Abstract
RNA localization is highly regulated, with subcellular organization driving context-dependent cell physiology. Although proximity-based labelling technologies that use highly reactive radicals or carbenes provide a powerful method for unbiased mapping of protein organization within a cell, methods for unbiased RNA mapping are scarce and comparably less robust. Here we develop α-alkoxy thioenol and chloroenol esters that function as potent acylating agents upon controlled ester unmasking. We pair these probes with subcellular-localized expression of a bioorthogonal esterase to establish a platform for spatial analysis of RNA: bioorthogonal acylating agents for proximity labelling and sequencing (BAP-seq). We demonstrate that, by selectively unmasking the enol probe in a locale of interest, we can map RNA distribution in membrane-bound and membrane-less organelles. The controlled-release acylating agent chemistry and corresponding BAP-seq method expand the scope of proximity labelling technologies and provide a powerful approach to interrogate the cellular organization of RNAs.
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Data availability
All BAP-seq data are available in the Sequence Read Archive through accession number GSE229451. All the raw images used for Supplementary Figs. 11–30 and Figs. 3 and 4 are provided with Mendeley Data (https://doi.org/10.17632/kzpvsdjrgy.1)67. All other data generated or analysed in this study are available within the main text and Supplementary Information. Links to vector maps are included in Supplementary Fig. 33. Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Institute of General Medical Sciences (GM119840), The National Institute of Mental Health (MH122142) and the National Human Genome Research Institute (RM1 HG008935) of the National Institutes of Health, as well as The G. Harold and Leila Y. Mathers Charitable Foundation. S.-A.A. was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (F30 DK125088). We thank A. Radakovic from J.W. Szostak’s laboratory for supplying synthetic RNA oligos and C. He for helpful discussions. We thank E. Wu and the University of Chicago CRI Bioinformatics Core for assistance with data acquisition and analysis. We also thank the University of Chicago Integrated Light Microscopy Core (RRID: SCR_019197) and the Chicago Immunoengineering Innovation Center at the Pritzker School of Molecular Engineering for training and support with confocal microscopy and Next-seq sequencing facilities, respectively. We thank S. Ahmadiantehrani for assistance with preparing this paper.
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S.P., T.Q., K.K. and B.C.D. conceived the project. S.P. and T.Q. synthesized all compounds and performed experiments. S.-A.A. assisted with experiments and data analysis. S.P., T.Q. and B.C.D. wrote the paper with input from all authors.
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B.C.D. is a founder and holds equity in Tornado Bio, Inc. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Measurement of the kinetics of mCP-coumarin unmasked by 5 nM of BS2 esterase.
a, Schematic of synthesis of mCP-coumarin and the unmasking reaction by BS2. b, Linear range of mCP ester unmasking measured with different concentrations of mCP-coumarin incubation. Y-axis represents the normalized concentration of coumarin generated from mCP unmasking. Error bars represent the standard deviation of the mean, n = 3 independent in vitro reactions. c, Michaelis-Menten plot showing the rate of mCP unmasking as a function of the substrate (mCP-coumarin) concentration. Error bars represent the standard deviation of the mean n = 3 independent in vitro reactions, and the curve represents the best fit. d, Kinetic parameters obtained by Michaelis-Menten plot of BS2 unmasking mCP-coumarin. All plots and curve fit were made using GraphPad Prism. Error bars represent the standard deviation, n = 3 independent in vitro reactions.
Extended Data Fig. 2 Optimization of catalyst conditions for regioselective carboxylic acid addition to synthesize α-alkoxy thioenol esters.
Silver-based catalysts yielded more than 90% of the undesired 1,2-addition product (entry 1). The screening of various metal catalysts revealed the iridium catalyst could provide desired 1,1-addition product (entries 2–8), especially the [Ir(OMe)(COD)]2. Further screening of several ligands suggested the ligand has minimal effects on yield (entries 9–16).
Extended Data Fig. 3 Confocal microscopy images showing BS2 dependent proximity labelling by AC-2 probe in the nucleus.
BS2 with NLS and V5 tag was transiently expressed in HEK293T cells, and the expression was visualized with V5 antibody; labelling activity was visualized after click reaction with azide-Alexa488. DAPI is a nuclear marker. The scale bar on Brightfield images is 10 µm.
Extended Data Fig. 4 Confocal microscopy images showing BS2 dependent proximity labelling by AC-2 probe in the cytosol.
BS2 with nuclear export signal (NES) and V5 tag was transiently expressed in HEK293T cells, and the expression was visualized with V5 antibody; labelling activity was visualized after click reaction with azide-Alexa488. DAPI is a nuclear marker. The scale bar on Brightfield images is 10 µm.
Extended Data Fig. 5 Confocal microscopy images showing BS2 dependent proximity labelling by AC-2 probe in the ERM.
BS2 with outer ER membrane localization via ER transmembrane anchor facing the cytosol was transiently expressed in HEK293T cells, and the expression was visualized with V5 antibody; labelling activity was visualized after click reaction with azide-Alexa488. DAPI is a nuclear marker. The scale bar on Brightfield images is 10 µm.
Extended Data Fig. 6 Confocal microscopy images showing BS2 dependent proximity labelling by AC-2 probe in the mitochondria.
BS2 with MTS-matrix (Mitochondria Targeting Signal) and V5 tag was transiently expressed in HEK293T cells, and the expression was visualized with V5 antibody; labelling activity was visualized after click reaction with azide-Alexa488. DAPI is a nuclear marker. The scale bar on Brightfield images is 10 µm.
Extended Data Fig. 7 Confocal microscopy images showing BS2 dependent proximity labelling by AC-2 probe in the nucleolus.
BS2 with nucleolar localization via three tandem nucleolar targeting sequences from NF-κB-inducing kinase (NIK) and V5 tag was transiently expressed in HEK293T cells, and the expression was visualized with V5 antibody; labelling activity was visualized after click reaction with azide-Alexa488. DAPI is a nuclear marker. The scale bar on Brightfield images is 10 µm.
Extended Data Fig. 8 Confocal microscopy images showing BS2 dependent proximity labelling by AC-2 probe in the nuclear pore.
BS2 fused with SENP2 protein and V5 tag was transiently expressed in HEK293T cells, and the expression was visualized with V5 antibody; labelling activity was visualized after click reaction with azide-Alexa488. DAPI is a nuclear marker. The scale bar on Brightfield images is 10 µm.
Extended Data Fig. 9 Enrichment of BS2 proximal RNA post AC-2 labelling.
a, Bioanalyzer quantification of enriched RNAs post AC-2 labelling. 25 µg of input RNA was taken for each sample for the enrichment, and the experiment was performed with 2 biological replicates. b, Principal component analysis (PCA) analysis of gene expression values for different samples of BAP-seq, n = 2-3 biological replicates. c, Comparison of sequencing counts post differentially expressed gene (DEG) analysis for Mt-genes across different compartments, n = 2 biological replicates. d, Comparison of intronic reads across different samples Numbers are presented as a percentage of total reads that includes exons and intergenic regions. Error bars represent the standard deviation of mean, n = 2 biological replicates. P-value was determined by two-tailed unpaired t-tests with Welch’s correction. ** represents p-value < 0.01 (p = 0.0099) and *** represents p-value < 0.001 (p = 0.0006).
Supplementary information
Supplementary Information
Supplementary Figs. 1–34, synthesis methods and characterization, detailed BAP-seq protocol and information-only references.
Supplementary Data 1
BAP-seq CPM reads of all samples.
Source data
Source Data Fig. 2
LC trace used for Fig. 2b,f. Both Excel workbooks are provided in the same folder.
Source Data Fig. 3
The data points used for the bar graph in Fig. 3b. The raw images used for Fig. 3a,c are provided with Mendeley. The DOI is mentioned in the ‘Data availability’ section.
Source Data Fig. 4
The raw images used are provided with Mendeley.
Source Data Fig. 5
Unprocessed gels and blots used in Fig. 5a,b.
Source Data Fig. 5
Cy2-fluorescence image for Fig. 5a.
Source Data Fig. 5
Streptavidin chemiluminiscence blot for Fig. 5b.
Source Data Fig. 5
Methylene blue staining for Fig. 5b.
Source Data Fig. 6
DEG tables used for plots in Fig. 6b–d.
Source Data Extended Data Fig. 1
Data used for the plots in the figure.
Source Data Extended Data Fig. 9
Data used for plots in the figure.
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Pani, S., Qiu, T., Kentala, K. et al. Bioorthogonal masked acylating agents for proximity-dependent RNA labelling. Nat. Chem. 16, 717–726 (2024). https://doi.org/10.1038/s41557-024-01493-1
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DOI: https://doi.org/10.1038/s41557-024-01493-1