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

> **NIH NIH R35** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2022 · $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 removal of
flexible regions often required for alternative techniques such as X-ray crystallography. Recent improvements
in microscope hardware and data-processing 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, cryo-EM is quite limited for structure determination of small (<100
kDa) macromolecules. Cryo-EM image data are low contrast, and small particles often lack well-defined
structural features required for the image alignment step of 3D reconstruction. Additionally, the method is
technically challenging, low-throughput, and expensive—further hindering its widespread adoption.
We propose to use DNA nanotechnology to develop a novel suite of tools to overcome the size and throughput
limitations of cryo-EM. DNA nanotechnology allows us to create soluble nanostructures with an unprecedented
combination of spatial resolution and chemical versatility. In principle, we can attach any moiety to our devices
as long as it can be coupled to DNA, or to a DNA-binding molecule. We can build structures with dimensions
ranging from 10 nanometers to 1 micrometer in size, but still create structures in which the location of every
atom is defined with atomic or near-atomic resolution.
We will design and optimize megadalton-sized DNA “hinge” nanostructures that will bind and orient small
macromolecules and serve as high-contrast fiducial markers for cryo-EM imaging and tomography. We will
also construct DNA “barcode” nanostructures and attach them to the DNA hinges for sample multiplexing. We
will validate our methods by determining the structure of a well-characterized DNA-binding protein that has
been previously crystalized. We will then work with collaborators to study several macromolecules of unknown
structure.
This technology will hugely improve our ability to solve near-atomic resolution cryo-EM structures of small
macromolecules in a high-throughput manner. We will apply our method to study macromolecules with
relevance to several human diseases, and expect that our efforts will ultimately enhance structure-based drug
design efforts to combat those diseases.

## Key facts

- **NIH application ID:** 10357671
- **Project number:** 5R35GM125027-05
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Shawn M Douglas
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $403,750
- **Award type:** 5
- **Project period:** 2018-03-01 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10357671

## Citation

> US National Institutes of Health, RePORTER application 10357671, DNA Nanostructures for High-Throughput Cryo-EM Studies of Small Macromolecules (5R35GM125027-05). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10357671. Licensed CC0.

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