# Chaperone-Assisted RNA Crystallography

> **NIH NIH R01** · UNIVERSITY OF CHICAGO · 2021 · $380,411

## Abstract

PROJECT SUMMARY
Biological systems possess a highly complex and dynamic cellular RNA population, collectively known as the
transcriptome. Many RNAs fold into complex three-dimensional structures, both intrinsically and as
ribonucleoprotein (RNP) complexes, and play fundamental roles in nearly every aspect of gene expression.
Understanding cell biology, health, and disease requires knowledge of how RNA structure mediates biological
function. X-ray crystallography provides a powerful method for structure determination, but RNA
crystallization represents a major bottleneck in the process, reflecting in part the limited surface chemistry for
mediating lattice interactions and repulsion among the phosphates. Considering the rapid pace of new RNA
discovery, there remains an acute need to develop methods to facilitate RNA structure acquisition. For difficult
protein targets, antibody fragments (Fab or scFv) have served as effective chaperones for crystallization, and
we hypothesized that the large size, conformational properties and surface chemistry of Fabs will facilitate
RNA crystallization as well. Using phage-display library selections we demonstrated that Fabs can bind RNA
with high affinity and specificity, mediate the majority of lattice interactions in Fab-RNA co-crystals, and
provide a molecular replacement model for solving the structures. The long-term goal of this project is to
facilitate resolution of the RNA crystallization bottleneck through development of a high-throughput pipeline
for antibody production against RNA. The objective of this application is to enable facile access to RNA-
binding Fabs and pursue them as reagents for RNA and RNP crystallization and structure determination. To
attain this objective we will (a) improve Fab libraries using phage display and molecular evolution approaches
to identify amino acid types that tailor complementary determining regions (CDRs) for RNA binding, (b)
develop general use crystallization modules with surface and conformational properties adjusted to facilitate
crystallization, and (c) use these techniques to create and use Fab complexes of RNA and RNP targets for
crystallization and structure determination. Completion of the research will allow facile access to RNA binding
Fabs, provide structural biologists with a suite of portable modules for generalized use in RNA/RNP
crystallization, and provide important new structural knowledge for understanding biological function.

## Key facts

- **NIH application ID:** 10058842
- **Project number:** 5R01GM102489-08
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** Joseph Anthony Piccirilli
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $380,411
- **Award type:** 5
- **Project period:** 2013-04-01 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10058842

## Citation

> US National Institutes of Health, RePORTER application 10058842, Chaperone-Assisted RNA Crystallography (5R01GM102489-08). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10058842. Licensed CC0.

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