cDNA-Display for High-Throughput Single-Molecule Measurements of Peptides and HLA-II Interactions

Abstract

Major histocompatibility complex (MHC) class II molecules play a crucial role within the immune system. They bind and present various peptides on the cell surfaces to T-cells, which activate the immune response upon recognition. Failure to recognize pathogens results in an insufficient immune response, but incorrectly recognizing self-antigens can cause an autoimmune response. Although T-cells ultimately determine the immmune response to peptides, MHC-II determines which peptides are presented, thus which peptides can be recognized. Therefore, studying the binding kinetics between MHC-II and peptides is crucial for understanding the immune response. However, studying the binding characteristics of MHC-II and peptides is challenging due to the infinite number of possible peptides and the thousands of MHC-II polymorphisms.
Single-molecule Parallel Analysis for Rapid eXploration of Sequence space (SPARXS) could address this challenge as it combines single molecule FRET (smFRET) experiments with Illumina sequencing to measure binding kinetics at high-throughput. Extending this technique to peptide libraries would enable high-throughput measurements of MHC-II peptide binding. SPARXS would be able to obtain high-throughput kinetic data of these interactions through a smFRET assay. To be able to use this data however, it is dependent on robust genotype-phenotype coupling. This ensures that the kinetic data extracted is matched with its corresponding DNA-sequence, which is extracted through Illumina sequencing. Robust genotype-phenotype coupling is possible when using cDNA-display, which links a peptide to its encoding DNA through a puromycin linker. Building on this principle, this thesis has established the groundwork for a cDNA-display designed, which is compatible with SPARXS to enable high-throughput characterization of peptide-MHC-II interactions.