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Hybridization Chain Reaction: Highly Multiplexed Quantitative RNA and Protein Imaging

Award Number: R44GM140796
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: 04/01/2021
PERIOD OF PERFORMANCE END DATE: 11/30/2023

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Hybridization Chain Reaction: Highly Multiplexed Quantitative RNA and Protein Imaging - Project Summary Hybridization Chain Reaction: Highly Multiplexed Quantitative RNA and Protein Imaging Encoded in the genome of each organism, biological circuits direct development, maintain integrity in the face of attacks, control responses to environmental stimuli, and sometimes malfunction to cause disease. RNA in situ hybridization (RNA-ISH) and immunohistochemistry (IHC) methods provide biologists, drug developers, and pathologists with critical windows into the spatial organization of this circuitry, enabling imaging of mRNA and protein expression in an anatomical context. While it is desirable to perform multiplexed experiments in which a panel of targets is imaged quantitatively at high resolution in a single specimen, using traditional RNA-ISH and IHC methods in highly autofluorescent samples including whole-mount vertebrate embryos and FFPE tissue sections, multiplexing is cumbersome or impractical, spatial resolution is frequently compromised by diffusion of reporter molecules, and staining is non-quantitative. These multi-decade technological shortcomings are signifi- cant impediments to biological research as well as to advancement in drug development and pathology assays, preventing high-dimensional quantitative analyses of developmental and disease-relevant regulatory networks in an anatomical context. To overcome these challenges, in situ amplification based on the mechanism of hybridization chain reaction (HCR) draws on concepts from the emerging discipline of dynamic nucleic acid nanotechnology to achieve four RNA-ISH breakthroughs in highly autofluorescent samples including whole-mount vertebrate embryos, thick brain slices, and FFPE tissue sections: 1) straightforward multiplexing with 1-step quantitative signal amplification for up to 5 target mRNAs simultaneously; 2) analog mRNA relative quantitation with subcellular resolution in an anatomical context; 3) digital mRNA absolute quantitation with single-molecule resolution in an anatomical context; 4) automatic background suppression throughout the protocol, dramatically enhancing performance and ease-of-use. The proposed research will build on the unique capabilities of HCR to enable next-generation levels of multiplexing for RNA-ISH, to extend the benefits of 1-step multiplexed quantitative enzyme-free HCR signal amplification to IHC, and to develop the first in situ amplification product for simultaneous RNA-ISH/IHC, performing 1-step HCR signal amplification for targets RNAs and proteins simultaneously. During Phase I, we will engineer molecular components to enable highly multiplexed signal amplification, and establish a unified framework for RNA and protein imaging. During Phase II, we will develop and commercialize robust technologies for highly multiplexed RNA-ISH/IHC in key sample types critical for academic research, drug development, and/or clinical diagnostics. These new HCR products will enable biologists, drug developers, and pathologists to perform previously impossible quantitative analyses on large panels of mRNAs and proteins at high resolution with full anatomical context.


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: 2024 ( Subtotal = -$1 )
2024	2023	MOLECULAR INSTRUMENTS, INC	5015 EAGLE ROCK BLVD STE 301	LOS ANGELES	CA	90041	LOS ANGELES	USA	Biomedical Research and Research Training	000	3	5/13/2024	NON-COMPETING CONTINUATION	-$1
														Subtotal = -$1

Issue Date FY: 2023 ( Subtotal = $500,000 )
2023	2023	MOLECULAR INSTRUMENTS, INC.	5015 EAGLE ROCK BLVD STE 301	LOS ANGELES	CA	90041	LOS ANGELES	USA	Biomedical Research and Research Training	000	3	11/10/2022	NON-COMPETING CONTINUATION	$500,000
2023	2022	MOLECULAR INSTRUMENTS, INC.	5015 EAGLE ROCK BLVD STE 301	LOS ANGELES	CA	90041	LOS ANGELES	USA	Biomedical Research and Research Training	001	2	1/12/2023	EXTENSION WITH OR WITHOUT FUNDS	$0
														Subtotal = $500,000

Issue Date FY: 2022 ( Subtotal = $500,000 )
2022	2022	MOLECULAR INSTRUMENTS, INC.	5015 EAGLE ROCK BLVD STE 301	LOS ANGELES	CA	90041	LOS ANGELES	USA	Biomedical Research and Research Training	003	2	7/26/2022	EXTENSION WITH OR WITHOUT FUNDS	$1
2022	2022	MOLECULAR INSTRUMENTS, INC.	5015 EAGLE ROCK BLVD STE 301	LOS ANGELES	CA	90041	LOS ANGELES	USA	Biomedical Research and Research Training	001	2	12/9/2021	EXTENSION WITH OR WITHOUT FUNDS	$500,000
2022	2021	MOLECULAR INSTRUMENTS, INC.	5015 EAGLE ROCK BLVD STE 301	LOS ANGELES	CA	90041	LOS ANGELES	USA	Biomedical Research and Research Training	000	1	10/15/2021	NEW	$0
2022	2021	MOLECULAR INSTRUMENTS, INC.	5015 EAGLE ROCK BLVD STE 301	LOS ANGELES	CA	90041	LOS ANGELES	USA	Biomedical Research and Research Training	002	1	7/26/2022	NEW	-$1
														Subtotal = $500,000

Issue Date FY: 2021 ( Subtotal = $150,000 )
2021	2021	MOLECULAR INSTRUMENTS, INC.	5015 EAGLE ROCK BLVD STE 301	LOS ANGELES	CA	90041	LOS ANGELES	USA	Biomedical Research and Research Training	001	1	4/16/2021	NEW	$0
2021	2021	MOLECULAR INSTRUMENTS, INC.	5015 EAGLE ROCK BLVD STE 301	LOS ANGELES	CA	90041	LOS ANGELES	USA	Biomedical Research and Research Training	000	1	4/1/2021	NEW	$150,000
														Subtotal = $150,000

Grand Total All Awards = $1,149,999

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Hybridization Chain Reaction: Highly Multiplexed Quantitative RNA and Protein Imaging

Award Number: R44GM140796

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: 04/01/2021

PERIOD OF PERFORMANCE END DATE: 11/30/2023

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