT-seq, the research unveiled an underlying mitochondrial translational remodeling pathway previously obscured in bulk transcriptomic studies. This discovery opens avenues to explore how mitochondrial transcriptomics influence immune responses and may aid in identifying novel therapeutic targets for inflammatory and infectious diseases.
A particularly remarkable aspect of this novel approach is the establishment of an orthogonal labeling system based on the distinctive chemistry of quinone methide warheads. By designing complementary chemistries that do not interfere with one another, the team achieved simultaneous labeling of both mitochondrial RNA and proteins within the same living cell sample. This synchronous multi-omics profiling provides a holistic view of mitochondrial molecular landscapes, linking transcriptomic information with proteomic insights to unravel coordinated regulatory networks. The ability to perform multi-omics investigations in situ and in live cells overcomes limitations of previous methods relying on cell disruption, fractionation, or genetic engineering.
This integrated multi-omics strategy significantly propels the options available for investigating complex biological phenomena where mitochondrial function is critical. For example, the interplay between mitochondrial gene expression and protein synthesis, critical for maintaining mitochondrial biogenesis and oxidative phosphorylation efficiency, can now be studied with remarkable spatiotemporal clarity. CAT-seq’s compatibility with intact primary living samples furthers its translational appeal, as conventional techniques often fail to capture the native mitochondrial transcriptomic state in these sensitive and heterogeneous biological matrices.
Furthermore, this study highlights the frontier interface of chemistry and cell biology, showcasing how innovative chemical biology tools can empower the life sciences community to answer long-standing questions about subcellular molecular organization. The use of photoactivatable quinone methide probes represents a paradigm shift, enabling precision manipulation and monitoring of RNA molecules localized within specific organelles under physiological conditions. This approach establishes a blueprint for future technologies aimed at resolving the complexity and dynamics of intracellular RNA populations with unparalleled resolution.
The implications of CAT-seq extend beyond mitochondrial studies as the fundamental principles of bioorthogonal photocatalytic labeling could be adapted to target other subcellular RNA populations and potentially other types of biomolecules in diverse living systems. This enhanced ability to dissect local transcriptomics will deepen insights into organelle-specific RNA processing, localization, and turnover, which are critical parameters in understanding cellular homeostasis, signaling, and disease progression.
On a technical note, the study details rigorous experimental controls validating the specificity of RNA labeling over DNA or protein counterparts and confirms minimal phototoxicity or perturbation of cellular viability. The authors also demonstrate the scalability of their technique, suggesting its compatibility with high-throughput sequencing workflows and its potential integration within existing omics pipelines. This scalability promises to accelerate widespread adoption and reproducibility across diverse research laboratories interested in subcellular omics.
The development of CAT-seq embodies the growing trend towards non-genetic and minimally invasive investigation techniques in cell biology, providing powerful alternatives to transgenic or viral labelling strategies, which carry inherent risks and technical barriers. Notably, the absence of genetic modification enhances the feasibility of applying CAT-seq directly to primary cells, stem cells, or clinical samples, thus bridging a significant gap between basic research and biomedical applications.
Moreover, the ability to capture real-time RNA profiles in live cells holds remarkable promise for studying temporal gene expression changes during dynamic biological processes such as differentiation, stress response, or disease progression. CAT-seq’s temporal resolution, governed by controllable photoactivation, allows for snapshots of RNA molecules at defined time points, enabling kinetic studies that were previously difficult to achieve with conventional RNA sequencing methods.
The versatility and precision of CAT-seq may also catalyze innovations in drug discovery and therapeutic monitoring, where mitochondrial dysfunction is implicated. By providing a sensitive readout of mitochondrial RNA alterations in response to pharmacological agents or environmental toxins, this method could facilitate the identification of mitochondrial biomarkers and enhance the screening of mitochondrial-targeted drugs.
This landmark study, therefore, not only provides a transformative tool for mitochondrial RNA research but also exemplifies how interdisciplinary approaches leveraging chemical biology, molecular biology, and advanced sequencing technologies can unveil hidden layers of cellular regulation. As the research community increasingly recognizes the importance of spatially resolved omics, CAT-seq stands out as a pioneering technique with vast potential to reshape our understanding of cellular architecture and function.
In summary, CAT-seq represents a monumental step forward in the capacity to profile mitochondrial RNA within living cells with high resolution, precision, and minimal invasiveness. By harnessing the power of bioorthogonal photocatalytic chemistry and innovative quinone methide probes, the method offers detailed insights into RNA localization, dynamics, and interplay with mitochondrial protein synthesis. This revolutionary technology promises to deepen our understanding of mitochondrial biology in health and disease and to foster novel discoveries across the biomedical sciences.
Subject of Research: Mitochondrial RNA profiling and synchronous multi-omics investigation using bioorthogonal photocatalytic labelling.
Article Title: Photocatalytic labelling-enabled subcellular-resolved RNA profiling and synchronous multi-omics investigation.
Article References:
Bi, Y., Yu, L., Deng, Q. et al. Photocatalytic labelling-enabled subcellular-resolved RNA profiling and synchronous multi-omics investigation. Nat. Chem. (2025). https://doi.org/10.1038/s41557-025-01946-1
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