The discovery and optimization of biomolecules that reliably function in metazoan cells is imperative for both the study of basic biology and the treatment of disease. We describe the development, characterization, and proof-of-concept application of a platform for directed evolution of diverse biomolecules of interest (BOIs) directly in human cells. The platform relies on a custom-designed adenovirus variant lacking multiple genes, including the essential DNA polymerase and protease genes, features that allow us to evolve BOIs encoded by genes as large as 7 kb while attaining the mutation rates and enforcing the selection pressure required for successful directed evolution. High mutagenesis rates are continuously attained by trans-complementation of a newly engineered, highly error-prone form of the adenoviral polymerase. Selection pressure that couples desired BOI functions to adenoviral propagation is achieved by linking the functionality of the encoded BOI to the production of adenoviral protease activity by the human cell. The dynamic range for directed evolution can be enhanced to several orders of magnitude via application of a small molecule-based adenoviral protease inhibitor to modulate selection pressure during directed evolution experiments. This platform makes it possible, in principle, to evolve any biomolecule activity that can be coupled to adenoviral protease expression or activation by simply serially passaging adenoviral populations carrying the BOI. As proof-of-concept, we use the platform to evolve, directly in the human cell environment, several transcription factor variants that maintain high levels of function while gaining resistance to a small molecule inhibitor. We anticipate that this platform will substantially expand the repertoire of biomolecules that can be reliably and robustly engineered for both research and therapeutic applications in metazoan systems.

Directed evolution experiments are typically carried out using in vitro systems, bacteria, or yeast -even when the goal is to probe or modulate mammalian biology. Performing directed evolution in systems that do not match the intended mammalian environment severely constrains the scope and functionality of targets that can be evolved. We review new platforms that are now making it possible to use the mammalian cell itself as the setting for directed evolution, and present an overview of frontier challenges and high-impact targets for this approach.

Electrochemical analysis is an important skill to teach in chemistry curricula because it is a critical tool in current high-impact chemical research. Electrochemistry enables researchers to analyze a variety of systems extending from molecules to materials that encompass research themes ranging from clean energy to substrate activation in biological systems. Specifically, it can reveal information about catalytic efficiency, the role of catalysts, and the nature of chemical reduction and oxidation (redox) processes. Researchers working in the area of catalysis rely heavily on electrochemistry, using it to identify effective catalysts and optimize reaction conditions. This laboratory experiment begins with an introductory tutorial to electrochemistry by guiding students through the use of several electrochemical techniques using the ferricyanide/ferrocyanide redox couple as a model system. The techniques covered in the tutorial include cyclic voltammetry, differential pulse voltammetry, Tafel analysis, and bulk electrolysis. The protocol then applies these techniques to electrocatalysis by identifying and characterizing catalysts for hydrogen and oxygen generation in the water electrolysis reaction. Electrochemical methods are connected to current chemical research by focusing on catalysis in the context of renewable energy, which is a current societal and curricular imperative. The entire laboratory protocol described herein is conducted in groups of two to three students in a four hour laboratory period and is suitable for upper-level physical, analytical, or inorganic chemistry.

A complete photophysical characterization of organic molecules designed for use as molecular materials is critical in the design and construction of devices such as organic photovoltaics (OPV). The nature of a molecule's excited state will be altered in molecules employing the same chromophoric units but possessing different molecular architectures. For this reason, we examine the photophysical reactions of two BODIPY-based D-A and A-D-A molecules, where D is the donor and A is the acceptor. A BODIPY (4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene) moiety serves as the A component and is connected through the meso position using a 3-hexylthiophene linker to a N-(2-ethylhexyl)dithieno[3,2-b:2',3'-d]pyrrole (DTP), which serves as the D component. An A-D-A motif is compared to its corresponding D-A dyad counterpart. We show a potential advantage to the A-D-A motif over the D-A motif in creating longer-lived excited states. Transient absorption (TA) spectroscopy is used to characterize the photophysical evolution of each molecule's excited state. Global analysis of TA data using singular value decomposition and target analysis is performed to identify decay-associated difference spectra (DADS). The DADS reveal the spectral features associated with charge-transfer excited states that evolve with different dynamics. A-D-A possess slightly longer excited-state lifetimes, 42 ps nonradiative decay, and 4.64 ns radiative decay compared to those of D-A, 24 ps nonradiative decay, and 3.95 ns radiative decay. A longer lived A-D-A component is observed with microsecond lifetimes, representing a small fraction of the total photophyscial product. Steady-state and time-resolved photoluminescence augment the insights from TA, while electrochemistry and spectroelectrochemistry are employed to identify the nature of the excited state. Density functional theory supports the observed electronic and electrochemical properties of the D-A and A-D-A molecules. These results form a complete picture of the electronic and photophysical properties of D-A and A-D-A and provide contextualization for structure-function relationships between molecules and OPV devices.

Targeted mutagenesis mediated by nucleotide base deaminase–T7 RNA polymerase fusions has recently emerged as a novel and broadly useful strategy to power genetic diversification in the context of in vivo directed evolution campaigns. Here, we expand the utility of this approach by introducing a highly active adenosine deaminase–T7 RNA polymerase fusion protein (eMutaT7A→G), resulting in higher mutation frequencies to enable more rapid directed evolution. We also assess the benefits and potential downsides of using this more active mutator. We go on to show in Escherichia coli that adenosine deaminase-bearing mutators (MutaT7A→G or eMutaT7A→G) can be employed in tandem with a cytidine deaminase-bearing mutator (MutaT7C→T) to introduce all possible transition mutations simultaneously. We illustrate the efficacy of this in vivo mutagenesis approach by exploring mutational routes to antibacterial drug resistance. This work sets the stage for general application of optimized MutaT7 tools able to induce all types of transition mutations during in vivo directed evolution campaigns across diverse organisms.