Since the discovery of DNA in the 19th century, biochemists have been elucidating not only the structure, but unique biochemical environment of each loci. Protein-DNA neighborhoods govern chromatin structure and cellular functions (transcription, replication, etc.). To investigate which proteins and oligonucleotides compose these microenvironments, our lab and collaborators developed DNA oligonucleotide-directed proximity-interactome mapping (DNA O-MAP), a locus purification method using oligo-based ISH probes to recruit horseradish peroxidase (HRP) activity to specific DNA intervals (Liu & McGann et. al. 2024). Once these secondary, HRP-conjugated probes are localized to loci of interest, hydrogen peroxide is added with biotin-tyramide. Hydrogen peroxide activates HRP, forming biotin-tyramide phenoxyl radicals that biotinylate proteins and nucleic acids within 10-75nm. This allows for a scalable, versatile method to investigate these microenvironments. Large scale DNA O-MAP, tiling across several genomic sites, can elucidate insights into biological questions. However, the upstream protocol remains a barrier to its throughput, sensitivity, and reproducibility. In order to ensure this for analysis of tagged proteins, we sought to automate the streptavidin affinity purification protocol onto the Opentrons OT-2 robot. This is where streptavidin-coated magnetic beads capture biotinylated species from lysate. Coupled beads are recaptured with a magnetic rack and pipetting-off of flow-through. Subsequently, several washes cleans up these beads before peptides are digested off via Trypsin/LysC, dried-down, resuspended, and loaded onto a Orbitrap Eclipse LC-MS for proteomic analysis. Purification of streptavidin beads is manually intensive, inherently leading to variation between runs. The Opentrons OT-2 is an open-source liquid handler, allowing our lab to easily transfer methods to others interested in DNA O-MAP. Automating this protocol launches us from technology development to biological application. Here, I present an automated protocol for streptavidin affinity purification and evaluation of its effectiveness via comparison of the automated protocol to our lab's current, manual methods.

Female mammals possess two X chromosomes in every cell, but one is silenced by condensing into a barr body, making its genetic information largely inaccessible. While X inactivation is stable in somatic cells, it is reversible in germ cells, raising the intriguing question of what proteins maintain this silenced state. My project aims to identify the protein composition of both active and inactive X chromosomes in mice. To achieve this, I will use in situ hybridization to target proximal labeling with biotin of X chromosome-associated proteins. This is accomplished by targeting a biotinylation enzyme, such as HRP, to the X chromosomal region, where it will selectively biotinylate neighboring proteins. After affinity purification, these proteins can be identified using mass spectrometry-based quantitative proteomics. To direct the enzyme to the correct location, a two-probe system is employed. The primary oligonucleotide probe complements a specific X chromosome region which also contains landing sites for a secondary probe. Hybridization of the secondary probe which is tagged with HRP enables precise labeling of chromosome-associated proteins. This approach enables in situ biotinylation, preserving proteins in their native context for accurate identification. Since the two X chromosomes are homologous, distinguishing between the active and inactive X requires careful probe design. By utilizing Single Nucleotide Polymorphisms (SNPs) that exist in the X chromosomes, the maternal and paternal X chromosomes can be differentially targeted by primary probes, allowing for homolog specific protein labeling and analysis of their distinct regulatory environments.