Expanded MutaT7 toolkit efficiently and simultaneously accesses all possible transition mutations in bacteria.

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.

The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope.

The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.

Directed evolution in mammalian cells.

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 the 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.

An Adaptable Platform for Directed Evolution in Human Cells.

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 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.

Photophysical and Electrochemical Characterization of BODIPY-Containing Dyads Comparing the Influence of an A-D-A versus D-A Motif on Excited-State Photophysics.

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.