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

Award Number: R35GM125027
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 03/01/2018
PERIOD OF PERFORMANCE END DATE: 02/29/2028

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


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $403,750 )
2025	2025	REGENTS OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, THE	1855 FOLSOM ST STE 425	SAN FRANCISCO	CA	94103	SAN FRANCISCO	USA	Biomedical Research and Research Training	001	8	4/8/2025	NON-COMPETING CONTINUATION	$403,750
2025	2024	REGENTS OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, THE	1855 FOLSOM ST STE 425	SAN FRANCISCO	CA	94103	SAN FRANCISCO	USA	Biomedical Research and Research Training	000	7	11/11/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $403,750

Issue Date FY: 2024 ( Subtotal = $403,750 )
2024	2024	REGENTS OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, THE	1855 FOLSOM ST STE 425	SAN FRANCISCO	CA	94103	SAN FRANCISCO	USA	Biomedical Research and Research Training	000	7	2/21/2024	NON-COMPETING CONTINUATION	$363,375
2024	2024	REGENTS OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, THE	1855 FOLSOM ST STE 425	SAN FRANCISCO	CA	94103	SAN FRANCISCO	USA	Biomedical Research and Research Training	001	7	6/19/2024	NON-COMPETING CONTINUATION	$40,375
2024	2023	REGENTS OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, THE	1855 FOLSOM ST STE 425	SAN FRANCISCO	CA	94103	SAN FRANCISCO	USA	Biomedical Research and Research Training	003	6	8/20/2024	COMPETING CONTINUATION	$0
2024	2022	REGENTS OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, THE	1855 FOLSOM ST STE 425	SAN FRANCISCO	CA	94103	SAN FRANCISCO	USA	Biomedical Research and Research Training	002	5	8/20/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $403,750

Issue Date FY: 2023 ( Subtotal = $419,826 )
2023	2023	REGENTS OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, THE	1855 FOLSOM ST STE 425	SAN FRANCISCO	CA	94143	SAN FRANCISCO	USA	Biomedical Research and Research Training	000	6	2/27/2023	COMPETING CONTINUATION	$419,826
														Subtotal = $419,826

Issue Date FY: 2022 ( Subtotal = $403,750 )
2022	2022	REGENTS OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, THE	1855 FOLSOM ST STE 425	SAN FRANCISCO	CA	94143	SAN FRANCISCO	USA	Biomedical Research and Research Training	000	5	2/17/2022	NON-COMPETING CONTINUATION	$363,373
2022	2022	REGENTS OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, THE	1855 FOLSOM ST STE 425	SAN FRANCISCO	CA	94143	SAN FRANCISCO	USA	Biomedical Research and Research Training	001	5	6/30/2022	NON-COMPETING CONTINUATION	$40,377
														Subtotal = $403,750

Issue Date FY: 2021 ( Subtotal = $403,750 )
2021	2021	REGENTS OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, THE	1855 FOLSOM ST STE 425	SAN FRANCISCO	CA	94143	SAN FRANCISCO	USA	Biomedical Research and Research Training	000	4	2/21/2021	NON-COMPETING CONTINUATION	$403,750
														Subtotal = $403,750

Issue Date FY: 2020 ( Subtotal = $402,916 )
2020	2020	REGENTS OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, THE	1855 FOLSOM ST STE 425	SAN FRANCISCO	CA	94143	SAN FRANCISCO	USA	Biomedical Research and Research Training	000	3	2/27/2020	NON-COMPETING CONTINUATION	$402,916
														Subtotal = $402,916

Issue Date FY: 2019 ( Subtotal = $400,834 )
2019	2019	REGENTS OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, THE	1855 FOLSOM ST STE 425	SAN FRANCISCO	CA	94143	SAN FRANCISCO	USA	Biomedical Research and Research Training	000	2	2/26/2019	NON-COMPETING CONTINUATION	$400,834
														Subtotal = $400,834

Issue Date FY: 2018 ( Subtotal = $399,583 )
2018	2018	REGENTS OF THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, THE	1855 FOLSOM ST STE 420	SAN FRANCISCO	CA	94103	SAN FRANCISCO	USA	Biomedical Research and Research Training	000	1	2/16/2018	NEW	$399,583
														Subtotal = $399,583

Grand Total All Awards = $3,238,159

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

Award Number: R35GM125027

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 03/01/2018

PERIOD OF PERFORMANCE END DATE: 02/29/2028

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