DNA origami: single cell analysis of T cell receptors without single cell sorting.

The immune system must be able to recognize virtually any pathogen (diversity) while maintaining enough cells specific for each pathogen in order to mount an effective response (protection). T cells generate diversity by imprecise joining of gene segments to generate alpha/beta heterodimeric receptors. Linking sequence information for TCRα and TCRβ from individual cells has been problematic due to the cost of single cell sorting and inadequate molecular approaches for linking mRNA encoding these proteins. We have developed novel DNA origami nanostructures to capture and protect both TCRα and TCRβ mRNA from individual cells, which can then be physically linked via a unique dual-primed reverse-transcription and ligation reaction to generate single amplicons containing both TCR from individual cells for use in next generation sequencing. We have demonstrated high efficiency transfection and recovery of DNA origami, optimized methods for purification with bound TCR mRNA, and validated this approach with transgenic T cells expressing a known TCR sequence. Surprisingly, we find that <1% of TCR are shared even between genetically identical individuals, challenging the idea of “public” TCR. This approach is directly amenable to single cell analysis of other immune receptors (or other species) by relatively simple modifications of the origami sequences, and could be applied to virtually any heterogeneous cell population for which sequence information on any two genes is required.