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Published in final edited form as: ACS Synth Biol. 2022 Mar 23;11(4):1488–1496. doi: 10.1021/acssynbio.1c00559 Charge Engineering Improves the Performance of Bst DNA Polymerase Fusions Inyup Paik Inyup Paik 1 Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, Austin, Texas 78712, United States; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas 78712, United States Find articles by Inyup Paik 1 , Sanchita Bhadra Sanchita Bhadra 2 Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, Austin, Texas 78712, United States; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas 78712, United States Find articles by Sanchita Bhadra 2 , Andrew D Ellington Andrew D Ellington 3 Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, Austin, Texas 78712, United States; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas 78712, United States Find articles by Andrew D Ellington 3 Author information Article notes Copyright and License information 1 Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, Austin, Texas 78712, United States; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas 78712, United States 2 Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, Austin, Texas 78712, United States; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas 78712, United States 3 Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, Austin, Texas 78712, United States; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas 78712, United States Author Contributions I.P. and S.B. designed the research, performed experiments, analyzed the data, and wrote the paper. A.D.E. designed the research, analyzed the data, and wrote the paper. ✉ Corresponding Author: Andrew D. Ellington – Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, Austin, Texas 78712, United States; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas 78712, United States; Phone: +1-512-232-3424; ellingtonlab@gmail.com ; Fax: +1-512-471-7014 Issue date 2022 Apr 15. PMC Copyright notice PMCID: PMC9514385 NIHMSID: NIHMS1836472 PMID: 35320674 The publisher's version of this article is available at ACS Synth Biol 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: supercharging, Br512, charge engineering, polymerase engineering, high-temperature LAMP, fusion domains Graphical Abstract INTRODUCTION The net and surface charges of proteins have been shown to be parameters that can dramatically alter their biophysical and kinetic properties. Initially, Lawrence, et al., among others, 1 showed that “supercharging” the surface of green fluorescent protein (GFP) with either large numbers of negatively or positively charged amino acid substitutions could greatly stabilize the protein to denaturation. 2 Since then, a variety of proteins have been supercharged, with similar results. This includes enzymes such as glutathione-S-transferase and human enteropeptidase, both of which exhibited improved thermostability and solubility after surface supercharging. 2 , 3 Beyond simple globular proteins, proteins with more complex structures such as antibodies have also displayed enhanced thermotolerance as well as antigen-binding affinity, 4 reduced aggregate formation, 5 and improved thermal stability across a wide pH range 6 following supercharging. One enzyme class that might greatly benefit from supercharging is polymerases, given that they routinely interact with “supercharged” (uniformly negatively charged) substrates. Indeed, it has previously been found that the entire domains can be added to polymerases to alter their nucleic acid-binding properties and hence their kinetics and performance in assays. For example, Sso7D 7 is a small (7 kDa), positively charged DNA-binding protein from the hyperthermophilic archaea Sulfolobus solfataricus , 8 and when used as a fusion partner for the otherwise low processive polymerase Pfu , it has been shown to greatly improve processivity and the speed, resulting in the widely used Phusion polymerase. 7 Sso7D is in fact a naturally supercharged protein where 14 out of 63 amino acids are lysine residues, 8 allowing both Sso7D and other members of the related Sul7d family to directly bind to the minor groove of the DNA. 9 More generally, multiple proteins with nucleic acid-binding properties are enriched with charged amino acids on their surfaces. 10 To improve the performance of the well-known Bst DNA polymerase (Bst DNAP), which shows robust strand-displacing activity and is widely used in diagnostic methods including loop-mediated isothermal amplification (LAMP), 11 – 14 we have attempted to develop a more general strategy for supercharging polymerases by engineering a fusion domain that already has a skewed charge distribution that alters interactions with nucleic acid templates. MATERIALS AND METHODS Reverse Transcription Loop-Mediated Isothermal Amplification Oligonucleotide Strand Displacement (RT-LAMP-OSD) Assays. Individual 25 μ L NB RT-LAMP-OSD assays were assembled in glucose-6-phosphate dehydrogenase (G6D) buffer (60 mM Tris–HCl, pH 8.0, 2 mm (NH 4 ) 2 SO 4 , 40 mM KCl, 8 mM MgCl 2 ) containing 20 pmol of Br512 and its variants. The buffer was supplemented with 1.4 mM dNTPs, 0.4 M betaine, and 2.4 μ M each of forward inner primer (FIP) and backward inner primer (BIP), 1.2 μ M of indicated loop primers, and 0.6 μ M each of F3 and B3 primers. Amplicon accumulation was measured by adding OSD probes. First, NB OSD probes were prepared by annealing 1 μ M of the fluorophore-labeled OSD strand with 5 μ M of the quencher-labeled strand in 1× isothermal buffer (NEB). Annealing was performed by denaturing the oligonucleotide mix at 95 °C for 1 min followed by slow cooling at a rate of 0.1 °C/s to 25 °C. Excess annealed probes were stored at −20 °C. Annealed NB OSD probes were added to RT-LAMP reactions at a final concentration of 100 nM of the fluorophore-bearing strand. The assays were seeded with indicated amounts of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene armored RNA (Asuragen, Austin, TX) in Tris–ethylenediaminetetraacetic acid (EDTA) (TE) buffer (10 mM Tris–HCl, pH 7.5, 0.1 mM EDTA) and transferred to a 96-well plate and incubated in the LightCycler 96 real-time polymerase chain reaction (PCR) machine programmed to maintain the reactions at 65 °C for 90 min followed by 21 min at 37 °C. OSD fluorescence in assays was measured every 3 min in the FAM channel for observation of both real-time amplification kinetics (at 65 °C) and endpoint signal versus noise (at 37 °C). All data were analyzed using LightCycler 96 software. Generation of Surface Charge Map. Surface charge maps of villin headpiece domain (vHP47) (PDB: 1YU5) were prepared by a Pymol built-in adaptive Poisson–Boltzmann solver (APBS) electrostatics plugin with the following parameters (method: pdb2pqr, calculate map with APBS: grid spacing 0.50, molecular surface visualization: range ±5). Supercharged versions of the surface maps were generated by the same method after editing the targeted amino acids in the 1YU5. Br512 and Enzyme Variant Purification Protocol. Br512 and its variant enzymes were prepared as described previously. Briefly, Br512 was cloned into an in-house made expression vector pKAR2 with a golden gate assembly with a 5′ flanking sequence TATG and 3′ ATCC (see Addgene ID: 161875 for details). The pKAR2–Br512 (Addgene ID: 161875) and its variants expressing plasmids were heat-shock-transformed into Escherichia coli BL21(DE3) (NEB, C2527H). The entire heat-shock-recovered cells were seed-cultured overnight in 5 mL of superior broth (Athena Enzyme Systems, 0105) in the presence of 100 μ M carbenicillin. The next day, 1 mL of seed culture was inoculated into 1 L of superior broth supplemented with 100 μ M carbenicillin and grown at 37 °C until it reached the late log phase with an OD 600 of 0.5–0.9. Protein expression was induced by adding 1 mM isopropyl β - d -1-thiogalactopyranoside (IPTG) and 100 ng/mL of anhydrous tetracycline (aTc) at 18 °C overnight. The next day, cells were collected by centrifuge at 5000g for 10 min at 4 °C. The pellet was resuspended in 30 mL of an ice-cold lysis buffer (50 mM phosphate buffer, pH 7.5, 300 mM NaCl, 20 mM imidazole, 0.1% Igepal CO-630, 5 mM MgSO 4 , 1× EDTA-free protease inhibitor tablet; Thermo Scientific; A32965). The cells were sonicated (1 s ON, 4 s OFF) with a sonifier for a total time of 4 min with 40% amplitude. The lysate was centrifuged at 35,000g for 30 min at 4 °C. The supernatant was poured into a 50 mL falcon tube and filtered through a 0.2 μ m syringe filter. All Br512 variants contain an N-terminal 8x His tag. Thus, we utilized metal affinity chromatography, a nickel-nitrilotri-acetic acid (Ni-NTA) column, for the first round of purification. In short, 1 mL of Ni-NTA agarose slurry was packed into a 10 mL disposable gravity column, and the column was equilibrated with 20 column volume (CV; 1 mL) of equilibration buffer (50 mM phosphate buffer, pH 7.5, 300 mM NaCl, 20 mM imidazole). The sample lysate was loaded, and gravity flowed through the column. The column was then washed with 20 CV of equilibration buffer and 5 CV of wash buffer (50 mM phosphate buffer, pH 7.5, 300 mM NaCl, 50 mM imidazole). Br512 and its variants were eluted with 5 mL of elution buffer (50 mM phosphate buffer, pH 7.5, 300 mM NaCl, 250 mM imidazole). The eluate protein was dialyzed twice with 2 L of Ni-NTA dialysis buffer (40 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT), 0.1% Igepal CO-630). The eluate was further purified by 2 × 5 mL heparin column (HiTrap Heparin HP) on an FPLC (AKTA pure, GE healthcare) and eluted using a linear NaCl gradient generated from heparin buffers A and B (40 mM Tris–HCl, pH 7.5, 100 mM NaCl for buffer A; 2 M NaCl for buffer B, 0.1% Igepal CO-630). The collected final eluate was dialyzed with 2 L of heparin dialysis buffer (50 mM Tris–HCl, pH 8.0, 50 mM KCl, 0.1% Tween-20) and then with 2 L of the final dialysis buffer (50% glycerol, 50 mM Tris–HCl, pH 8.0, 50 mM KCl, 0.1% Tween-20, 0.1% Igepal CO-630, 1 mM DTT). At this stage, we usually obtain ~1 mL of Br512 variants with varying concentrations. The final concentrations of the purified polymerases were determined by the Bradford assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel staining with bovine serum albumin (BSA) as a standard. Site-Directed Mutagenesis. Site-directed mutagenesis was performed with the Q5 Site-Directed Mutagenesis Kit (NEB; E0554S) according to the manufacturer’s instructions. The pKAR2–Br512 (Addgene ID: 161875) and other variant plasmids were used as a template to generate single-, double-, and high-order mutations determined by supercharging analysis. All primer sequences used in this study are shown in Supporting Information Table 1 . All plasmid sequences were verified by Sanger sequencing. Heat Challenge and High-Temperature LAMP. Heat challenge LAMP assays were prepared in total 25 μ L volume reactions using 6 × 10 7 copies of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA templates with a final concentration of 1.6 μ M each of BIP and FIP primers, 0.4 μ M each of B3 and F3 primers, and 0.8 μ M of loop primer. A total of 20 pmol of supercharged and g3 enzyme variants were added to each well. Amplification was performed in a 1× LAMP heat challenge buffer (40 mM Tris–HCl, pH 8.0, 10 mM (NH 4 ) 2 SO 4 , 80 mM KCl, 4 mM MgCl 2 ) supplemented with 0.4 mM dNTP, 1× EvaGreen dye, and 0.4 M betaine, all final concentrations. For heat challenge assays, 8-well PCR tube strips that contain the entire reaction mixtures (with 20 pmol of Br512 enzyme variants) were heated on a preheated PCR machine (75 and 80 °C). The exact time on the heat block was measured by a stopwatch, and the tubes were immediately removed from the PCR machine and cooled on an ice-cooled metal rack at least for another 5 min. The subsequent LAMP amplifications were performed at 65 °C for 2 h. For high-temperature LAMP assays, the same LAMP reaction mixtures were prepared and incubated at various high temperatures (72, 73, and 74 °C) indicated in the figures during the entire process. Dye-Based Protein Thermal Shift Assay. The transition midpoint ( T m , melting temperature) measurement of the Br512 variants was performed using protein thermal shift reagents (Thermo Fisher; catalog number: 4461146) according to the manufacturer’s instruction. A total of 10 μ g (5 μ g/ μ L) of each Br512 variant in the final dialysis buffer (50% glycerol, 50 mM Tris–HCl, pH 8.0, 50 mM KCl, 0.1% Tween-20, 0.1% Igepal CO-630, 1 mM DTT) were added into a reaction mixture (20 μ L) containing a 1× protein thermal shift buffer and a 1× protein thermal shift dye. Changes in fluorescence signals were captured by the Texas Red channel provided by LightCycler 96 software during a shift of temperature from 37 to 95 °C with a 0.1 °C/s ramp speed. The recorded values (delta fluorescence/delta temperature) were plotted on a graph, and the peaks of Δ F /Δ T were drawn manually. Electrophoretic Mobility Shift Assay (EMSA). For the EMSA, the polymerases were prepared as detailed in the Br512 and Enzyme Variant Purification Protocol section in this method. Probes were ordered as forward and reverse primers (see Supporting Information Table 1 for the sequences), annealed by heating at 95 °C for 1 min before cooling down to room temperature on the PCR machine. A total of 10 pmol of double-stranded DNA probes were incubated with indicated amounts of polymerases for 20 min at room temperature in binding buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 10 mM MgCl 2 , 0.1 mM EDTA, 1 mM DTT, 5% glycerol, 100 ng/ μ L bovine serum albumin, 0.1% Igepal CO-630) in 10 μ L of the reaction volume. Then, 2 μ L of a 5× EMSA loading dye (a component in the electrophoretic mobility shift assay (EMSA) kit, with SYBR Green & SYPRO Ruby EMSA stains, Thermo, Cat: E33075) was added to the reaction. The complexes were separated on a 6% DNA retardation gel (Thermo, Cat: EC6365BOX) with 125 V in a cold room to prevent heat dissociation of the complexes. An hour later, the gel was incubated with a 1× SBYR green gel staining dye (a component in the electrophoretic mobility shift assay (EMSA) kit, with SYBR Green & SYPRO Ruby EMSA stains, Thermo, Cat: E33075) for 10 min, visualized on a Bio-rad gel doc machine. RESULTS AND DISCUSSION Designing Fusion Domains with Altered Charges. We had previously generated an improved variant of the well-known strand-displacing DNA polymerase I from Bacillus stearothermophilus 15 (now classified as a Geobacillus 16 ) in which the core enzyme was augmented with an amino terminal fusion domain containing the carboxyl terminal 47 amino acids of the actin-binding protein villin from chicken ( Gallus gallus ) (HP47; Figure 1 ). It had previously been shown that this domain was fast-folding 17 and could potentially improve protein stability and solubility. 18 The augmented polymerase with the HP47 fusion domain was termed Br512 and was indeed stabilized and showed higher activity. Figure 1. Open in a new tab 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 . We further speculated that these improved properties might be due, in part, to the charged surfaces present on the villin headpiece. An examination of its crystal structure (PDB: 1YU5) and a surface charge map generated using the adaptive Poisson-–Boltzmann solver (APBS) algorithm in Pymol ( Figure 1b ) revealed a large cluster of positively charged amino acids on one side of the domain, opposite a smaller cluster of negatively charged amino acids. In this regard, we anticipated that the villin headpiece might be acting as an “anchor” for the polymerase, similar to fusions with other DNA-binding domains (such as Sso7d), that have proved successful in generating enzymes (such as the commercially used Phusion 7 ) with increased processivity and resistance to PCR inhibitors. The positively charged surface might be improving the overall DNA binding, while the negatively charged surface would help with orientation during binding. 19 , 20 We hypothesized that further changes to the charge distribution on HP47 might lead to additional improvements in the functionality of Br512, just as supercharging of other proteins has proven to improve their biophysical and other properties. 2 , 4 , 21 , 22 In particular, to further enhance the “Janus” nature of this domain presenting two oppositely charged surfaces, we initially wished to add positive charges to the already positively charged face and negative charges to the opposing face. We sought to avoid amino acids that were conserved among HP47 homologues ( Figure S1 ) to avoid compromising structural integrity. For example, while HP47 homologues overall have highly conserved sequences, showing only a few phylogenetic variations, the N40 position is asparagine in G. gallus but is lysine in roughly half the homologues ( Figure S1 ). We ultimately selected the eight positions depicted in Figure 1a for the charge engineering, including four substitutions (SC1–4) that can add negative charges to the negatively charged surface and four substitutions (SC5–8) that can add positive charges to the positively charged surface ( Figure 1b ). Thermal Challenge Assays. We initially examined the effect of all eight individual charge substitutions (SC1–8) on the Br512 enzymatic activity via thermal challenge and subsequent LAMP reactions with GAPDH DNA templates ( Figure S2 ), as we believed this assay would provide the best grounding for subsequent diagnostics development. We initially scanned several temperatures and times and chose to perform thermal challenge assays between 75 °C for 3 min and 82 °C for 30 s because these showed the greatest discrimination between activities. While the four, individual, negatively charged substitutions exhibited no enhanced activities, the four positively charged substitutions (SC5, SC6, SC7, SC8) all showed enhanced activities with heat challenges ( Figure S2 ). To examine the combined effect of the individual substitutions, we generated various double and triple combinations ( Figure S3 ). The higher-order, positively charged substitutions showed roughly additive improvements to heat challenges, suggesting that the increased surface charge imparts either greater overall activity or stability to vHP47 ( Figures 1c , S3 , and S4 ). The two best variants (SC5,6,7,8 and SC6,7,8) showed robust target amplification starting as early as 18 min after a heat challenge at 75° C for 3 min and at 33 min after a heat challenge at 80 °C for 0.5 min. Both variants were able to amplify targets at least half an hour earlier than was the case for the wild-type enzyme (Br512) at similar temperatures ( Figures 1c and S4 ). Combining Charge Substitutions with Other Stabilizing Mutations. We had previously built a convolutional neural network (CNN) that was trained by ~1.6 M microenvironments sampled from ~19k protein structures across the Protein Data Bank. 23 This algorithm assessed the “fit” of individual amino acids at every position in a protein structure to their local chemical microenvironments. We have previously shown that substitutions identified via the CNN can enhance the Br512 activity in response to thermal challenges. 24 We hypothesized that the “surface” variants on the HP47 fusion domain might complement the “buried” variants on the polymerase domain, leading to additive stabilization. Using a stabilized, triple mutant version of the polymerase domain of Br512 (Mut235; T493N, A552G, S371D; PDB ID: 3TAN) as a starting point, we added the best, combined supercharged mutations (SC678 and SC5678) discovered via our charge engineering efforts, generating Mut235-SC678 (renamed as Br512g3.1) and Mut235-SC5678 (renamed as Br512g3.2; Figure 2 ). Figure 2. Open in a new tab 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. LAMP assays were again performed with these two combined variants, but rather than looking at the thermal challenge, we further quantitated improvements by looking at the amplification threshold cycle ( C t ), which indicates the time to a significant signal above background. The combined variants had C t values of 10.8 min, approximately 5 min faster than the parental Br512 wt ( C t of 15.6 min) ( Figure 2a ). This is significant given the need for extremely rapid point-of-care (POC) diagnostic assays for consumer and public health applications. Not only were the combined variants faster but the combined variants again showed additive and quantitative improvements in the face of heat challenges ( Figure 2b – d ). For instance, the combined variants were faster following a thermal challenge at 75 °C for 3 min ( C t 10.1 min) than either of the parental mutations (13 and 16.2 min for Mut235 and SC678, respectively) and at least 24 min faster than the parental Br512 enzyme ( Figure 2b ). After a heat challenge at 80 °C for 30 s, the combined variants again showed improvements relative to either the parental mutations (Mut235, SC678, SC5678) or to the parental enzyme ( Figure 2c ). Surprisingly, the combined variants Br512g3.1 and Br512g3.2 remained largely active up to 82 °C ( Figure 2d ), a temperature that slowed or completely inactivated the various parental enzymes. The lack of activity of the parental enzymes in the LAMP assay may indicate complete heat denaturation of the enzymes under the temperature tested. 24 While the increased thermostability of the jointly engineered variants was strongly indicated by the thermal challenge assays, we also used a dye-based protein thermal shift assay (TSA) to determine the melting temperatures of the proteins ( Figure S5 ). In the thermal shift assay, an environmentally sensitive fluorescence dye binds to the exposed hydrophobic regions of heat-denatured proteins, resulting in an increased fluorescence emission. The parental fusion construct Br512 showed a slightly higher T m value (76.1 °C) compared to the wild-type Bst-LF (75.8 °C), but the two best-combined variants (Br512g3.1 and Br512g3.2) showed greatly improved T m values: 78.3 and 78.0 °C, respectively. Stabilized Variants Enable High-Temperature Ultra-fast Isothermal Amplification. LAMP and other isothermal amplification methods rely on a relatively small number of DNA polymerase with unique strand displacement properties. 11 , 25 , 26 The overall speed of LAMP reactions is generally limited by the formation of initiating hairpin loops and polymerization through increasingly longer concatemers. The fact that we have now engineered enzymes with greater thermal tolerance should also allow us to carry out the entire amplification cycle at higher temperatures, which might in and of itself increase the likelihood of hairpin loop formation and strand separation and therefore lead to more efficient and faster reactions. To identify optimal temperatures for the new, stabilized enzymes, where thermal dissociation and strand displacement could best work together in isothermal amplification reactions, we carried out LAMP reactions at several different temperatures ( Figure S6 ). In general, the speed of LAMP detection was facilitated by higher temperatures: the times to detection (time to reach C t ) for Br512 and g3.1 were ~10 and ~6 min at 72 °C, respectively, which are roughly 5 minutes faster than at 65 °C ( Figures 2a and S6 ). While the parental enzyme Br512 could only carry out LAMP at temperatures up to 72 °C, the optimized variants were fully capable of LAMP at temperatures as high as 74 °C. Indeed, we found that the 74 °C LAMP reaction with Br512g3.1 and g3.2 could produce a readily detected signal as soon as 6 minutes with 6 × 10 7 copies of template DNA ( Figure 3a , b ). While these engineered enzymes showed robust amplification at higher temperatures, they could also generate spurious amplicons ( Figures 3c , S7 , and S8 ). For example, while an easily detectable true amplicon was generated with the g3 polymerases at 74 °C LAMP assays ( Figure 3a ) within 10 min, a spurious amplicon was detected at around 20 min ( Figure 3c ). Such background amplification has also been observed with other engineered LAMP polymerases, such as Bst 3.0. 27 Therefore, similar to the widespread use of TaqManTM probes in gold-standard diagnostic qPCR assays, we consistently recommend 28 – 31 that LAMP assays should be read with oligonucleotide strand displacement (OSD) probes ( Figure 5 ), especially when designing clinically relevant assays. Figure 3. Open in a new tab 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 × 10 7 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 5. Open in a new tab 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. Stabilization Strategies Also Lead to Chaotrope Resistance. As previous efforts to engineer polymerases often led to a resistance to inhibitors, we examined activities of the stabilized variants in the presence of a chaotrope, urea. We found that the g3.1 and g3.2 variants were resistant up to 2 M urea concentrations (final) in the GAPDH LAMP assay, whereas the parental Br512 showed delayed amplification in the presence of 1 M urea and no amplification in 2 M urea concentration ( Figure 4 ). Interestingly, we observed exponential amplifications starting at ~10 min with g3.1 and g3.2 variants even in the presence of 1 M urea ( Figure 4b ), which is about 4 min faster than LAMP performed without chaotropes ( Figure 4a ). This is consistent with the fact that temperature can improve strand separation, and hence the speed of LAMP, and with previous data that show urea can increase the sensitivity of LAMP. 32 Moreover, this advance opens the way to the further development of LAMP assays without sample preparation, 33 with the possibility of carrying out assays directly from samples that have been inactivated in viral transport media, 34 which contains substantial concentrations of guanidinium. 35 Figure 4. Open in a new tab 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. Charge Engineering Alters Polymerase:DNA Interactions. We have hypothesized that the enhanced activity and thermostability of our polymerases were due to elaborations of our initial suggestion that HP47 anchors the polymerase to its nucleic acid substrate. To demonstrate this more directly, we carried out an electrophoretic mobility shift assay (EMSA) based on the wild-type and engineered variants ( Figure S9 ). While the parental Bst-LF polymerase exhibited a weak bandshift with a double-stranded probe (70 bp, based on the GAPDH amplicon), Br512 and the supercharged variant (SC567) showed most robust bandshifts, suggesting a stabilized interaction between the probes and the polymerases. To better differentiate DNA-binding affinities of Br512 and SC567, we performed an EMSA with a range of amounts of polymerase concentrations ( Figure S9b ). As anticipated, the charged variants (SC567) showed stronger bandshifts than Br512 at lower concentrations and revealed additional bands at higher polymerase concentrations, likely representing DNA–protein complexes of different stoichiometries. 36 Thus, it is safe to conclude that charge engineering has led to better interactions between the polymerase and its substrate, including the possibility of multiple binding events in an ever-diversifying concatemeric LAMP amplicon, which presents multiple nodes for strand displacement polymerization, including continuously created foldback priming structures that are created upon the binding of new inner and loop primers to amplicon loop sequences. 26 , 37 Modulating Charge Impacts Relative Polymerase Activities. While our efforts to engineer Br512 resulted in greatly improved stabilities and activities, we unexpectedly noticed that the inherent reverse transcription activity of Bst-LF was reduced in the engineered enzymes ( Figure 5 ). While the parental Br512 still showed considerable reverse transcription activity in NB RT-LAMP-OSD assays ( Figure 5a ) and could detect as low as 500 copies of SARS-Cov-2 RNA templates, the g3.1 variant showed reduced or sometimes no reverse transcription activity ( Figure 5d ). To dissect the underlying cause of RT inhibition, we also carried out RT-LAMP-OSD assays using either only the charge-engineered variant (g2.1; SC6,7,8) or only the machine learning-engineered variant (g2.2; Mut2,3,5) ( Figure 5b , c ). While the machine learning-engineered variant still exhibited robust reverse transcription activity ( Figure 5c ), the charge-engineered variant (g2.1) showed reduced reverse transcription activity ( Figure 5b ). Since our charge engineering effort added positive charges only to one face of the Janus-like HP47 ( Figure 1b ), we speculated that this may have changed interaction kinetics between the HP47 and nucleic acids in a way that reduced the reverse transcription activity. Thus, in an attempt to restore RT activity while retaining higher stability and activity, we attempted to charge-balance the HP47 surface by adding additional, negative substitutions on the opposite face. While the four, previously identified negative substitutions did not show any altered reverse transcription activities compared to their parental Br512 enzyme ( Figure S10a – d ), when SC4 was introduced to the combined variant g3.1, it largely restored the reverse transcription activity ( Figure 5e ). An additional negative charge (SC4) also did not alter the thermostability ( Figure S11 ) or inhibitor resistance of the g3.1 enzyme ( Figures S12 and S15 ). These results suggest that the charge balance of the anchor domain is a further parameter that can be modulated to fine-tune the activities of polymerases. To better demonstrate the practical application of the thermostable engineered enzymes, we have additionally performed a Mycobacterium tuberculosis-specific real-time rpoB LAMP-OSD assay at a high temperature (74 °C) ( Figure S13 ). In this assay, we observed the robust amplification of 6000 copies of rpoB DNA by g3.1 and g3.1+SC4 variants within 8 and 5 min, respectively, while other enzyme variants were completely inactive at the higher temperature. This result suggests an immediate use of the engineered enzymes in the detection of clinically relevant DNA targets. CONCLUSIONS Supercharging has previously been used for the stabilization of a variety of proteins (1, 3, 5, 17, and 27). By extending these findings to a fusion domain (HP47) that has also shown a great value on its own for enhancing protein folding and stabilization, we potentially provide a new array of supercharged domains that can be added to polymerase and other protein classes for enhancing their functions. Moreover, we show for the first time that charge engineering of a protein can be carried out to not only stabilize the protein but also modulate its overall function. The anchor provided by the positively supercharged HP47 domain led to greatly improved binding and performance of Bst-LF in LAMP assays that could be carried out at higher temperatures and in the presence of chaotropes, yielding shorter times-to-answer. By balancing the positively charged residues with additional negative residues, we were also ultimately able to better “release” the anchor and reduce inhibition during reverse transcription. We have established a multimodal protein engineering pipeline that has the potential to be adapted for engineering other polymerases, nucleic acid-modifying enzymes, and binding proteins. The demonstration that charge engineering can simultaneously improve thermostability and nucleic acid-binding affinity of the fusion domain could also lead to the further engineering of extant fusion polymerases and other enzymes. From a practical point of view, the inhibitor-resistant polymerases could be used for direct nucleic detection from clinical samples such as saliva and urine, as well as sample collection matrices, such as viral transport media (VTM). As shown in Figure S14 , engineered variants could readily detect the femtomolar-to-attomolar range of SARS-CoV-2 virions in LAMP assays comprising as much as 20% (v/v) VTM. Amplification assays at high temperatures might also enable heat inactivation of pathogens and of inhibitors, such as nucleases, in clinical samples, leading to direct detection from sample collection matrices, such as viral transport medium or blood ( Figure S14 ). The ability to perform LAMP assays at a high temperature can be useful from several points of view. First, we have observed that the time to signal is usually accelerated in high-temperature LAMP, likely due to enhanced strand displacement activity at higher temperatures. For instance, with our g3 polymerases, we could detect the same amounts of target analytes of ~5 min faster in 74 °C LAMP compared to those in 65 °C LAMP ( Figure 2a ). This faster detection, which, we believe, can be further improved by combining the engineered enzymes with temperature-optimized primers and buffer additives, is extremely valuable for the adoption of LAMP assays in point-of-care diagnostics where speedier diagnostic results are critical. Second, the ability to operate LAMP at higher temperatures may facilitate direct sample analysis, thus obviating sample processing by favoring heat lysis of target analytes and inactivation of inhibitors. This diagnostic procedure minimization would not only make testing user-friendly and less infrastructure-dependent but also reduce the generation of biohazardous waste. Supplementary Material Supplementary Material NIHMS1836472-supplement-Supplementary_Material.pdf (2.7MB, pdf) ACKNOWLEDGMENTS This work was supported by grants from the National Science Foundation (2027169), the National Institutes of Health (1R01EB027202-01A1, 3R01EB027202-01A1S1), the Welch Foundation (F-1654), and the National Aeronautics and Space Administration (NNX15AF46G). Footnotes Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.1c00559 . vHP47 and its orthologue sequences; heat challenge results for various supercharged versions of Br512 variants; T m measurement for Br512 variants; high-temperature GAPDH LAMP assay results; nontemplate control results; EMSA results showing polymerase–nucleic acid interactions; inhibitor resistance LAMP; high-temperature LAMP assay results; and primers used in this study ( PDF ) Accession Codes Bst DNA polymerase I (Uniprot id: Q45458 , PDB id:3TAN), Villin-1 (Uniprot id: P02640 ). Complete contact information is available at: https://pubs.acs.org/10.1021/acssynbio.1c00559 The authors declare the following competing financial interest(s): IP, SB, and AE are named inventors on an IP filing relating to methods and compositions described in this manuscript. 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