# Nanobodies targeting stress granule components

> **NIH NIH R21** · UNIVERSITY OF CONNECTICUT STORRS · 2023 · $460,800

## Abstract

Transcriptomic and proteomic studies on AD/ADRD consistently show alterations of pathways involved in
immunity, lipid metabolism, tau-binding protein network, and RNA metabolism. Recent advances in
understanding the proteins involved in RNA metabolism, including RNA-binding proteins (RBPs) involved in
stress granule (SG) formation provided new insights into the pathogenesis of AD. SGs formed of RNA and RBPs
such as TDP-43, hnRNPA2B1, and TIA1 are biomolecular condensates (BMCs) that can form a separate liquid
phase in cells. Mislocalization of RBPs to the cytoplasm increases the liquid-liquid phase separation (LLPS)
propensity, leading to increased SG formation. Under chronic stress, the SGs mature into a more solid or gel-
like assembly, sequestering the SG components. Over the past years, we and others have identified the
sequestration of RBPs as a critical mechanism of dysfunction in frontotemporal dementia-TDP-43, ALS, and AD.
We discovered that stress response is linked to oligomeric tau (o-tau) accumulation through the RBP
hnRNPA2B1, which preferentially interacts with tau when it is oligomerized. Since hnRNPA2B1 binds to the m6A
RNA methylation, this study also revealed the relevance of RNA modification in AD. As the interest in RBPs and
SGs grows, there is an increasing need to validate these assemblies in vivo. However, we lack the ability to
monitor SG dynamics without altering the intracellular concentration of SG components. We hypothesize that
endogenous SG components can be detected without perturbing their LLPS propensity through specific,
monovalent binders to SG components. Here we demonstrate for the first time that nanobodies (Nbs), single-
domain intracellular binding proteins, specific to RBPs can be identified through a high-throughput screen
approach. We aim to use the Nbs to detect SG components without altering their intracellular concentration and
demonstrate their use in a novel 3D human induced pluripotent stem cell (iPSC) model of AD that recapitulates
the tau-associated SG pathogenesis. We also demonstrate targeted degradation of hnRNPA2B1 using Nb fused
to an E3 ligase adaptor domain, and aim to validate the reversibility of SGs in primary neurons and the 3D human
iPSC model. Finally, we will screen Nbs specific to the m6A RNA methylation to enable imaging m6A RNA
methylation in SGs.

## Key facts

- **NIH application ID:** 10739370
- **Project number:** 1R21AG083761-01
- **Recipient organization:** UNIVERSITY OF CONNECTICUT STORRS
- **Principal Investigator:** Yongku Peter Cho
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $460,800
- **Award type:** 1
- **Project period:** 2023-09-01 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10739370

## Citation

> US National Institutes of Health, RePORTER application 10739370, Nanobodies targeting stress granule components (1R21AG083761-01). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10739370. Licensed CC0.

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