Journal: Nucleic Acids Research

Article Title: Deep learning-guided dual-fitness evolution of T7 RNA polymerase for enhanced stability and activity

doi: 10.1093/nar/gkag259

Figure Lengend Snippet: Comparison between WT and mutant T7 RNAP. ( a ) Selection of the improved dual-fitness mutant. Comparison of thermal unfolding curves of the WT and M1 determined by ( b ) far-UV CD spectroscopy and ( c ) DSC. Effects of temperature on activity of M1 under ~100 bp ( d ) and ~1000 bp ( e ) templates. ( f ) The dependence of dsRNA impurities for M1 on temperature. ( g ) The RNA yield (bar) and dsRNA impurities (scatter) under the ~1000 bp template for the WT and mutant. ( h ) The kinetic stability of the proteins is determined by monitoring residual enzymatic activity after incubation for 15 min at various temperatures. The \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $T_{50}^{15}$\end{document} values for the WT, M8, M10, and M11 are 42.1, 51.1, 52.4, and 52.2°C, respectively. ( i ) RNA yield (bar) and dsRNA content (scatter) of the WT, M10, and M11 under different length templates. ( j ) Diagram illustrating the process from transcription to preparation of mRNA–LNPs, followed by cell transfection and characterization of expression efficiency. ( k ) The expression efficiency in HEK293T cells of mRNA encoding luciferase transcribed by the WT at 37°C and M10 at 50°C. The relative activity shown in (a) and (d) is based on the WT at 37°C. The residual activity shown in (h) is based on the maximum activity of each protein.

Article Snippet: dsRNA content was determined using the dsRNA ELISA kit from Hzymes Biotech. mRNA samples were diluted to 0.5 ng μl −1 , and dsRNA standards were prepared in a gradient ranging from 1 to 0 pg μl −1 (1, 0.5, 0.25, 0.125, 0.0625, 0.0312, 0.0156, and 0 pg μl −1 ).

Techniques: Comparison, Mutagenesis, Selection, Circular Dichroism, Activity Assay, Incubation, Transfection, Expressing, Luciferase

Journal: Nucleic Acids Research

Article Title: Deep learning-guided dual-fitness evolution of T7 RNA polymerase for enhanced stability and activity

doi: 10.1093/nar/gkag259

Figure Lengend Snippet: Comparison between WT and mutant T7 RNAP. ( a ) Selection of the improved dual-fitness mutant. Comparison of thermal unfolding curves of the WT and M1 determined by ( b ) far-UV CD spectroscopy and ( c ) DSC. Effects of temperature on activity of M1 under ~100 bp ( d ) and ~1000 bp ( e ) templates. ( f ) The dependence of dsRNA impurities for M1 on temperature. ( g ) The RNA yield (bar) and dsRNA impurities (scatter) under the ~1000 bp template for the WT and mutant. ( h ) The kinetic stability of the proteins is determined by monitoring residual enzymatic activity after incubation for 15 min at various temperatures. The \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $T_{50}^{15}$\end{document} values for the WT, M8, M10, and M11 are 42.1, 51.1, 52.4, and 52.2°C, respectively. ( i ) RNA yield (bar) and dsRNA content (scatter) of the WT, M10, and M11 under different length templates. ( j ) Diagram illustrating the process from transcription to preparation of mRNA–LNPs, followed by cell transfection and characterization of expression efficiency. ( k ) The expression efficiency in HEK293T cells of mRNA encoding luciferase transcribed by the WT at 37°C and M10 at 50°C. The relative activity shown in (a) and (d) is based on the WT at 37°C. The residual activity shown in (h) is based on the maximum activity of each protein.

Article Snippet: The Cap1 capping kit and dsRNA ELISA (enzyme-linked immunosorbent assay) kit were acquired from Hzymes Biotech.

Techniques: Comparison, Mutagenesis, Selection, Circular Dichroism, Activity Assay, Incubation, Transfection, Expressing, Luciferase