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Life is orchestrated by biomolecules interacting in complex networks of biological circuitry with emerging function. Progress in different areas of chemistry has made the design of systems that can recapitulate elements of such circuitry possible. Herein we review prominent examples of networks, the methodologies available to translate an input into various outputs, and speculate on potential applications and directions for the field. The programmability of nucleic acid hybridization has inspired applications beyond its function in heredity. At the circuitry level, DNA provides a powerful platform to design dynamic systems that respond to nucleic acid input sequences with output sequences through diverse logic gates, enabling the design of ever more complex circuitry. In order to interface with more diverse biomolecular inputs and yield outputs other than oligonucleotide sequences, an array of nucleic acid conjugates have been reported that can engage proteins as their input and yield a turn-on of enzymatic activity, a bioactive small molecule, or morphological changes in nanoobjects. While the programmability of DNA makes it an obvious starting point to design circuits, other biosupramolecular interactions have also been demonstrated, and harnessing progress in protein design is bound to deliver further integration of macromolecules in artificial circuits.

Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. As tracks for cargo delivery, kinesin-1 uses a subset of microtubules within the dense microtubule network. It is still debated what defines the specific binding of kinesin-1 to a subset of microtubules. Therefore, the ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate for kinesin-1 that yields a precipitating dye. The activity of kinesin-1 thus leaves a fluorescent trail along its walking path and can be visualized without loss of signal due to diffusion. Kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time.

Dimeric proteins are prominent in biology, and receptor dimerization (homo- or heterodimerization) is central to signal transduction. Herein, we report a network that responds to a membrane-associated dimeric protein with the uncaging of a powerful cytotoxic. The network is based on two ligands functionalized with peptide nucleic acids (PNAs) (templating strand and catalyst-functionalized strand, respectively) and a substrate with the caged cytotoxic (monomethyl auristatin E: MMAE; a high-affinity tubulin ligand). In the presence of the dimeric protein, the network yields a cooperative supramolecular assembly with a hybridization architecture that enhances the templated reaction and enables the uncaging of a substrate. The network was tested on cells that express a cancer biomarker, carbonic anhydrase IX, in response to hypoxia. The output of the network correlates with the expression of carbonic anhydrase IX, and this biomarker was harnessed to uncage a potent cytotoxic agent.

DNA-based circuitry empowers logic gated operations and amplifications but is restricted to nucleic acid output. Templated reactions enable the translation of nucleic acid cues into diverse small-molecule outputs but are more limited in their amplification. Herein, we demonstrate the coupling of a DNA circuit to templated reactions in order to achieve high levels of amplification in the output of small molecules, in response to nucleic acid input. We demonstrate that the coupling of the DNA circuit to templated reactions allows for the detection of the fM concentration of analyte and can respond with the release of a cytotoxic drug.

Bioluminescence resonance energy transfer (BRET) has been widely used for studying dynamic processes in biological systems such as protein-protein interactions and other signaling events. Aside from acting as a reporter, BRET can also turn on functions in living systems. Herein, we report the application of BRET to performing a biorthogonal reaction in living cells; namely, releasing functional molecules through energy transfer to a coumarin molecule, a process termed bioluminolysis. An efficient BRET from Nanoluc-Halotag chimera protein (H-Luc) to a coumarin substrate yields the excited state of coumarin, which in turn triggers hydrolysis to uncage a target molecule. Compared to the conventional methods, this novel uncaging system requires no external light source and shows fast kinetics (t1/2

The development of bioorthogonal reactions have had a transformative impact in chemical biology and the quest to expand this toolbox continues. Herein we review recent applications of ruthenium-catalyzed photoredox reactions used in chemical biology.

Bioluminescence resonance energy transfer (BRET) is extensively used to study dynamic systems and has been utilized in sensors for studying protein proximity, metabolites, and drug concentrations. Herein, we demonstrate that BRET can activate a ruthenium-based photocatalyst which performs bioorthogonal reactions. BRET from luciferase to the ruthenium photocatalyst is used to uncage effector molecules with up to 64 turnovers of the catalyst, achieving concentrations >0.6 μM effector with 10 nM luciferase construct. Using a BRET sensor, we further demonstrate that the catalysis can be modulated in response to an analyte, analogous to allosterically controlled enzymes. The BRET-induced reaction is used to uncage small-molecule drugs (ibrutinib and duocarmycin) at biologically effective concentrations in cellulo., Bioluminescence resonance energy transfer (BRET) has been mostly employed in imaging applications. Here the authors use BRET to activate a ruthenium-based photocatalyst and perform a bioorthogonal chemical reaction, which can be used to uncage small molecule drugs in a cellular context.