Origami Nanoprobes for Large-Scale Single-Cell Analysis of Lymphocytes

Project Summary Abstract Next generation sequencing platforms have revolutionized modern approaches for understanding a wide variety of biological processesincluding immune responses and cancerHoweverincreasing evidence shows that the diversity of the cells involved in these processes has important implications for understanding biologic outcomesFor instancethe diversity of T cell receptors on lymphocytes during responses to virus or cancer can have dramatic effects on disease outcomeConverselythe diversity of cancer cells has important implications for successful control of diseaseThereforea critical hurdle in these situations is the ability to provide single cell analysis techniques coupled with high throughput next generation sequencingto adequately measure the diversity of cellsUnfortunatelycurrent single cell analysis approaches are either unfeasible for large cell populationstoo expensiveand or require specialized equipment that is not available to most labs while bulk sequencing approaches provide no information on individual cellsTo address this problemwe have engineered DNA origami nanostructures that are able to specifically bind and protect two different mRNA within transfected cellsand used DNA origami specific matching barcodes upstream of capture sequences to generate to identify in HT NGS output sequences which sequences came from the same nanostructure and therefore from the same transfected cellIn this proposal we develop this approach for quantitating the diversity of clonally distributed TCRand TCRT cell receptors in human T lymphocyte populations and directly compare the throughputefficiencycost and error rates to both conventional bulksequencing and single cell sequencing approachesWe have previously developed this approach for analysis of mouse TCR pairs from primary T cellsand expect little difficulty in adapting this modular platform to human T cellsThis technology will be useful for downstream application to a wide variety of biologic processesby relatively simple modifications to the DNA origami nanostructure probe sequencesincluding single cell analysis of other diverse lymphocyte populationsincluding other T cell subsets or antibody producing B cellsas well as single cells analysis of heterogeneous tumors or diverse microbial communitiesMoreoverbecause this approach utilizes equipment found in most modern molecular biology laboratoriesit could be easily adopted by many researchers for these analyses Project Narrative Gemneo Biosciences has developed a novel approach for obtaining single cell sequence information on gene pairs while maintaining the high throughputlow costand high efficiency of bulk sequencing approachesWe will apply this method to analysis of human TCRpairs from primary cellsand directly compare the costefficiencythroughputand error rates to conventional single cell RNA or bulk DNA analysis methodsThis will facilitate rapid transition to market with product for analysis of TCR in a multitude of immune oncology projects