Project Summary
Reactive oxygen species (ROS) are a family of small-molecules in living systems that serve vital
roles in both signaling and stress. Hydrogen peroxide (H2O2), superoxide (O2•-), and hypochlorous
acid (HOCl), among others, are all examples of ROS that have been traditionally viewed as
sources of oxidative stress and damage. Aberrant ROS production contributes to a multitude of
pathologies such as neurodegeneration, cancer, and cardiovascular disorders. However, ROS
are also critical for maintaining metabolic homeostasis through activation of multiple classes of
proteins. This signal-stress dichotomy, coupled with the small and transient nature of ROS,
presents a challenge when attempting to decode the complex landscape of cellular redox
homeostasis. Fluorescent probes are frequently employed to visualize ROS in living systems
through fluorescence microscopy, however these probes are prone to diffusion after ROS
detection. This leads to inaccurate determination of ROS localization and poor signal-to-noise
responses. As such, there is a need to create probes amenable to the permanent recording of
ROS via fluorescence imaging. We hypothesize that activity-based cell-trappable fluorescent
probes can be used as a platform to gain further understanding of ROS-mediated inter- and intra-
cellular signaling. We propose three specific aims to test this hypothesis. First, we will synthesize
fluorophores caged with activity-based triggers and proximal fluoromethyl groups to serve as
latent equivalents of quinone methide upon ROS sensing. ROS responsive uncaging will allow
for the fluorescent labeling of adjacent biomolecules. Second, we will apply our probes across
multiple model live cell lines to monitor ROS fluxes. We will also map cell-to-cell communication
mediated by ROS using microglia-neuron co-culture as a biological model. This system will allow
us to probe transcellular redox signaling as microglia can be selectively activated in the presence
of neurons thereby dispatching H2O2 to nearby neurons. The third aim involves developing a
fluorescent polymer amplification strategy to increase signal-to-noise responses of tandem
activity-based sensing/labeling probes and will primarily be carried out in the R00 phase. Small-
molecule polymer initiators will be caged in a similar manner to the previously described
fluorescent probes. After ROS sensing and biomolecule labeling, polymerization will be performed
to generate fluorescent polymers from biomolecule surfaces thus enabling signal amplification
and visualization. This strategy will be carried over into live cell lines described above. This
research fits into the applicant’s goal of establishing a program which uses polymer chemistry to
probe fundamental questions in biological systems.
