ABSTRACT
Recent COVID-19 vaccines unleashed the potential of mRNA-based therapeutics. A common bottleneck across mRNA-based therapeutic approaches is the rapid design of mRNA sequences that are translationally efficient, long-lived and non-immunogenic. Currently, an accessible software tool to aid in the design of such high-quality mRNA is lacking. Here, we present mRNAid, an open-source platform for therapeutic mRNA optimization, design and visualization that offers a variety of optimization strategies for sequence and structural features, allowing one to customize desired properties into their mRNA sequence. We experimentally demonstrate that transcripts optimized by mRNAid have characteristics comparable with commercially available sequences. To encompass additional aspects of mRNA design, we experimentally show that incorporation of certain uridine analogs and untranslated regions can further enhance stability, boost protein output and mitigate undesired immunogenicity effects. Finally, this study provides a roadmap for rational design of therapeutic mRNA transcripts.
ABSTRACT
Protein engineering is the discipline of developing useful proteins for applications in research, therapeutic, and industrial processes by modification of naturally occurring proteins or by invention of de novo proteins. Modern protein engineering relies on the ability to rapidly generate and screen diverse libraries of mutant proteins. However, design of mutant libraries is typically hampered by scale and complexity, necessitating development of advanced automation and optimization tools that can improve efficiency and accuracy. At present, automated library design tools are functionally limited or not freely available. To address these issues, we developed Mutation Maker, an open source mutagenic oligo design software for large-scale protein engineering experiments. Mutation Maker is not only specifically tailored to multisite random and directed mutagenesis protocols, but also pioneers bespoke mutagenic oligo design for de novo gene synthesis workflows. Enabled by a novel bundle of orchestrated heuristics, optimization, constraint-satisfaction and backtracking algorithms, Mutation Maker offers a versatile toolbox for gene diversification design at industrial scale. Supported by in silico simulations and compelling experimental validation data, Mutation Maker oligos produce diverse gene libraries at high success rates irrespective of genes or vectors used. Finally, Mutation Maker was created as an extensible platform on the notion that directed evolution techniques will continue to evolve and revolutionize current and future-oriented applications.
Subject(s)
Mutagenesis, Site-Directed/methods , Mutagenesis , Mutation , Oligonucleotides/genetics , Proteins/genetics , Software , Algorithms , Codon/genetics , Computer Simulation , Directed Molecular Evolution/methods , Escherichia coli/genetics , Gene Library , Mutant ProteinsABSTRACT
We propose a setup allowing to entangle two directly non-interacting radiation modes applying four sequential pulsed quantum resonant interactions with a noisy vibrational mode of a mechanical oscillator which plays the role of the mediator. We analyze Gaussian entanglement of the radiation modes generated by the transducer and confirm that the noisy mechanical mode can mediate generation of entanglement. The entanglement, however, is limited if the interaction gains are not individually optimized. We prove the robustness of the transducer to optical losses and the influence of the mechanical bath and propose the ways to achieve maximal performance through the individual optimization.
