D-Amino-Acid-Powered Enzyme Cascade Maps Biomolecules Across Living Animals

For decades, biologists have dreamed of watching the molecular machinery of life not in a dish, but inside a living animal — seeing which proteins cluster at a synapse, which RNAs fold into which shapes, and how these arrangements shift as tissues grow, age, or turn cancerous. A new technique reported in Nature Chemical Biology brings that vision substantially closer. The method, called PRADA — peroxidase reactions activated by D-amino acids — lets researchers tag and map proteins and RNA molecules in their immediate molecular neighborhoods inside fruit flies, roundworms, zebrafish and mice, all with remarkably low toxicity and background noise.

The core challenge that PRADA addresses is a long-standing bottleneck in proximity labeling. The most widely used tools, such as APEX2 and TurboID, genetically fuse an engineered enzyme to a protein of interest. The enzyme then converts a supplied small molecule into a reactive species that tags nearby biomolecules, which can later be fished out and identified by mass spectrometry or sequencing. The catch is the trigger. APEX2 requires a bolus of exogenous hydrogen peroxide, a reactive oxidant that damages cells and diffuses poorly through intact tissues. TurboID requires biotin, which works in vivo but generates its own background labeling. Neither approach has proven ideal for the delicate, three-dimensional environment of a living organism.

The Shanghai- and Singapore-based team, led by Shuo Han of the Chinese Academy of Sciences together with Yue Wan of the Genome Institute of Singapore, took a different route: instead of supplying hydrogen peroxide directly, they engineered the system to make it on the spot. PRADA pairs a genetically fused peroxidase with an engineered D-amino acid oxidase, or DAAO, derived from the yeast Rhodotorula gracilis. When researchers deliver a nonproteinogenic D-amino acid such as D-phenylalanine or D-alanine — molecules that animal cells largely ignore — the oxidase converts them into hydrogen peroxide right next to the peroxidase. That locally generated oxidant then activates the peroxidase, which converts phenol probes such as biotin-phenol into short-lived radicals that covalently tag whatever proteins or nucleic acids sit within roughly a few tens of nanometers.

The engineering itself was nontrivial. The team used structural modeling and rational mutagenesis to delete a flexible C-terminal loop of the R. gracilis oxidase, producing a monomeric variant that fuses cleanly with APEX2 without disrupting either enzyme’s activity. Extended data show the researchers systematically tested fusion orientations, split-enzyme designs and D-amino acid concentrations, using fluorescence assays to confirm that the cascade produces hydrogen peroxide efficiently and that the labeling signal colocalizes precisely with the tagged protein’s known subcellular address. When the two enzymes were expressed separately rather than fused, labeling dropped sharply, confirming that the reaction is genuinely confined to the immediate vicinity of the fusion protein.

That spatial confinement is what makes the method safe enough for living animals. Because the hydrogen peroxide is generated in situ at nanometer scales and consumed almost immediately by the peroxidase, it never accumulates to toxic levels. The researchers measured malondialdehyde levels, a marker of lipid peroxidation, and protein carbonylation after PRADA labeling and found both below detection limits or unchanged compared with controls. Mitochondrial superoxide, a sensitive indicator of oxidative stress, was actually lower in PRADA-labeled cells than in cells labeled conventionally with APEX2 and exogenous peroxide. Cell viability assays showed no measurable harm from the D-amino acid treatment itself. In effect, PRADA converts the most dangerous step of proximity labeling into a self-limiting, locally contained reaction.

The versatility of the platform is striking. In cultured cells, the team targeted PRADA to the nucleus, nucleolus, endoplasmic reticulum, plasma membrane and mitochondria, and in each case recovered proteomic profiles consistent with the known composition of those compartments. They also showed that PRADA can drive functional polymer assembly — the genetically targeted chemical assembly of conducting or insulating materials inside living cells — extending the technique beyond mapping into materials synthesis. And, crucially, they demonstrated that the peroxidase step can label RNA as well as protein, opening a door that most proximity-labeling systems leave closed.

That RNA reactivity became the foundation of the study’s most inventive application. When peroxidase radicals oxidize RNA, they leave behind chemical lesions that cause reverse transcriptase to misread bases during cDNA synthesis. The team realized these misincorporations could be read as a molecular fingerprint of RNA structure: nucleotides that are chemically accessible — that is, single-stranded and exposed — accumulate more mutations than nucleotides buried inside base-paired helices. Building on the logic of mutational profiling sequencing, they developed PRADA-MaPseq, a strategy that converts peroxidase labeling directly into a spatiotemporally resolved map of RNA secondary structure inside living cells.

They benchmarked PRADA-MaPseq against the well-established mitochondrial DMS-MaPseq datasets, showing that uracil and guanine reactivity scores accurately reproduce known structures of mitochondrial messenger RNAs, with area-under-the-curve values reaching 0.60 to 0.82 across transcripts and inter-experimental correlation coefficients as high as 0.96 for individual transcripts. The approach worked with both 4-thiouridine and 6-selenoguanosine metabolic labeling, and the team optimized reverse transcriptase choice to maximize mutational signal. In short, they turned a proximity-labeling enzyme into a structure-probing reagent that reports on RNA folding in a defined cellular compartment at a defined moment in time.

The payoff came in a mouse xenograft model. The researchers implanted HEK293T cells expressing mitochondria-targeted PRADA into immunodeficient mice, then administered D-amino acids and phenol probes systemically. Labeling proceeded efficiently inside the tumors in living animals. Proteomic analysis recovered the expected mitochondrial proteome, transcriptomic enrichment captured mitochondrial RNAs with high specificity, and PRADA-MaPseq yielded the first in vivo structurome of mitochondrial RNA within a living mammalian tumor. Comparing these in vivo structure maps with in vitro measurements of the same transcripts revealed systematic differences — evidence that RNA folding inside a living organism is actively shaped by its environment rather than being a fixed property of the sequence.

Those differences carried biological meaning. The team focused on LRPPRC, a mitochondrial RNA-binding protein known to organize the folding of the mitochondrial transcriptome. When they knocked down LRPPRC in the xenografts, changes in RNA structure propagated to changes in mitochondrial gene expression, demonstrating that RNA architecture functions as a regulatory layer controlling how mitochondrial genes are read. This finding elevates the structurome from a descriptive atlas to a mechanistic insight: the three-dimensional shapes of RNAs inside mitochondria help determine how much protein the organelle makes, and those shapes are tunable in vivo.

Beyond mitochondria, the breadth of validated applications suggests PRADA could become a workhorse across biology. The team demonstrated labeling in the cytoplasm and nuclei of body wall muscle cells in Caenorhabditis elegans, recovering nuclear-enriched transcripts and detecting retained introns as expected; in rat neurons and Drosophila; and in zebrafish. Because the trigger molecule — a simple D-amino acid — is cell-permeable, inexpensive and largely inert, the method should scale to tissues and organisms where peroxide bolus delivery is impractical, such as dense neural tissue, developing embryos or intact tumors. The authors have filed patent applications, and the analysis code for RNA structuromics is freely available on GitHub under an MIT license, with sequencing and proteomic data deposited in public repositories.

Limitations remain, as with any new technology. The labeling window is measured in tens of minutes rather than seconds, so fast molecular events may still escape capture. The peroxidase chemistry, while gentler than exogenous peroxide, still relies on radical intermediates whose diffusion radius sets the spatial resolution. And the D-amino acid oxidase must be carefully engineered for each fusion context to avoid perturbing the protein under study. Nevertheless, PRADA represents a genuine conceptual advance: it decouples proximity labeling from externally supplied oxidants, unifies protein, RNA and polymer chemistries under one enzymatic umbrella, and — for the first time — brings RNA structure mapping into living animals. If the platform generalizes as its developers hope, the spatial organization of biomolecules in health and disease may soon be readable not just in culture dishes, but in the bodies of living organisms, one D-amino acid at a time.

Subject of Research: An engineered D-amino-acid-activated peroxidase cascade for in vivo multiomic proximity labeling of proteins and RNA structures

Article Title: Multiomic proximity labeling in vivo by D-amino-acid-activated peroxidase reaction

Article References: Liu, J., Han, J., Wang, Y., Song, M., Zhu, J., Liang, Y., Chen, F., Liu, Z., Yan, X., Wang, Z., Peng, W., Tu, R., Zhang, Z., Zhong, B., Sun, H., Yin, J., Chang, J., Long, Y., Men, Y., … Han, S. (2026). Multiomic proximity labeling in vivo by D-amino-acid-activated peroxidase reaction. Nature Chemical Biology. https://doi.org/10.1038/s41589-026-02336-5

Image Credits: AI Generated

DOI: 10.1038/s41589-026-02336-5

Keywords: proximity labeling, PRADA, D-amino acid oxidase, APEX2, RNA structure, structurome, mitochondria, spatial proteomics, chemical biology, in vivo imaging, xenograft, LRPPRC