About T4 DNA Ligase
Fig 1 Schematic Diagram of the Reaction Principle of T4 DNA Ligase[1]
However, T4 DNA Ligase still faces some challenges in practical applications, including poor stability, high adapter-adapter ligation, or low DNA library yield. These issues may lead to reduced experimental efficiency, decreased data quality, poor repeatability of experimental results, and low product purity, thereby affecting the overall effectiveness of molecular biology experiments and the accuracy of sequencing data. To address these problems, Yeasen, based on its subsidiary Molefuture's innovative ZymeEditor™ enzyme evolution platform has carried out enzyme evolution on the T4 DNA ligase.
Iterative wet and dry experimental screening to obtain high-performance T4 DNA Ligase
T4 DNA ligase, an ATP-dependent ligase enzyme, comprises a compact-helical DNA-binding domain (DBD), a nucleotidyl-transferase (NTase) domain, and an OB-fold domain. Based on the protein structure, Yeasen's team analyzed the residues spanning distances of 5 to 20Å around the DNA substrate, followed by in silico screening and structure-function analysis using advanced computational tools. During this process, the team observed a flexible loop situated distant from the active pocket. Guided by energy calculations, the team designed truncations in this loop region, hypothesizing that it might not significantly impact ligation function but could affect overall structural stability. Furthermore, the team aligned consensus sequences to design mutants, coupling this analysis with structural-functional considerations, as illustrated in the figure below.
Besides rational design, the directed evolution of T4 DNA ligase was conducted with the MTPS method by screening the stability, activity, and residual activity. And a library of over 104 mutants was screened. Following a thorough analysis of mutation data, promising candidates were selected for purification and rigorous testing. Then all the winners obtained by rational design and directed evolution were underwent detailed characterization after refined purification, encompassing assessments of activity, stability, adapter self-ligation, DNA fragment self-ligation, and DNA yield. Based on G1 mutants, the team conducted DNA shuffling and combined directed mutation design to get exceptional mutants that cater to diverse application requirements.
From Mutant to Market Product with Multiple Rounds of Sequencing Validation
The ZymeEditorTM enzyme evolution platform has successfully evolved a T4 DNA ligase mutant with high thermal stability, high library yield, and low adapter self-ligation through enzyme evolution technology. The currently improved T4 DNA Ligase has been successfully launched- Hieff® Versatile T4 DNA Ligase (600 U/μL) (Cat#12996ES). This product outperforms the traditional wild-type T4 DNA Ligase in terms of ligation efficiency and thermal stability, and it has been verified by high-throughput sequencing, ensuring excellent quality.
Data Presentation
1.Higher Thermal Stability
The T4 DNA Ligase mutants from Yeasen were subjected to heat treatment at 42℃ for 0, 2, and 4 hours, and were compared with T4 DNA Ligases from Vendor Q, N, A, and T. Subsequently, a whole-genome DNA library was constructed using 1 µg of fragmented bovine gDNA with the Yeasen DNA Library Construction Kit (Cat#12927ES) to compare the thermal stability of the T4 DNA Ligase.
Fig 3 Thermal Stability Test at 42℃
Results:
Thermal Stability: Yeasen ≈ Vendor N ≈ Vendor A > Vendor T > Vendor Q.
2.Higher Library Yield
The T4 DNA Ligase mutant from Yeasen was tested for library yield at both high and low DNA input levels, compared with T4 DNA Ligases from Vendor Q, N, A, and T. The Yeasen DNA Library Construction Kit (Cat#12927ES) was used, with 1 µg and 0.5 ng of fragmented bovine gDNA being input to construct DNA libraries.
3.Lower Adapter Self-Ligation
The T4 DNA Ligase mutant from Yeasen was compared with T4 DNA Ligases from Vendor Q, N, A, and T for adapter self-ligation. The Yeasen DNA Library Construction Kit (Cat#12927ES) was used, with 0 ng and 0.5 ng of fragmented bovine gDNA being input to construct whole-genome DNA libraries.
Contact Us
Yeasen has successfully overcome the limitations of traditional T4 DNA ligases, offering a T4 DNA ligase product that excels in thermal stability, library yield, and adapter self-ligation. In this process, the company has accumulated a rich library of mutants, providing researchers with a variety of options to meet different experimental needs. If you are interested in our products or T4 DNA ligase mutants, please feel free to contact us through the following methods.Yeasen T4 DNA Ligase Product Series
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product positioning |
Product Name |
Cat. No. |
|
High thermal stability and low self-ligation rate |
Hieff® Versatile T4 DNA Ligase (600 U/μL) |
12996ES |
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Rapid and efficient ligation, low residue, perfectly suited for pathogen detection needs. |
Fast T4 DNA Ligase (400 U/µL) |
10299ES |
|
universal model |
Quick T4 DNA Ligase (400 U/µL) |
10301ES |