Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2·107 independent random genes of fluorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of ~ 8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as C.elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multi-color imaging of small model organisms.

Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2x107 independent random genes of fluorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of ~8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as C.elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.

Known Evolutionary Paths Are Accessible to Engineered ß-Lactamases Having Altered Protein Motions at the Timescale of Catalytic Turnover

The evolution of new protein functions is dependent upon inherent biophysical features of proteins. Whereas, it has been shown that changes in protein dynamics can occur in the course of directed molecular evolution trajectories and contribute to new function, it is not known whether varying protein dynamics modify the course of evolution. We investigate this question using three related ß-lactamases displaying dynamics that differ broadly at the slow timescale that corresponds to catalytic turnover yet have similar fast dynamics, thermal stability, catalytic, and substrate recognition profiles. Introduction of substitutions E104K and G238S, that are known to have a synergistic effect on function in the parent ß-lactamase, showed similar increases in catalytic efficiency toward cefotaxime in the related ß-lactamases. Molecular simulations using Protein Energy Landscape Exploration reveal that this results from stabilizing the catalytically-productive conformations, demonstrating the dominance of the synergistic effect of the E014K and G238S substitutions in vitro in contexts that vary in terms of sequence and dynamics. Furthermore, three rounds of directed molecular evolution demonstrated that known cefotaximase-enhancing mutations were accessible regardless of the differences in dynamics. Interestingly, specific sequence differences between the related ß-lactamases were shown to have a higher effect in evolutionary outcomes than did differences in dynamics. Overall, these ß-lactamase models show tolerance to protein dynamics at the timescale of catalytic turnover in the evolution of a new function.

Correction of a Factor VIII genomic inversion with designer-recombinases

Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct a 140 kb genomic inversion of the F8 gene, which is frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we developed a fused heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, resulted in prominent correction of the inversion and restored Factor VIII mRNA expression. Our data suggests that designer-recombinases may represent efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.

Directed Evolution of Therapeutic Antibodies Targeting Glycosylation in Cancer

Glycosylation patterns commonly change in cancer, resulting in expression of tumor-associated carbohydrate antigens (TACA). While promising, currently available anti-glycan antibodies are not useful for clinical cancer therapy. Here, we show that potent anti-glycan antibodies can be engineered to acquire cancer therapeutic efficacy. We designed yeast surface display to generate and select for therapeutic antibodies against the TACA SLea (CA19−9) in colon and pancreatic cancers. Elite clones showed increased affinity, better specificity, improved binding of human pancreatic and colon cancer cell lines, and increased complement-dependent therapeutic efficacy. Molecular modeling explained the structural basis for improved antibody functionality at the molecular level. These new tools of directed molecular evolution and selection for effective anti-glycan antibodies, provide insights into the mechanisms of cancer therapy targeting glycosylation, and provide major methodological advances that are likely to open up innovative avenues of research in the field of cancer theranostics.

Organophosphorus Nerve Agents: Types, Toxicity, and Treatments

Organophosphorus compounds are extensively used worldwide as pesticides which cause great hazards to human health. Nerve agents, a subcategory of the organophosphorus compounds, have been produced and used during wars, and they have also been used in terrorist activities. These compounds possess physiological threats by interacting and inhibiting acetylcholinesterase enzyme which leads to the cholinergic crisis. After a general introduction, this review elucidates the mechanisms underlying cholinergic and noncholinergic effects of organophosphorus compounds. The conceivable treatment strategies for organophosphate poisoning are different types of bioscavengers which include stoichiometric, catalytic, and pseudocatalytic. The current research on the promising treatments specifically the catalytic bioscavengers including several wild-type organophosphate hydrolases such as paraoxonase and phosphotriesterase, phosphotriesterase-like lactonase, methyl parathion hydrolase, organophosphate acid anhydrolase, diisopropyl fluorophosphatase, human triphosphate nucleotidohydrolase, and senescence marker protein has been widely discussed. Organophosphorus compounds are reported to be the nonphysiological substrate for many mammalian organophosphate hydrolysing enzymes; therefore, the efficiency of these enzymes toward these compounds is inadequate. Hence, studies have been conducted to create mutants with an enhanced rate of hydrolysis and high specificity. Several mutants have been created by applying directed molecular evolution and/or targeted mutagenesis, and catalytic efficiency has been characterized. Generally, organophosphorus compounds are chiral in nature. The development of mutant enzymes for providing superior stereoselective degradation of toxic organophosphorus compounds has also been widely accounted for in this review. Existing enzymes have shown limited efficiency; hence, more effective treatment strategies have also been critically analyzed.

Combinatorial Avidity Selection of Mosaic Landscape Phages Targeted at Breast Cancer Cells—An Alternative Mechanism of Directed Molecular Evolution

Low performance of actively targeted nanomedicines required revision of the traditional drug targeting paradigm and stimulated the development of novel phage-programmed, self-navigating drug delivery vehicles. In the proposed smart vehicles, targeting peptides, selected from phage libraries using traditional principles of affinity selection, are substituted for phage proteins discovered through combinatorial avidity selection. Here, we substantiate the potential of combinatorial avidity selection using landscape phage in the discovery of Short Linear Motifs (SLiMs) and their partner domains. We proved an algorithm for analysis of phage populations evolved through multistage screening of landscape phage libraries against the MDA-MB-231 breast cancer cell line. The suggested combinatorial avidity selection model proposes a multistage accumulation of Elementary Binding Units (EBU), or Core Motifs (CorMs), in landscape phage fusion peptides, serving as evolutionary initiators for formation of SLiMs. Combinatorial selection has the potential to harness directed molecular evolution to create novel smart materials with diverse novel, emergent properties.