Multiplexed DeNAno Protein Assay and Quantitation: Sequencing Based Proteomics

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 DESCRIPTION (provided by applicant): Proteomics studies have the power to deliver pivotal new insight into cancer cells, biomarkers, and host responses. Unlike genomics, however, the field of proteomics has been constrained by a lack of simple and affordable analytical tools. Massively multiplex proteomic analysis methods remain expensive, cumbersome, and specialized. Here we propose the development of a novel assay termed MuDPAQ to occupy the niche for high throughput, highly multiplexed protein or biomarker assays that is currently poorly served with existing technologies. There is a growing need for assays of this type as tissue banks become more extensive, patterns of markers rather than single markers become validated, and as pharmacologic responses to therapy become more utilized. By reverse translating a protein detection event into a DNA signal, the MuDPAQ assay leverages the power and commoditization of next generation sequencing to enable massively parallel analyses that can be cheaply outsourced to any academic or commercial facility. The core of the technology is our "DeNAno" DNA nanoparticle, a novel biomolecular affinity reagent that replaces single or bi-valent affinity with hyper-avidity. DeNAno particles are produced by rolling circle replication of circular oligonucleotide templates. We have previously shown that when random templates are used to produce highly diverse libraries, particles that bind specifically to antibody coated beads can be easily recovered by biopanning. These particles, composed of concatameric repeats, bind to the cognate antibody coated bead through highly avid but individually low affinity interactions and can thus be competitively displaced by the appropriate antigen. DeNAno particles released from the beads can be sequenced, and the count frequency of the unique sequence of a given DeNAno particle translated into the quantity of its competitive analyte. When coupled with sequencing library tagging, this technology will enable high throughput analysis of multiple, multiplexed samples at a cost significantly below current technology and with no additional instrumentation required. For this Phase I proof-of-concept study, our aims will be to 1) select and validate masking DeNAno particles for each of ten mAb coated beads from a Luminex panel of cancer relevant biomarkers, and 2) demonstrate multiplexed analysis of all ten analytes. Successful completion of this study will validate the core concept behind MuDPAQ and lead to development of expanded panels of MuDPAQ reagents as well as further optimization of the assay performance. The long term goal of this proposal is to develop a catalog of MuDPAQ reagents that can be sold directly to researchers or licensed to sequencing companies or reagent manufacturers.