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## pnas.071052198
- 标题：
- 期刊/年： ｜DOI: 10.1073/pnas.071052198｜PMCID: ｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1073_pnas.071052198.txt

**摘要（直引节选）**：Directed evolution of polymerase function by compartmentalized self-replication. PMCID: PMC31872 DOI: 10.1073/pnas.071052198 Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Proc Natl Acad Sci U S A . 2001 Mar 27;98(8):4552–4557. doi: 10.1073/pnas.071052198 Directed evolution of polymerase function by compartmentalized self-replication Farid J Ghadessy Farid J Ghadessy 1 Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom Find articles by Farid J Ghadessy 1 , Jennifer L Ong Jennifer L Ong 1 Medical Research Council Laboratory of Molecular Biology, Hills Road

**含数值句（直引）**：
- Copyright © 2001, The National Academy of Sciences PMC Copyright notice PMCID: PMC31872 PMID: 11274352 Abstract We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases.
- By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin.
- Reactions take place in individual aqueous compartments of a water-in-oil emulsion that are stable for prolonged periods at temperatures exceeding 90°C.
- Cells were harvested and washed once in 1× Taq buffer [50 mM KCl/10 mM Tris⋅HCl (pH 9.0)/0.1% Triton X-100/1.5 mM MgCl 2 ].
- After addition of the aqueous phase, stirring continued for 5 min more before thermocycling.
- For selecting thermostable mutants, emulsions were incubated at 99°C for increasing amounts of time (up to 15 min) before CSR.
- CSRs (94°C for 5 min, 20 times at 94°C for 1 min, 55°C for 1 min, and 72°C for 5 min) were carried out as above by using primers 4 (5′-CAG GAA ACA GCT ATG ACA AAA ATC TAG ATA ACG AGG GCAA-3′) and 5 (5′-GTA AAA CGA CGG CCA GTA CCA CCG AAC TGC GGG TGA CGC CAA GCG-3′).
- Briefly, 2 μl of induced cells were added to 30 μl of PCR mix and amplification of a 0.4-kb fragment was compared under standard and selection conditions (e.g., increasing amounts of heparin).
- Briefly, a 68-mer ( 18 ), biotinylated at the 5′ end, was bound to a streptavidin (SA) sensorchip and binding of polymerases was measured in 1× Taq buffer (see above) at 20°C.
- Steady-state kinetics were measured as described ( 19 ) by using the homopolymeric substrate poly(dA) 200 (Amersham Pharmacia) and an oligo(dT) 40 primer at 50°C.
- Cooperative CSRs (65°C for 10 min, 37°C for 10 min, and 15 times at 94°C for 0.25 min, 55°C for 0.5 min, and 72°C for 1.5 min) were performed as above except at an oil-to-water ratio of 3:1 by using primers 6 and 7 in 1× Taq buffer including 100 μM of each dNDP, 400 μM ATP, and 0.1 unit/μl Taq .
- Compartments had average diameters of 15 μm and proved heat-stable, with no coalescence or changes in compartment size after 20 cycles of PCR as judged by laser diffraction and light microscopy (Fig.
- 1 and 2 represent the same frame, imaged at 535 nm for GFP fluorescence ( 1 ) and in visible light to visualize bacterial cells within compartments ( 2 ).
- Two standard PCRs, differing in template size [R1 (0.9 kb) and R2 (0.3 kb)] and presence of Taq enzyme (R1, + Taq and R2, no enzyme), are amplified (25 cycles) either individually or combined.
- Bacterial cells expressing wt Taq polymerase (2.7 kb) or the less active sf Taq (1.8 kb) are mixed 1:1 before emulsification.
- coli cells (diameter 1–5 μm) fit readily into the emulsion compartments while leaving room for sufficient amounts of PCR reagents like nucleoside triphosphates and primers (Fig.
- In a trial selection, a single round of CSR was sufficient to isolate wt Taq from a 10 6 -fold excess of an inactive Taq mutant (not shown).
- The half-life of enzymatic activity at 97.5°C of wt Taq is 1.5 min and of T8 is 16.4 min.
- Heparin concentration increases stepwise by a factor of 4 starting at 1.3 units/ml.
- The activity of wt Taq is inhibited at concentrations >5.2 units/ml, whereas H15 remains active at concentrations of up to 1,350 units/ml.
- To explore the potential of CSR for the evolution of polymerases with novel properties, we used two different methods of error-prone PCR (see Materials and Methods ) to generate two repertoires (L1 and L2) of random Taq mutants with a diversity of 5 × 10 7 clones each.
- Taq clones in L1 and L2 had an average of 4–5 mutations and only 1–5% were active in PCR.
- A single round of CSR selection for polymerase activity under standard conditions increased the proportion of active clones to 81% (L1*) and 77% (L2*) (not shown).
- We first used CSR for the directed evolution of Taq variants with increased thermostability, starting from preselected libraries (L1* and L2*) and progressively increasing the duration of the initial thermal denaturation at 99°C.
- After three rounds of CSR selection, we isolated T8 (F73S, R205K, K219E, M236T, E434D, A608V), a Taq clone with an 11-fold longer half-life at 97.5°C compared with the already thermostable wt Taq enzyme (Fig.
- When preincubated at different temperatures for 20 min before PCR, T8 remained active after incubation at 99°C, whereas wt Taq lost detectable activity at 96°C (not shown).

**工艺/辅料句（直引）**：
- For purification, Taq clones were subcloned with an N-terminal hexahistidine tag by using primers 2 and 3 (5′-GGC GGC TCT AGA TAA CGA GGG CAA AAA ATG CAT CAT CAT CAC CAT CAC GGT GCC ATG GCT CTT CCT CTT TTT GAG CCC AAG GGC-3′) expressed as above, lysed as described ( 13 ), and purified by using Ni-NTA Agarose (Qiagen, Chatsworth, CA).

## pnas.0910781107
- 标题：Ultrahigh-throughput screening in drop-based microfluidics for directed evolution.
- 期刊/年：Proceedings of the National Academy of Sciences of the Unite 2010｜DOI: 10.1073/pnas.0910781107｜PMCID: PMC2840095｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1073_pnas.0910781107.txt

**摘要（直引节选）**：The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify ∼100 variants comparable in activity to the parent from an initial population of ∼10 7 . After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen ∼10 8 individual enzyme reactions in only 10 h, using < 150 μL of total reagent volume; compared to state-of-the-art robotic

**含数值句（直引）**：
- doi: 10.1073/pnas.0910781107 Ultrahigh-throughput screening in drop-based microfluidics for directed evolution Jeremy J Agresti Jeremy J Agresti a Harvard School of Engineering and Applied Sciences, Cambridge, MA 02138;
- PMC Copyright notice PMCID: PMC2840095 PMID: 20142500 This article has been corrected.
- In total, we screen ∼10 8 individual enzyme reactions in only 10 h, using < 150 μL of total reagent volume;
- compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.
- New developments exploiting microfluidic lab-on-a-chip technology are significantly improving screening capabilities, decreasing the reaction volume by roughly 1,000-fold while increasing the speed by ∼10-fold ( 12 ).
- We sort these drops at rates of thousands per second, allowing us to screen a library of 10 8 in about 10 h, using a total reagent volume of < 150 μL.
- This enables us to discover variants of HRP that are more than 10-fold faster than the parent and that approach the diffusion-limited rate.
- Compared to state-of-the-art robotic screening, this is 1,000-fold faster and uses 10-million-fold less volume of reagent, representing a cost savings estimated to be about 10-million-fold.
- We use a long and narrow channel to produce drops with > 64% aqueous volume fraction.
- The yeast are at a concentration of 1 × 10 8 cells per milliliter, which gives an average of 0.3 cells per 6-pL drop after being diluted by half by the substrate stream.
- The aqueous drops are formed at a flow-focusing junction ( 33 ) in a fluorocarbon oil at a rate of 2 kHz, and the number of cells per drop follows a Poisson distribution: ∼22% have a single cell ( 16 ) ( SI Text ).
- ( C ) The drops flow out of this device into a tube that acts as an incubation line where they incubate for ∼5 min.
- the light drops contain 1 mM fluorescein, and the dark contain 1% bromophenol blue.
- (Scale bar, 80 μm.) The drops then flow into the second microfluidic device, a high-speed drop sorter.
- The detection limit of this system is ∼1 nm Amplex Ultrared (AUR) (∼3,500 molecules in 6 pL).
- Upon induction with galactose, each cell displays on its surface ∼10,000 copies of a single mutant HRP protein (μHRP).
- To perform each round of screening, we induce the yeast to express the surface-displayed HRP, encapsulate the cells in drops, and sort drops at 2,000/s for up to 3 h, screening as many as 2 × 10 7 cells.
- The right panel shows a dot plot of the activities of the 50 unique first-generation (g1) mutants and 31 second-generation (g2) mutants.
- From the distributions of the numbers of identical sequences ( 26 ), we estimate there are a total of ∼200 unique active sequences in the starting library of 2 × 10 7 ( Fig.
- We screen as before, but sort the brightest 5–10% of the drops, which contain the enzymes with the highest catalytic activity.
- We then plate the cells, measure the activity of 184 mutants, and sequence the top 50%.
- All are more than twice as efficient as the wild type, and the best mutant is nearly seven times more efficient, with k cat / K M ∼ 2.5 × 10 7 M -1 s -1 ;
- this is approaching the maximum rate allowed by diffusion-limited encounters of ∼10 8 M -1 s -1 ( SI Text ).
- Even though the screen was performed at a substrate concentration of 100 μM, near the wild-type K M , we find that the K M increases for all mutants;
- however, this is compensated for by even larger increases in k cat , of up to 11-fold, yielding the higher catalytic efficiencies observed.
- by comparison, the microfluidic device requires only 5 h for the full screen.

**工艺/辅料句（直引）**：
- HRP libraries were grown, induced, washed twice with PBS containing 0.5% BSA and 1 mM Mannose, and then filtered through a 10 um nylon mesh syringe filter (Millipore).
- The optical density of the cells was measured, and the cells were centrifuged and resuspended at the appropriate density for loading into drops in a PBS solution containing 0.2% BSA and 1 mM mannose.

## pnas.1901979116
- 标题：Machine learning-assisted directed protein evolution with combinatorial libraries.
- 期刊/年：Proceedings of the National Academy of Sciences of the Unite 2019｜DOI: 10.1073/pnas.1901979116｜PMCID: PMC6500146｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1073_pnas.1901979116.txt

**摘要（直引节选）**：Machine learning-assisted directed protein evolution with combinatorial libraries. PMCID: PMC6500146 DOI: 10.1073/pnas.1901979116 Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Proc Natl Acad Sci U S A . 2019 Apr 12;116(18):8852–8858. doi: 10.1073/pnas.1901979116 Machine learning-assisted directed protein evolution with combinatorial libraries Zachary Wu Zachary Wu a Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125; Find articles by Zachary Wu a , S B Jennifer Kan S B Jennifer Kan a Division of Chemistry and Chemical Engineering, California Institute of 

**含数值句（直引）**：
- PMC Copyright notice PMCID: PMC6500146 PMID: 30979809 This article has been corrected.
- The approach predicted libraries enriched in functional enzymes and fixed seven mutations in two rounds of evolution to identify variants for selective catalysis with 93% and 79% ee (enantiomeric excess).
- The landscape contains a fitness maximum at 8.76, with a fitness value of 1 set for the parent sequence and 19.7% of variants at a reported value of 0.
- On this landscape, the simulated single-mutant walk (described later) reached 869 fitness peaks, 533 of which outperformed the wild-type (WT) sequence and 138 of which had fitness less than 5% of the WT fitness.
- Assuming each amino acid occurs with equal frequency and that the library has complete coverage, applying the threefold oversampling rule to obtain approximately 95% library coverage ( 18 , 19 ) results in a total of 570 variants screened ( SI Appendix , Library Coverage ).
- 2 , with the same number of variants screened, machine learning-assisted evolution reaches the global optimum fitness value in 8.2% of 600 simulations, compared with 4.9% of all starting sequences reaching the same value through a single-mutant walk and 4.0% of simulated recombination runs.
- Additionally, on this landscape, the machine-learning approach requires approximately 30% fewer variants to achieve final results similar to the single-mutant walk with this analysis.
- Carbon–silicon bond formation is a new-to-nature enzyme reaction ( 20 ), and Rma NOD with mutations Y32K and V97L catalyzes this reaction with 76% ee (enantiomeric excess) for the ( S )-enantiomer in whole-cell reactions ( SI Appendix , Table S1 ).
- We chose the parent Rma NOD (UniProt ID D0MGT2) ( 26 ) enzyme for two reasons.
- Second, although we previously engineered a cytochrome c ( Rma cyt c ) to >99% ee for the ( R )-enantiomer, WT Rma cyt c serendipitously started with 97% ee ( 20 ).
- We hypothesized that a parent enzyme with less enantiopreference [76% ee for the ( S )-enantiomer in whole cells] would be a better starting point for engineering enantiodivergent variants.
- In the first round, Rma NOD Y32K V97L (76% ee ) was used as a parent for NDT mutagenesis at the set I positions.
- From the predicted libraries for both enantiomers, two variants, called VCHV (86% ee ) and GSSG (62% ee ) for their amino acids at positions 32, 46, 56, and 97, were identified by screening 90 variants for each.
- From those predicted libraries, we obtained two variants with measured enantioselectivities of 93% and 79% ee for the ( S )- and ( R )-enantiomers, respectively.
- The improvement in total activity measured in whole cells compared with the starting variant (32K, 46F, 56L, 97L) obtained after two rounds of machine learning-assisted directed evolution is also shown in Table 2 .
- The two variants most selective for the ( S )-enantiomer differ by less than 1% in ee .
- Activity increase over the starting variant, 32K, 46F, 56L, 97L (KFLL), is shown for the final variants.
- For reference, dotted lines are shown for no enantiopreference (i.e., 0% ee ).
- However, tyrosine and arginine are also tolerated at position 49 with less than 1% loss in enantioselectivity.
- With 570 total variants ( SI Appendix , Library Coverage ), this leaves 470 variants for the input library in ( 1 ) for an equal screening burden, assuming 95% coverage of 19 mutations from WT at each position.
- Expression was performed in 96 deep-well plates in 1 mL HyperBroth (AthenaES) using Escherichia coli BL21 E.
- cloni EXPRESS (Lucigen) with 100 μg/mL ampicillin from a 20-fold dilution of overnight culture.
- Expression cultures were induced after 2.5 h of outgrowth with 0.5 mM isopropyl β-D-1-thiogalactopyranoside, and heme production was enhanced with supplementation of 1 mM 5-aminolevulinic acid.
- The relative product activity was measured by using 10 mM Me-EDA and 10 mM PhMe 2 SiH with whole cells resuspended in 400 μL nitrogen-free M9-N buffer, pH 7.4 (47.7 mM Na 2 HPO 4 , 22.0 mM KH 2 PO 4 , 8.6 mM NaCl, 2.0 mM MgSO 4 , and 0.1 mM CaCl 2 ).
- Reactions were incubated anaerobically at room temperature for 6 h before extraction into 600 μL cyclohexane.
- Enantiomeric excess was measured by running the organic solution on a Jasco 2000 series supercritical fluid chromatography system with a CHIRALCEL OD-H (4.6 mm × 25 cm) chiral column (95% CO 2 , 5% isopropanol, 3 min).

## pnas.91.22.10747
- 标题：DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution.
- 期刊/年：Proceedings of the National Academy of Sciences of the Unite 1994｜DOI: 10.1073/pnas.91.22.10747｜PMCID: PMC45099｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1073_pnas.91.22.10747.txt

**摘要（直引节选）**：DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. PMCID: PMC45099 DOI: 10.1073/pnas.91.22.10747 Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Proc Natl Acad Sci U S A . 1994 Oct 25;91(22):10747–10751. doi: 10.1073/pnas.91.22.10747 DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. W P Stemmer W P Stemmer 1 Affymax Research Institute, Palo Alto, CA 94304. Find articles by W P Stemmer 1 Author information Copyright and License information 1 Affymax Research Institute, Palo Alto, CA 94304. PMC Copyright noti

**含数值句（直引）**：
- PMC Copyright notice PMCID: PMC45099 PMID: 7938023 Abstract Computer simulations of the evolution of linear sequences have demonstrated the importance of recombination of blocks of sequence rather than point mutagenesis alone.
- Complete recombination was obtained between two markers separated by 75 bp;

## ijms27104261
- 标题：Bst DNA Polymerase: Structure, Properties and Engineering Strategies in LAMP.
- 期刊/年：International journal of molecular sciences 2026｜DOI: 10.3390/ijms27104261｜PMCID: PMC13207698｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.3390_ijms27104261.txt

**摘要（直引节选）**：Bst DNA polymerase is a biotechnologically modified thermostable enzyme from the thermophilic Gram-positive bacterium Geobacillus stearothermophilus . The unique structure of Bst DNA polymerase determines its thermal stability, ability to replace a DNA strand and specificity. The high specificity of Bst DNA polymerase ensures the efficiency, sensitivity, and high rate of loop-mediated isothermal amplification (LAMP), which is widely used in vitro biotechnology. The review reveals the structural and functional features of the enzyme, its application in LAMP and methods of improvement of thermal stability (including directed evolution, site-directed mutagenesis, fusion constructs, and chemical modifications). The terminal transferase activity and ab initio synthesis are discussed regarding problems of Bst DNA polymerase and the ways to eliminate them. The questions of introducing modified nucleotides and primers to expand the diagnostic capabilities of LAMP are also discussed. Modern advances in Bst DNA polymerase engineering pave the way for the creation of reliable, thermostable, and highly specific test systems suitable for widespread diagnostic applications.

**含数值句（直引）**：
- The essence of this method ( Figure 2 ) is to perform analysis at a constant temperature of 60–65 °C using a set of six primers targeting eight regions (F3, R3, FIP, BIP, LF, LB).
- Due to its higher activity at 65 °C compared to the original DNA polymerase I, Bst DNA polymerase has been used in LAMP since 2000 [ 13 ].
- Complete removal of the 3′-5′ exonuclease domain led to a 10-fold decrease in the catalytic activity of Klenow DNA polymerase, indicating its contribution to polymerase processivity [ 26 , 27 ].
- stearothermophilus at (( A ), PDB ID 6DSU) and the time-resolved structure of Bst DNA polymerase, 4 hr post dATP and dCTP addition (( B ), PDB ID 7K5S), where domains are highlighted according to the InterPro classification https://www.ebi.ac.uk/interpro/ (accessed on 25 October 2025): 3′-5′ exonuclease domain (red);
- The enzyme exhibits the maximum of the polymerase activity in the temperature range from 60 to 65 °C [ 21 ].
- Conducting the reaction at temperatures below 55 °C leads to a significant decrease in the synthesis rate and a sharp increase in non-specific products due to the weakening of primer annealing stringency.
- Complete and irreversible inactivation of the enzyme is observed during prolonged incubation (more than 15 min) at temperatures exceeding 80 °C.
- However, a progressive decrease in enzymatic activity is already observed at temperatures above 70 °C.
- High concentrations of salts, for example, (NH 4 ) 2 SO 4 > 50 mM, NaCl > 100 mM, have an inhibitory effect by disrupting the ionic and hydrophobic interactions that stabilize the enzyme structure.
- Its specific activity is 1.5 × 10 5 U/mg on Calf thymus DNA and 4.9 × 10 5 U/mg on Primed M13, Kcat is 191.2 (39.3) total dNTP/sec, Km DNA is 3.4 (1.6) nM, Km dNTP is 13 (5.5) μM and processivity is 111 nt.
- However, it has been found that during cDNA synthesis of more than 65 bp, the efficiency of reverse transcription using Bst DNA polymerase is significantly reduced compared to Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT) [ 40 ].
- The authors of study [ 51 ] managed to produce up to 20% of Bst DNA polymerase from the total cell mass in the E.
- The highest protein yield was obtained after 4 h of cultivation at 23 °C following induction with isopropyl-β-D-1-thiogalactopyranoside (IPTG).
- However, the overall protein yield decreased—4.38 ± 0.21 mg/L of E.
- coli culture compared to 9.77 ± 0.09 mg/L obtained using nickel resin purification.
- However, this approach requires either a thermostable reverse transcriptase capable of functioning at 60–65 °C—the optimal temperature range for LAMP, or a Bst DNA polymerase with high reverse transcriptase activity.
- For instance, the use of phosphorothioate oligonucleotides in combination with urea and single-strand binding proteins (SSBs) enabled LAMP to be performed at 40 °C with sensitivity and specificity comparable to those achieved at 65 °C [ 66 ].
- PNA-mediated LAMP has been successfully employed for detecting macrolide-resistant Treponema pallidum harboring mutations in the 23S rRNA gene [ 56 ].
- The authors examined the effect of helix–hairpin–helix (HhH) domains on the thermostability of DNA polymerases, using variants of the C-terminal domain of topoisomerase V (Topo V) from Methanopyrus kandleri (hyperthermophile, with optimal growth at 98 °C).
- The addition of the C2 domain to the C-terminus of Bst DNA polymerase increased its half-life eightfold at temperatures up to 95 °C [ 67 ].
- The first enzyme exhibited enhanced thermostability compared to native Bst DNA polymerase and withstood brief incubations at up to 92.5 °C but showed reduced LAMP efficiency.
- In contrast, the second variant demonstrated greater activity than the wild-type enzyme but was completely inactivated at 85 °C [ 68 ].
- For instance, introducing the K431E substitution and adding a DNA-binding domain (DBD) from Pyrococcus abyssi (hyperthermophilic archaeon with optimum growth temperature at 96 °C) to the N-terminus of Bst DNA polymerase increased the enzyme’s tolerance to LAMP inhibitors and its thermostability, while also doubling its catalytic activity [ 29 ].
- These modifications enabled LAMP reactions to be conducted at 73 °C, substantially increasing reaction speed [ 69 ].
- For example, Bst DNA polymerase chemically modified with mPEG-ALD (methoxy-polyethylene-glycol-aldehyde) retained up to 80% activity at 70 °C and displayed increased tolerance to LAMP inhibitors such as urea, ethanol, sodium chloride, and potassium chloride.
- These variants exhibited increased thermostability, improved strand displacement activity, and accelerated LAMP kinetics—reducing the reaction time from 40 min to 10 min.

**工艺/辅料句（直引）**：
- In contrast, the 5′→3′ exonuclease activity of Taq DNA polymerase is compatible with PCR conditions, as cyclic thermal denaturation enables repeated rounds of primer annealing and extension.
- PNAs are synthetic DNA analogs in which the natural phosphodiester backbone is replaced with a non-hydrolyzable 2-aminoethylglycine chain.
- Finally, a major source of nonspecific products is the formation of primer dimers and hairpin structures: stable, double-stranded DNA regions formed through self-annealing or cross-annealing of primers.
- It was found that the rate-limiting step in LAMP initiation is primer annealing [ 78 ].
- The enhanced stability of the enzyme under harsh conditions was attributed to conformational changes induced by PEGylation;
- The enzyme also remained stable in lyophilized form for up to two months at 50 °C and enabled high-temperature LAMP assays at 70 °C [ 71 ].