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Transcriptome Analysis with RNA-Reactive Probes

Award Number: R35GM145357
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 05/01/2022
PERIOD OF PERFORMANCE END DATE: 04/30/2027

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Transcriptome Analysis with RNA-Reactive Probes - Work in the last decade from many labs has underlined the critical importance of RNA-mediated cellular pathways, and clear connections of specific RNAs to human health. RNAs are increasingly viewed both as appealing therapeutic targets, and as therapeutic agents themselves. We hypothesize that obtaining a deeper and broader understanding about how ligands interact with the many RNA species of the cell will provide important new insights into RNA networks and functions, provide new understanding of how current drugs cause cellular toxicity, and lend novel insights into improving RNA therapies. We are convinced that the analysis of RNA interactions transcriptome-wide is essential to future biomedicine. Unfortunately, methods for assessing RNA interactions directly in the cell lag well behind those for protein and proteome analysis. Recent work from this laboratory has established numerous new molecular tools for analysis of biologically and clinically relevant RNAs. We developed the first high-yield reaction strategy for functionalizing RNA 2'-OH groups, establishing broad utility of acylimidazole reagents. We designed the cell-permeable and broadly used structure-mapping reagents NAI and NAI-N3 - now commercially available - and applying them with RNA Seq, we mapped folded structures of 16000 mRNAs in mammalian cells. We developed rapid and simple chemical approaches for functionalizing RNA with fluorescent labels, biotin, hydrophobic groups, crosslinkers, and caging groups. Further, we designed strategies for labeling RNA either broadly or at specific sites. Unlike recent enzymatic approaches for RNA labeling, our methods require no engineered structure or sequence, and thus can be employed rapidly and easily with native RNAs of any origin or length. The proposed project will consolidate our RNA work into a broad program that will develop a new set of RNA-reactive reagents and methods, and will apply them to provide specific, quantitative information about ligand interactions with the transcriptome. We will develop first-in-class methods for functionalizing native RNAs at specific sites, and novel strategies for controlling RNAs with red light. Combining our reactive acyl tools and methods with next-gen sequencing, we will pinpoint and quantify ligand binding sites in the whole transcriptome. These methodologies, together termed Reactivity-Based RNA Profiling (RBRP), will be applied to analyzing off-target RNA binding by known small-molecule drugs with clinically limiting toxicity, to profiling RNA interactions of endogenous secondary metabolites, and to the analysis of how modified bases in next-generation mRNA vaccines and therapeutics affect their structures and interactions in the cell. This work is significant because it seeks answers to system-wide clinically-relevant questions regarding RNA interactions. Further, it develops the 2'-OH group as a nearly universal handle for manipulation, conjugation, and study of RNAs, introducing enabling molecular technologies that will broadly benefit researchers in the fields of RNA biology and contribute to improving future RNA therapies.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $623,371 )
2026	2026	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	5	5/1/2026	NON-COMPETING CONTINUATION	$623,371
														Subtotal = $623,371

Issue Date FY: 2025 ( Subtotal = $623,371 )
2025	2025	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	001	4	4/7/2025	NON-COMPETING CONTINUATION	$623,371
2025	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	3	11/7/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $623,371

Issue Date FY: 2024 ( Subtotal = $635,555 )
2024	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	002	3	8/27/2024	SUPPLEMENT FOR EXPANSION	$12,184
2024	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	001	3	7/30/2024	NON-COMPETING CONTINUATION	$62,337
2024	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	3	4/4/2024	NON-COMPETING CONTINUATION	$561,034
														Subtotal = $635,555

Issue Date FY: 2023 ( Subtotal = $669,203 )
2023	2023	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	2	5/3/2023	NON-COMPETING CONTINUATION	$623,371
2023	2023	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	001	2	6/1/2023	SUPPLEMENT FOR EXPANSION	$45,832
														Subtotal = $669,203

Issue Date FY: 2022 ( Subtotal = $425,319 )
2022	2022	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	1	4/5/2022	NEW	$425,319
														Subtotal = $425,319

Grand Total All Awards = $2,976,819

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Transcriptome Analysis with RNA-Reactive Probes

Award Number: R35GM145357

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 05/01/2022

PERIOD OF PERFORMANCE END DATE: 04/30/2027

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