Single-molecule platform for drug discovery: methods and apparatuses for drug discovery, including discovery of anticancer and antiviral agents
First Claim
1. A method of identifying one or more nucleic acid polymerization modifying drug candidates that affect a nucleic acid polymerase, wherein the method comprises:
- contacting a target nucleic acid polymerase with an immobilized substrate of the target polymerase in the absence of one or more nucleic acid polymerization modifying drug candidates, said immobilized substrate being maintained under constant tension by at least partial stretching of the immobilized substrate using feedback control;
detecting and recording with instrumentation a baseline feedback response signal of the applied force necessary to maintain the tension on the immobilized substrate of the target polymerase in the presence of the target polymerase, the baseline feedback response signal being correlated with the velocity of the target polymerase along the bound substrate of the target polymerase, but in the absence of one or more nucleic acid polymerization modifying drug candidates and recording the baseline feedback response signal;
contacting the target polymerase and the substrate of the target polymerase with a nucleic acid polymerization modifying drug candidate; and
detecting and recording with instrumentation a feedback response signal of the applied force necessary to maintain the tension on the immobilized substrate of the target polymerase and further in the presence of the one or more nucleic acid polymerization modifying drug candidates, the feedback response signal being correlated with the velocity of the target polymerase along the bound substrate of the target polymerase in the presence of the one or more nucleic acid polymerization modifying drug candidates and recording the feedback measurement response signal.
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Abstract
The present application discloses methods and apparatuses for single molecule drug screening, discovery and validation. These methods and apparatuses allow a user to detect rapidly, using observation of single molecules, whether and how a drug candidate interferes with a target enzyme involved in a particular disease pathway. The methods and apparatuses described herein utilize single molecule manipulation and detection technologies (e.g., optical or magnetic tweezers) to directly detect whether the characteristic dynamics, or “mechanical signature,” of the target enzyme-substrate interaction are substantially altered or modulated by a drug candidate. Furthermore, the methods and apparatuses are useful for analyzing the modulation of the mechanical signature in order to identify potential interference mechanisms of a drug candidate. In one aspect of the invention, the methods and apparatuses disclosed herein relate to monitoring the real-time dynamic mechanical signatures of individual polymerase molecules (e.g. DNA polymerases, RNA polymerases, and reverse transcriptases) along a polynucleotide substrate in the presence of drug candidates that either inhibit or otherwise modulate the polymerization process. Identification and analysis of such drug candidates is critical for anti-viral, anti-cancer, and antibiotic drug development.
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Citations
16 Claims
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1. A method of identifying one or more nucleic acid polymerization modifying drug candidates that affect a nucleic acid polymerase, wherein the method comprises:
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contacting a target nucleic acid polymerase with an immobilized substrate of the target polymerase in the absence of one or more nucleic acid polymerization modifying drug candidates, said immobilized substrate being maintained under constant tension by at least partial stretching of the immobilized substrate using feedback control; detecting and recording with instrumentation a baseline feedback response signal of the applied force necessary to maintain the tension on the immobilized substrate of the target polymerase in the presence of the target polymerase, the baseline feedback response signal being correlated with the velocity of the target polymerase along the bound substrate of the target polymerase, but in the absence of one or more nucleic acid polymerization modifying drug candidates and recording the baseline feedback response signal; contacting the target polymerase and the substrate of the target polymerase with a nucleic acid polymerization modifying drug candidate; and detecting and recording with instrumentation a feedback response signal of the applied force necessary to maintain the tension on the immobilized substrate of the target polymerase and further in the presence of the one or more nucleic acid polymerization modifying drug candidates, the feedback response signal being correlated with the velocity of the target polymerase along the bound substrate of the target polymerase in the presence of the one or more nucleic acid polymerization modifying drug candidates and recording the feedback measurement response signal. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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Specification