# Characterization of ncRNAs' post-transcriptional modifications by antisense affinity capture and MS analysis

> **NIH NIH R01** · UNIVERSITY OF CONNECTICUT STORRS · 2021 · $395,865

## Abstract

Project Summary
This proposal aims at the development of a platform for the comprehensive classification and
characterization of post-transcriptional modifications (PTMs) in non-protein coding RNAs (ncRNAs). The
availability of a convenient approach for the detection of N6-methyladenosine (m6A), which relies on
specific antibodies and RNA-seq, has enabled groundbreaking studies that revealed the significance of
this PTM in essential regulatory processes. In particular, seminal reports on viral replication and cocaine
addiction have clearly shown that m6A pathways are very promising targets for the development of new
therapeutic strategies. The implementation of a more versatile approach based on mass spectrometry
allowed us to show that the genome of many RNA viruses is decorated by different types of RNA
modifications in addition to m6A. We also found that long non-coding RNAs involved in stress response
and cancer contain constellations of ribonucleotide variants. Based on the m6A precedents, we anticipate
that elucidating their roles in the activities of the respective parent RNAs will open countless new
avenues for therapeutic intervention.
 This project will develop tools for determining the incidence and distribution of PTMs, which is
essential for their functional elucidation. Libraries of antisense DNA-probes will be employed to capture
desired classes of RNAs identified by genetic screens, which will be immediately analyzed for PTM
content. Following a divide-and-conquer strategy, the libraries will target progressively narrower pools to
enable the classification of PTM-bearing RNAs. The multi-fold sample enrichment afforded by the
capture process will allow the isolation and concentration of individual RNAs to be submitted to mass
mapping and sequencing. The sample preparation steps will be carried out on microtiter well plates to
multiplex the entire process, reduce sample losses, support unattended operations, minimize the
duration of each analysis, and eliminate any delay between consecutive analyses.
 The platform development will initially employ standards consisting of synthetic and recombinant
RNAs, which will be promptly replaced with actual biological samples from different cellular systems. The
latter will primarily consist of long non-coding RNAs obtained from human monocytes and yeast cultures
under different stress conditions. The results will provide new precious insights into the role of PTMs in
the stress response mediated in humans and yeast by the homologous p38-MAPK and HOG pathways,
respectively. The enabling technologies developed here will immediately benefit several ongoing
collaborations in the fields of genetics, epigenetics, human virology, and cancer biology. The ability to
take these projects in new unimaginable directions substantiates the excellent innovative impact of these
technologies. These projects are representative of much broader communities with an enormous stake in
understanding the effects of PTM...

## Key facts

- **NIH application ID:** 10246533
- **Project number:** 5R01GM121844-05
- **Recipient organization:** UNIVERSITY OF CONNECTICUT STORRS
- **Principal Investigator:** Daniele Fabris
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $395,865
- **Award type:** 5
- **Project period:** 2017-09-01 → 2025-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10246533

## Citation

> US National Institutes of Health, RePORTER application 10246533, Characterization of ncRNAs' post-transcriptional modifications by antisense affinity capture and MS analysis (5R01GM121844-05). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10246533. Licensed CC0.

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