Charge Engineering Improves the Performance of Bst DNA Polymerase Fusions

Abstract

The charge states of proteins can greatly influence their stabilities and interactions with substrates, and the addition of multiple charges (supercharging) has been shown to be a successful approach for engineering protein stability and function. The addition of a fast-folding fusion domain to the Bacillus stearothermophilus DNA polymerase improved its functionality in isothermal amplification assays, and further charge engineering of this domain has increased both protein stability and diagnostics performance. When combined with mutations that stabilize the core of the protein, the charge-engineered fusion domain leads to the ability to carry out loop-mediated isothermal amplification (LAMP) at temperatures up to 74° C or in the presence of high concentrations of urea, with detection times under 10 min. Adding both positive and negative charges to the fusion domain led to changes in the relative reverse transcriptase and DNA polymerase activities of the polymerase. Overall, the development of a modular fusion domain whose charged surface can be modified at will should prove to be of use in the engineering of other polymerases and, in general, may prove useful for protein stabilization.

Keywords: Br512; charge engineering; fusion domains; high-temperature LAMP; polymerase engineering; supercharging.

Figure 1.

Supercharging the villin headpiece domain (vHP47) imparts thermostability on Br512. (a) The villin headpiece (vHP47) amino acid sequence and its corresponding supercharging mutations. Neutral, negatively charged, and positively charged amino acids are depicted by green, red, and blue letter designations, respectively. (b) Surface charge models of the wild-type (wt) vHP47 domain and its supercharged variants generated, as described in Figure 1. A total of eight amino acids of vHP47 designated as SC1–8 were mutated into either negatively (SC1,2,3,4) (aspartate D/glutamate E) or positively charged amino acids (SC5,6,7,8) (lysine; K/arginine; R). (c) Effect of thermal challenge on triple and quadruple positively supercharged mutants of Br512. Identical GAPDH LAMP assays assembled using the same amount of indicated enzymes were subjected to either no heat challenge (top panel), 3 min at 75 °C (middle panel), or 30 s at 80 °C (bottom panel) prior to the real-time measurement of GAPDH DNA amplification kinetics at 65 °C. Three independent GAPDH LAMP assays were performed and showed similar results in thermostability. Representative amplification curves generated by measuring increases in EvaGreen dye fluorescence (Y-axis) over time (X-axis; time in hh:mm) are depicted as blue (Br512 wild type), burnt orange (SC5,6,7,8), gray (SC6,7,8), and yellow (SC5,7,8) traces. The effects of various single, double, and triple mutations are shown in Supporting Information Figures S2 and S3.

Figure 2.

Effect of combining MutCompute and supercharging mutations on Br512 thermal stabilities. Identical GAPDH LAMP assays assembled using either wild-type (wt), supercharged-villin headpiece (SC), MutCompute (Mut), or combined SC+Mut Br512 variants were subjected to either no heat challenge (a), 3 min at 75 °C (b), 30 s at 80 °C (c), or 30 s at 82 °C (d) prior to real-time measurement of GAPDH DNA amplification kinetics at 65 °C. Threshold cycle (min; time to detection) values for the amplification of 20 pg (6 × 107 copies) GAPDH DNA templates were calculated using LightCycler 96 software and plotted as bar graphs (blue: Br512 wild type; burnt orange: Mut235; gray: SC678; black: SC678+Mut235; green: SC5678+Mut235). Mut235, T493N, A552G, S371D; PDB ID: 3TAN. The time to reach the threshold cycles (mins; time to detection) is shown as bar graphs in minutes. The standard deviation in Ct values calculated from three replicate experiments is depicted as error bars. N/D, amplification not detected. Student’s t-test has been performed between samples and p values *p < 0.05 and **p < 0.005 are depicted in the graphs.

Figure 3.

Generation-3 combined variants can perform high-temperature LAMP at 74 °C. (a, b) Identical GAPDH LAMP assays were assembled using the same amounts of either wild-type (blue) or mutant Br512 variants (burnt orange: Mut235; gray: SC678; yellow: SC5678; black: SC678+Mut235 (Br512g3.1); green: SC5678+Mut235 (Br512g3.2)) and incubated at 74 °C for up to 2 h. Amplification kinetics of GAPDH DNA templates were determined by the real-time measurement of EvaGreen dye fluorescence, and the threshold cycles (min; time to detection) for amplification of 20 pg (6 × 10⁷ copies) GAPDH DNA templates were calculated using LightCycler 96 software. Representative changes in fluorescence intensities (Y-axis) over time are depicted as amplification curves (a), while average C_t values from three replicate experiments are plotted as bar graphs (panel b: N/D, amplification not detected; error bar = S.D.; n = 3). (c) Amplification curves for a nontemplate control (NTC). Identical GAPDH LAMP assays were performed at 74 °C for up to 2 h.

Figure 4.

g3 variants exhibit chaotrope resistance in LAMP assay. (a–c) Various concentrations of urea (0–2 M, final) were added to the GAPDH DNA LAMP assay. Three graphs show amplification curves in the presence of the indicated final concentration of urea in the LAMP reaction (Y-axis: fluorescence intensity; X-axis: time of incubation): blue (Br512 wt), burnt orange (SC678+Mut235, g3.1), and gray (SC5678+Mut235, g3.2) traces are shown.

Figure 5.

Comparison of variants in RT-LAMP-OSD assays. Performance of 20 pm of g2.1 ((b) positively supercharged variant), g2.2 ((c) machine learning variant), g3.1 ((d) positively supercharged + machine learning variant), and g3.1+SC4 ((e) g3.1 + negatively supercharging mutation) was compared to the activity of unmodified “parental” Br512 (a) by amplifying SARS-CoV-2 N gene armored RNA templates using the NB RT-LAMP-OSD assay. Note fluorescence curves spiking up during 37 °C post amplification readouts indicate true positive OSD signals. OSD fluorescence values measured during (65 °C) and post (37 °C) amplification are depicted as black (5000 template copies/reaction), red (500 template copies/reaction), blue (50 template copies/reaction), and gray (0 template copies/reaction) traces. Results representative of two biological replicates are depicted.