PROJECT SUMMARY Living systems choreograph molecular events with precise control—place, time, kinetics, and intensity —and record memories of those occurrences in synaptic networks, gene expression, epigenetic marks, and myriad other circuits that govern the where&when of biologic responses. Visualizing this choreography and tracing these histories are tasks that chemists and biologists can accomplish with only partial accuracy, considerable effort, and limited temporal range. Our research agendas are thus focused on constructing new chemical tools for temporospatial analysis of living systems, and organized around the emergent properties that result from deploying next-level bioorthogonal chemistries within multi-layered (bio)molecular architectures. The resulting hybrid systems circumvent perennial challenges, achieving: i) simultaneous speed/stability, for efficient real-time molecular machinery and longitudinal performance in vivo; ii) sensitivity for detection of rare/unique events; and iii) specificity/multiplicity, for accurate detection and fine-grained molecular encoding of (sub)cellular histories across time. Building on the momentum of ongoing mechanistic investigations and the success of our recent effort to achieve multiplexed imaging of living cells and tissues, our goals for the next five years extend bioorthogonal chemistry in applications that exploit two/three dimensional topologies, rather than singular ligation/cleavage events, and in architectures that leverage nucleic acid hybridization to encode sequence recognition, accelerate reaction kinetics, and enable signal amplification. In one set of projects, we aim to create self-amplifying programmable bioorthogonal reactions, elaborate the capabilities of this new toolkit, and apply them to transform our methods for visualizing living cells and tissues. In another, we have envisioned sequence-generating architectures that convert biocompatible chemical reactivity into amplifiable biological information, establishing the concepts of bioorthogonal translation and sequegenicity. With scaffolds that readily integrate into the workflows of existing high- performance nucleic acid biotechnologies, we anticipate broad applicability and rapid downstream development of a new generation of tools for tracking (bio)molecules, individual cells, and populations.

PROJECT NARRATIVE We seek to construct biocompatible chemical tools that can interrogate the dynamic molecular events of living systems across space and time. By embedding highly efficient reactions within 2D/3D nucleic acid scaffolds, we will build toolkits with the speed and sensitivity to capture the deep complexity of living cells and tissues. With architectures that can translate chemical events into new sequence information, we aim to erase boundaries between in vivo chemistry and nucleic acid biotechnologies, transforming (bio)molecular tracking and sensing.