Project Grant R44GM151897

SINGLE-MOLECULE PROTEIN SEQUENCING: RESOLVING INDIVIDUAL AMINO ACIDS VIA COMMERCIALLY ACCESSIBLE NANOPORES - PROJECT SUMMARY THE PREMISE OF SINGLE-MOLECULE PROTEIN SEQUENCING (SMPS) IS TO DETERMINE THE AMINO ACID SEQUENCE OF A PROTEIN ONE MOLECULE AT A TIME. SMPS THUS ALLOWS ACCESS TO A WEALTH OF INFORMATION ACROSS BASIC BIOLOGY AND HEALTH- RELATED SCIENCE, OTHERWISE UNREACHABLE BY THE AGGREGATE READOUT OF KNOWN PROTEIN/PEPTIDE IDENTITIES. THUS, AS AN ALTERNATIVE TO THE ALREADY ESTABLISHED METHODS OF MEASURING PROTEINS, THIS TECHNOLOGY HOLDS GREAT PROMISE FOR NICHE APPLICATIONS, SUCH AS ENHANCING PERSONALIZED MEDICINE THROUGH IDENTIFYING KEY (NOVEL AND/OR LOW- ABUNDANCE) PEPTIDE-BASED PLAYERS INVOLVED IN MODULATING IMMUNE RESPONSE OR REGULATING ACCESS TO THE GENOME. HOWEVER, THERE IS CURRENTLY NO EFFICIENT STRATEGY TO SEQUENCE MIXTURES OF UNKNOWN PROTEIN MOLECULES AT A SINGLE AMINO ACID RESOLUTION THAT IS ALSO ACCESSIBLE AND READY TO USE FOR STREAMLINED AND SCALABLE SMPS. NANOPORE TECHNOLOGY UTILIZES NANOMETER-SIZED PORES TO READ THE STRUCTURAL FEATURES OF INDIVIDUAL BIOPOLYMER MOLECULES AS THEY TRANSLOCATE THROUGH THE NANOPORE CONSTRICTION AND MODULATE IONIC CURRENTS. OXFORD NANOPORE TECHNOLOGIES' (ONT) SYSTEM HAS SHOWN THAT NANOPORES CAN BE HIGHLY PARALLELIZED TO SUCCESSFULLY READ LONG STRETCHES OF NATIVE DNA AND RNA MOLECULES, INCLUDING EPIGENETIC MARKERS. SMPS UTILIZING NANOPORES WOULD ALLOW FOR SCALABLE AND DIRECT READOUT OF SINGLE MOLECULES: INDIVIDUAL PEPTIDES AND PROTEINS AND THEIR MODIFICATIONS. HOWEVER, NATIVE PROTEINS DO NOT READILY TRAVEL THROUGH COMMERCIAL NANOPORES AND NANOPORES CANNOT DISCRIMINATE BETWEEN AMINO ACIDS WHEN IN THE CONTEXT OF A PROTEIN. SUPPORTED BY A PHASE I SBIR, GLYPHIC BIOTECHNOLOGIES HAS DEVELOPED, TESTED, AND IMPLEMENTED A NOVEL SMPS STRATEGY, IN WHICH AMINO ACIDS ARE SPATIALLY SEPARATED, DISRUPTING THE INTRAMOLECULAR INTERACTIONS THAT HINDER NANOPORE AMINO ACID RESOLUTION WHILE PRESERVING THE SAME SEQUENCE AND MODIFICATIONS. THIS "BIOPOLYMER" CAN TRAVEL THROUGH VARIOUS COMMERCIAL NANOPORES. THE INDIVIDUAL AMINO ACIDS CAN THEN BE RESOLVED WHILE SIMULTANEOUSLY PERFORMING QUALITY CONTROL BY ALSO READING THE BIOPOLYMER BACKBONE, USING GLYPHIC-DEVELOPED SOFTWARE. IN THIS PHASE II RENEWAL PROPOSAL, GLYPHIC SEEKS TO COMMERCIALIZE THIS TECHNOLOGY BY AGGREGATING THE AUTOMATIC BIOPOLYMER GENERATION AND SOFTWARE FOR THE INTERPRETATION OF THE NANOPORE-READ SEQUENCES AND COUPLING IT WITH A SAMPLE PREPARATION SPECIFIC TO ~10 AMINO ACID PEPTIDES TOWARD ITS FIRST COMMERCIAL UNIT, ENABLING PERFORMING COMPLEX IMMUNOPEPTIDOMICS EXPERIMENTS IN A ROUTINE SETTING OF A LAB BENCH. THE WHOLE SET-UP WILL BE TESTED IN REAL-WORLD CONDITIONS, COLLECTING VALUABLE FEEDBACK TOWARD DEVELOPING A BETA VERSION OF THE GLYPHIC SMPS PRIOR TO LAUNCH. ONCE TESTED, GLYPHIC SMPS CAN BE RAPIDLY PIVOTED TOWARD OTHER APPLICATIONS, WITH EMPHASIS ON UNDERSTANDING THE SEQUENCE OF LOW ABUNDANCE UNIQUE PEPTIDES, SIMPLY BY ADJUSTING SAMPLE PREPARATION PROTOCOLS AND OPTIMIZING THE RUNS AND CLASSIFICATIONS TO FIT THE FIELD-SPECIFIC TARGET MILESTONES. COMMERCIALIZING THIS TECHNOLOGY WILL MAKE PROTEIN SEQUENCING FEASIBLE, INEXPENSIVE, AND ROUTINE.