# DNA Nanostructures for High-Throughput Cryo-EM Studies of Small Macromolecules

> **NIH NIH R35** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2024 · $403,750

## Abstract

DNA Nanostructures for High-Throughput Cryo-EM Studies of Small Macromolecules
Single particle cryo-electron microscopy (cryo-EM) is an approach for visualizing structures of
macromolecules and their complexes at near-native conditions without the need for large sample
quantities or the removal of flexible regions often required for alternative techniques such as X-ray
crystallography. Improvements in microscopy hardware and software have helped to achieve near-
atomic structure determination of macromolecules by cryo-EM allowing, for example, the visualization
of individual amino-acid side chains of protein targets. However, structure determination by cryo-EM
remains challenging for many macromolecules. Small (<100 kDa) particles can be especially difficult
to study because they lack well-defined structural features required for the image alignment step of
3D reconstruction.
To address these challenges, we constructed tiny goniometers—instruments that precisely orient
objects—using DNA nanotechnology. Our molecular goniometers can dock a single DNA-binding
protein onto a linear double-helix stage. With the power of DNA origami design, we can make several
goniometer variants, each with user-defined tilt and rotation angles of the protein-DNA complex. The
goniometers include asymmetric barcode domains. Thus, we can bind and precisely orient small
DNA-binding proteins, easily identify them on the grid, correctly assign the angle priors, and rapidly
solve their structure. We have validated our approach by obtaining a 6.5-Å structure of an 82-kDa
DNA-binding protein whose helical pseudosymmetry prevents accurate image orientation using
traditional cryo-EM.
We propose to apply our DNA origami molecular goniometers to characterize several biomedically
important DNA-binding proteins of unknown structure that remain recalcitrant to cryo-EM analysis.
We will study nuclear receptors linked to pancreatic and breast cancer, inflammatory bowel disease,
diabetes, and nonalcoholic fatty liver disease. We will also study small transcription factors that
regulate pleiotropic drug resistance in yeast. That structural understanding may aid in developing
future antifungal therapies. We will build custom molecular goniometers for each protein target with
the appropriate DNA-binding site and any necessary structural modifications.
We will continue to optimize our first-generation designs to address outstanding challenges related to
sample preparation and extend the devices for general use with non-DNA binding proteins. These
engineering efforts will be aided by computational modeling and simulation tools that our lab has
developed and will adapt for each target system.

## Key facts

- **NIH application ID:** 10795961
- **Project number:** 5R35GM125027-07
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Shawn M Douglas
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $403,750
- **Award type:** 5
- **Project period:** 2018-03-01 → 2028-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10795961

## Citation

> US National Institutes of Health, RePORTER application 10795961, DNA Nanostructures for High-Throughput Cryo-EM Studies of Small Macromolecules (5R35GM125027-07). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10795961. Licensed CC0.

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