| Popis: |
When we consider the roles biopolymers play in our cells, the central dogma of biology generally comes to mind: DNA stores information to be read out by cellular machinery transcribing it into RNA which encodes proteins responsible for catalysis. While these functions are critical, they do not account for the numerous secondary and tertiary phenomena responsible for the bulk of cellular function: all three biopolymers can be decorated with various modifications, and, in addition to or in concert with secondary structural changes, interact transiently with each other through protein-protein, RNA-protein, and DNA-protein interactions. These dynamic processes dictate whether DNA is accessible for transcription, whether resultant RNA will be processed normally or degraded/upregulated, and which competition-mediated signaling pathways might be activated downstream, to name a few. Due to this complexity, it has been historically challenging to measure or harness such interactions. Therefore, in this thesis we will cover the development of several tools designed to tackle these challenges. The first exploits the RNA output created by two small molecule-sensing T7 polymerase-based biosensors in order to control Cas9 activity in cells. The second harnesses the interchangeability-potential of ester-caged imaging and bioactive molecules using a split esterase biosensor to study protein-protein interactions. The third further employs the esterase-based technology through the creation of an RNA proximity labeling method via a unique ester-masked enol ester acylating reagent. Finally, we will briefly discuss efforts toward an esterase-based “synthetic” bioluminescent sensitive in vivo imaging system. Taken together, this work generated technologies that will help better understand biomolecular interactions and harness them to control cellular processes. |
