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Generalizable Nanosensors for Probing Highly Specific Interactions of Protein Kinases

Award Number: R01GM151299
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: 09/23/2023
PERIOD OF PERFORMANCE END DATE: 08/31/2027

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Generalizable Nanosensors for Probing Highly Specific Interactions of Protein Kinases - Project Summary Developing novel technologies for identifying and quantifying transient protein-protein interactions is critical in basic research and medical biotechnology. Protein kinases represent a focal group among strategic drug targets for treating numerous hematological malignancies and solid tumors. Yet, creating high-resolution sensors to detect, quantify, and analyze the plasticity of diverse kinome members in a broad dynamic range of interactions remains difficult. This challenge is exacerbated because the kinase superfamily members vary drastically in their complexity. To address this long-standing technological shortcoming, we will formulate, develop, and validate a new class of generalizable and highly specific nanopore sensors (nanosensors) for kinase analytics. The key innovating aspect of this design is fusing a generic protein recognition ligand with a transmembrane protein nanopore. This approach will employ a robust nanostructure made of a single polypeptide entity with no requirement for an additional tail or other exogenous tags. The binding interface of the protein recognition ligand is interchangeable to accommodate the required specificity for a targeted kinase, whereas the nanopore facilitates the generation of a reporting electrical signal. A protein kinase analyte in solution produces a unique electrical signature that varies with its identity and quantity. The reporting signal is mediated by the ligand-kinase assembly at the nanopore tip. In these studies, kinase recognition events will be discriminated at single-molecule precision without the necessity of using complex data analysis algorithms. This engineering strategy substantially broadens the spectrum of applications of these nanosensors to various kinases and their interactions. Our preliminary studies prove the power of this approach by creating a single- molecule nanosensor platform that probes and quantifies structurally and functionally diverse proteins beyond the fundamental limit of sensing inside the nanopore. In addition, such a tactic will enable the detection of competing binding interactions of kinase isoforms against the same recognition ligand. These generalizable nanosensors permit integration into scalable devices, representing versatile elements for small-molecule inhibitor screening and drug discovery pipelines. Further project developments will be aimed at maintaining a high performance of these nanosensors in a complex biofluid. Therefore, they can be utilized using realistic samples, having prospects in molecular diagnostics. The expected immediate outcomes of this project will be the following: (i) the development of high-affinity nanosensors for ultrasensitive analysis of receptor tyrosine kinases (RTKs); (ii) the creation of genetically-encoded nanosensors for probing serine-threonine kinases (STKs); (iii) the detection and analysis of kinases in multiplexed settings and biofluids. These studies will impact healthcare by providing tools and a fundamental framework in biosensor technology, synthetic biology, and single-molecule enzymology.


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 = $397,599 )
2025	2025	SYRACUSE UNIVERSITY	900 S CROUSE AVE	SYRACUSE	NY	13244	ONONDAGA	USA	Biomedical Research and Research Training	000	3	8/19/2025	NON-COMPETING CONTINUATION	$397,599
														Subtotal = $397,599

Issue Date FY: 2024 ( Subtotal = $397,590 )
2024	2024	SYRACUSE UNIVERSITY	900 S CROUSE AVE	SYRACUSE	NY	13244	ONONDAGA	USA	Biomedical Research and Research Training	000	2	8/31/2024	NON-COMPETING CONTINUATION	$397,590
														Subtotal = $397,590

Issue Date FY: 2023 ( Subtotal = $422,233 )
2023	2023	SYRACUSE UNIVERSITY	900 S CROUSE AVE	SYRACUSE	NY	13244	ONONDAGA	USA	Biomedical Research and Research Training	000	1	9/21/2023	NEW	$422,233
														Subtotal = $422,233

Grand Total All Awards = $1,217,422

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Generalizable Nanosensors for Probing Highly Specific Interactions of Protein Kinases

Award Number: R01GM151299

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: 09/23/2023

PERIOD OF PERFORMANCE END DATE: 08/31/2027

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