Wednesday, July 2, 2025 7/2/2025

Synthetic mRNA Control Set for Nanopore-Based Pseudouridine Modification Profiling in Human Transcriptomes

Award Number: R01HG012856
ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 04/01/2023
PERIOD OF PERFORMANCE END DATE: 01/31/2027

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Synthetic mRNA Control Set for Nanopore-Based Pseudouridine Modification Profiling in Human Transcriptomes - PROJECT SUMMARY/ABSTRACT Mammalian cells expend large amounts of energy into generating enzyme-mediated RNA chemical modifications that can change the base-pairing, RNA structure, or recruitment of RNA-binding proteins, among other elusive roles. Pseudouridine (ψ)-modified mRNAs are more thermodynamically stable, more resistant to RNAse-mediated degradation, and have the potential to modulate immunogenicity and enhance translation in vivo. However, ψ detection is extremely challenging: ψ modifications do not affect Watson-Crick base pairing and are indistinguishable from uridine when using hybridization-based methods. Further, since ψ is an isomer of uridine, detection using mass spectrometry requires non-quantitative chemical derivatization methods. While recent studies have shown that RNA modifications can be detected through direct RNA nanopore sequencing by monitoring basecalling errors, we have recently shown that the accuracy and fidelity of this approach is relatively low and sequence dependent. Our team has recently used a ligation approach to produce synthetic mRNA controls that contain single ψ sites within relevant transcripts mammalian cells. Using these synthetic controls we performed nanopore-based RNA sequencing and developed computational tools that increase the accuracy of ψ-calling to 90+%, depending on the specific sequence. We are basing our work on our recent finding that achieving ψ quantification requires sequence-specific training using unique signal parameters. The initial success of our team has laid the foundation to 1) generate an expanded set of barcoded synthetic RNA constructs that contain single ψ sites, 2) obtain a rigorous set of quadruplicate nanopore runs with ~50,000 single-molecule reads per construct, 3) develop computational tools to allow highly accurate sequence-specific ψ-calling. We will develop a gold-standard set of synthetic mRNA transcripts as a training molecular set for quantitative ψ profiling in direct RNA nanopore sequencing of human transcriptomes. The molecular set will allow quantitative profiling of hundreds of putative ψ sites across mammalian samples. This proposal will serve an unmet need by addressing a critical bottleneck: the lack of available modified RNA modification gold standards, i.e., RNA molecules that contain a site-specific and structure-specific modification. In this collaborative project we will develop a complete pipeline for synthesis of gold standard molecules; use these molecules to measure the nanopore signals that ψ modifications produce; develop a machine-learning tool to accurately quantify these modifications; profile site-specific ψ modifications in various cell lines to obtain ψ-maps that can be used to assess relationships of ψ modifications with phenotypes.


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: 2025 ( Subtotal = $845,562 )
2025	2025	NORTHEASTERN UNIVERSITY	360 HUNTINGTON AVE	BOSTON	MA	02115	SUFFOLK	USA	Human Genome Research	001	3	4/24/2025	NON-COMPETING CONTINUATION	$84,555
2025	2025	NORTHEASTERN UNIVERSITY	360 HUNTINGTON AVE	BOSTON	MA	02115	SUFFOLK	USA	Human Genome Research	000	3	2/20/2025	NON-COMPETING CONTINUATION	$761,007
														Subtotal = $845,562

Issue Date FY: 2024 ( Subtotal = $845,562 )
2024	2024	NORTHEASTERN UNIVERSITY	360 HUNTINGTON AVE	BOSTON	MA	02115	SUFFOLK	USA	Human Genome Research	001	2	6/17/2024	NON-COMPETING CONTINUATION	$67,644
2024	2024	NORTHEASTERN UNIVERSITY	360 HUNTINGTON AVE	BOSTON	MA	02115	SUFFOLK	USA	Human Genome Research	002	2	7/23/2024	NON-COMPETING CONTINUATION	$16,911
2024	2024	NORTHEASTERN UNIVERSITY	360 HUNTINGTON AVE	BOSTON	MA	02115	SUFFOLK	USA	Human Genome Research	000	2	1/24/2024	NON-COMPETING CONTINUATION	$761,007
														Subtotal = $845,562

Issue Date FY: 2023 ( Subtotal = $848,307 )
2023	2023	NORTHEASTERN UNIVERSITY	360 HUNTINGTON AVE	BOSTON	MA	02115	SUFFOLK	USA	Human Genome Research	000	1	3/31/2023	NEW	$848,307
														Subtotal = $848,307

Grand Total All Awards = $2,539,431

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Synthetic mRNA Control Set for Nanopore-Based Pseudouridine Modification Profiling in Human Transcriptomes

Award Number: R01HG012856

ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 04/01/2023

PERIOD OF PERFORMANCE END DATE: 01/31/2027

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