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


Year of Award:
2018
Status:
Complete
Award Type:
R43
Project Number:
CA236142
RFA Number:
PAR-18-303
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
SCHOETTLE, LOUIS
Other PI or Project Leader:
BLATTMAN, JOSEPH N
Institution:
GEMNEO BIOSCIENCE, INC.
Project Summary/AbstractNext generation sequencing platforms have revolutionized modern approaches for understanding a widevariety of biological processes, including immune responses and cancer. However, increasing evidence showsthat the diversity of the cells involved in these processes has important implications for understanding biologicoutcomes. For instance, the diversity of T cell receptors on lymphocytes during responses to virus or cancercan have dramatic effects on disease outcome. Conversely, the diversity of cancer cells has importantimplications for successful control of disease. Therefore, a critical hurdle in these situations is the ability toprovide single-cell analysis techniques coupled with high-throughput next generation sequencing, toadequately measure the diversity of cells. Unfortunately, current single-cell analysis approaches are eitherunfeasible for large cell populations, too expensive, and/or require specialized equipment that is not availableto most labs while bulk sequencing approaches provide no information on individual cells. To address thisproblem, we have engineered DNA origami nanostructures that are able to specifically bind and protect twodifferent mRNA within transfected cells, and used DNA origami-specific matching barcodes upstream ofcapture sequences to generate to identify in HT-NGS output sequences which sequences came from the samenanostructure and therefore from the same transfected cell. In this proposal we develop this approach forquantitating the diversity of clonally-distributed TCR? and TCR? T cell receptors in human T lymphocytepopulations and directly compare the throughput, efficiency, cost and error rates to both conventional bulk-sequencing and single-cell sequencing approaches. We have previously developed this approach for analysisof mouse TCR pairs from primary T cells, and expect little difficulty in adapting this modular platform to humanT cells. This technology will be useful for downstream application to a wide variety of biologic processes, byrelatively simple modifications to the DNA origami nanostructure probe sequences, including single-cellanalysis of other diverse lymphocyte populations, including other T cell subsets or antibody producing B cells,as well as single cells analysis of heterogeneous tumors or diverse microbial communities. Moreover, becausethis approach utilizes equipment found in most modern molecular biology laboratories, it could be easilyadopted by many researchers for these analyses.