Protease chain reactions for molecular analysis of cancer markers
DESCRIPTION (provided by applicant): Our objective is to develop a protease chain reaction technology (ProCR) which will enable ultra- sensitive molecular detection. A protease chain reaction has four basic components: 1) a protease conjugated to a bindingmolecule, 2) an unconjugated protease, 3) an inhibitor protein which contains a proteolytic cleavage site and 4) a protease substrate which generates a signal upon its cleavage. Fundamentally, a protein chain reaction is a powerful analogue computer withtwo key characteristics which greatly facilitate the detection of target molecules. 1) It can convert the concentration of a specific target molecule into a time signature. 2) It can create enormous signal amplification, analogous to the amplification of DNA by a polymerase chain reaction (PCR). Thus detection is enabled because the final observable signal produced by a target molecule can be very large and the time lag until onset of the signal is precisely correlated with the concentration of target molecule. The critical elements for controlling the protease chain reaction (and ultimately determining the sensitivity of assays linked to it) are very tight inhibition of the protease by the intact inhibitor and cleavage of the inhibitor by free protease. Accordingly, the three experimental Aims are: 1) Engineering the subtilisin prodomain to maximize inhibition; 2) Design and construct prodomain inhibitors with cleavable loops; 3) Construct and characterize a prototype chain reaction useful for detection. ThePhase I goal is to demonstrate proof of principle for protease chain reactions and their applicability for molecular detection. The three milestones are: 1) Develop tight inhibitors with KI d 100pM; 2) Engineer cleavable loops into the inhibitors of Aim 1which are cleaved a rate gt 1000 M-1s-1; 3) Detect 1 femtomole of protease- conjugated antibody by ProCR in a microtiter dish assay. The long term goal is to develop protease- inhibitor complexes as enzymatic nano-processors which can be combined to detect multiple signals and to control output with multiple logic gates. PUBLIC HEALTH RELEVANCE: The development of polymerase chain reaction (PCR) technology demonstrated the extraordinary power of harnessing an enzyme to perform novel, programmable reactions. Our objective is here to develop an analogous protease chain reaction technology (ProCR) which will enable ultra-sensitive molecular detection. The long term goal is to improve cancer detection and prevention by enabling accurate quantitation of multiple, low abundance molecular markers.
Small Business Information at Submission:
POTOMAC AFFINITY PROTEINS, LLC
11305 DUNLEITH PL NORTH POTOMAC, MD 20878-
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