# Using nanobodies to increase the sensitivity and resolution of chromatin profiling through uliCUT&RUN

> **NIH NIH R21** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2021 · $220,075

## Abstract

PROJECT SUMMARY/ABSTRACT
 DNA-binding proteins play crucial roles in all DNA templated processes, such as transcription, splicing,
replication, and DNA repair. DNA binding proteins include transcription factors that bind preferentially to certain
DNA sequences, and histone proteins that form the core of nucleosomes. Importantly, genomic location of
factors or histone proteins cannot be predicted in cell types by DNA sequence alone. Therefore, protein profiling
technologies are used to identify cell specific characteristics of functional binding.
 The importance of DNA-binding proteins has motivated the continued development of experimental and
analytical methods to better identify and characterize these interactions. Genome-wide profiling by ChIP-seq is
a widely-used technique that has assisted in the characterization of countless chromatin binding proteins.
However, this technique is limited in its ability to characterize factor occupancy in samples with small cell
numbers and by the availability of specific and robust antibodies. These limitations have necessitated the
development of complementary methods and extensions of ChIP-seq to provide a more complete of biological
processes in the cell. Very recently, we optimized CUT&RUN, a new localization method, to profile factor
occupancy in extremely low cell populations, down to single cells and individual mouse blastocyst embryos
(termed uliCUT&RUN). This technical advancement has opened the opportunity to profile factor occupancy in
rare cell populations, such as patient biopsies. Furthermore, it permits for testing cell heterogeneity that occurs
in cell populations. However, practical limitations of this technology still include antibody development and
efficiency.
 Camelid single-chain VHH antibodies or Nanobodies (Nbs) are a compelling new class of antibodies
characterized by exceptionally high solubility and thermostability. We have recently developed a robust pipeline
for the discovery and characterization of high-quality antigen-specific Nb repertoires. This pipeline has been
extensively tested and optimized for a dozen of antigens with different structures and immune responses. With
this approach, a large cohort of high-quality conformational Nb binders can be identified.
 Here we propose to couple our expertise on Nb development and uliCUT&RUN to develop nanobody
specific CUT&RUN for low cell populations and apply this technology to single cells and rare cell populations.
The development and application of Nb-based uliCUT&RUN will be of wide use to the community and we are
well poised to develop this technology given our optimization of CUT&RUN is the first time single cell transcription
factor profiling has been accomplished and our expertise in the new field of Nb development. Further, results
from applying this technology to samples will continue to further our understanding of normal cell biology, but
also provide crucial information that will benefit efforts to determine the causes...

## Key facts

- **NIH application ID:** 10272481
- **Project number:** 1R21CA261737-01
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Sarah Jane Hainer
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $220,075
- **Award type:** 1
- **Project period:** 2021-09-06 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10272481

## Citation

> US National Institutes of Health, RePORTER application 10272481, Using nanobodies to increase the sensitivity and resolution of chromatin profiling through uliCUT&RUN (1R21CA261737-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10272481. Licensed CC0.

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