Measuring Apoptotic Sensitivity Through Oligonucleotide Mediated Peptide Delivery

Abstract

Apoptosis is a process of cell death triggered by cellular stress or extrinsic signals. In apoptosis, pro-death proteins like Bcl-2 interacting mediator of cell death (BIM) activate pore-forming proteins BAX and BAK at the mitochondria, leading to mitochondrial outer membrane permeabilization (MOMP) and downstream activation of apoptotic caspases. BH3 profiling is a method used to measure the signals required to initiate this cell death pathway. Cells are treated with peptides mimicking the Bcl-2 homology 3 (BH3) domain of pro-apoptotic proteins. By measuring responses to these peptides, BH3 profiling can compare peptide sensitivity, or “apoptotic priming,” across cell types, genotypes, or treatments. However, the requirement for membrane permeabilization limits physiological relevance and prevents longitudinal analysis. My thesis develops an alternative method for delivering BH3 peptides to living cells without membrane permeabilization. We used a trimethoprim (TMP)-inducible degron system based on a destabilized dihydrofolate reductase (DHFR) tag that enables rapid, post-translational control of protein stability. For system validation, Citrine-DHFR fusion protein was utilized, and TMP treatment induced Citrine expression in over 90% of cells at concentrations as low as 625 nM. In parallel, we optimized transfection conditions across HeLa lines with varying apoptotic sensitivity. Through in silico design, site-directed mutagenesis and Golden Gate cloning were used to replace Citrine with BIM and integrate the sequence into a new plasmid construct to allow future experimental validation. This system supports new extensions of BH3 profiling, including rapid nucleotide changes to expressed peptides and downstream applications such as single-cell RNA sequencing in unpermeabilized cells.