T4 RNA Ligase 2 Explained: Structure, Mechanism & Applications

In the highly precise domain of molecular biology research, enzyme molecules function as exquisitely engineered "molecular tools," underpinning the successful execution of numerous critical experimental procedures. T4 RNA Ligase 2, an ATP-dependent double-stranded RNA ligase (dsRNA ligase), plays an indispensable role in RNA research due to its unique molecular architecture and efficient catalytic mechanism. This enzyme exhibits both intermolecular and intramolecular RNA strand-joining activities, enabling it not only to precisely seal nick sites within double-stranded RNA (dsRNA) but also to catalyze nick-joining reactions between the 3´-hydroxyl terminus of RNA and the 5´-phosphate group of DNA within duplex structures. Compared to its homolog T4 RNA Ligase 1, T4 RNA Ligase 2 demonstrates markedly higher activity in ligating nicks in double-stranded RNA than in joining the ends of single-stranded RNA, thereby establishing itself as a central enzymatic tool for studies involving dsRNA nick ligation.

Figure 1. Schematic illustration of substrates for T4 RNA Ligase 2

• Structural Analysis of T4 RNA Ligase 2

T4 RNA Ligase 2 is encoded by gene 24.1 of bacteriophage T4 and has a molecular weight of approximately 37 kDa, belonging to the ATP-dependent ligase family. Its molecular architecture comprises multiple highly conserved domains, including I, III, IIIa, IV, and V, which are precisely arranged in three-dimensional space to form a stable nucleotide-binding pocket, providing a robust structural foundation for catalysis. Crucially,

the precise spatial positioning of key amino acid residues—such as Glu-34, Arg-55, and Lys-209—within the enzyme's active site enables T4 RNA Ligase 2 to exhibit both high catalytic activity and substrate specificity during RNA and DNA ligation reactions, ensuring accurate and efficient execution of the enzymatic process.^[1]

Figure 2. Structural illustration of T4 RNA Ligase 2^[2]

• Catalytic Mechanism of T4 RNA Ligase 2

T4 RNA Ligase 2 accomplishes the ligation of adjacent nucleotides through a three-step, ordered catalytic reaction, exemplifying precise molecular-level regulation:

1. The ligase reacts with ATP to form an enzyme-AMP intermediate complex;

2. AMP is transferred from the enzyme-AMP complex to the 5′-phosphate group of RNA, generating a 5′-adenylylated RNA intermediate;

3. The 5′-adenylylated RNA reacts with the 3′-hydroxyl group of the adjacent nucleotide to form a phosphodiester bond, with concomitant release of AMP.

Figure 3. Schematic diagram of the catalytic mechanism of T4 RNA Ligase 2[3]

T4 RNA Ligase 2 (Cat#14652) of Yeasen

Based on the core structure and catalytic mechanism of T4 RNA Ligase 2, Yeasen Biotech has successfully developed a high-performance T4 RNA Ligase 2 (Cat#14652) through optimization of the expression system and purification process. This product retains the enzyme's native catalytic activity while eliminating interference from contaminating nucleases via a multi-step purification protocol, thereby providing researchers with a stable and reliable experimental tool. The following section presents its key performance characteristics with respect to ligation activity and purity control.

• High Ligation Efficiency

T4 RNA Ligase 2 from Yeasen and Supplier A* were tested at varying enzyme concentrations for their ability to ligate a double-stranded RNA (dsRNA) substrate. The results demonstrate that Yeasen's enzyme effectively catalyzes the ligation of dsRNA, with ligation efficiency comparable to that of theSupplier A* enzyme across all tested concentrations.

Figure 4. Validation of T4 RNA Ligase 2 Ligation Efficiency.

Note: The 20 μL reaction system contains a final concentration of 20 μM dsRNA ligation substrate.

• High Purity

T4 RNA Ligase 2 (100 U) from three different batches of Yeasen were individually incubated with nucleic acid substrates, and the resulting samples were analyzed by agarose gel electrophoresis to assess banding patterns. The results demonstrate that all three batches are free of contaminating exonuclease, nicking enzyme, and RNase activities. This absence of residual nucleases effectively eliminates the risk of interference from non-specific enzymatic activities, ensuring high fidelity and reliability in experimental applications.

Figure 5. Detection results of exonuclease, nicking enzyme, and RNase contamination in T4 RNA Ligase 2

Ordering Information

Product Name	Applications	Catalog No.
T4 RNA Ligase 1	1. Intramolecular and intermolecular ligation and cyclization of ssRNA; 2. Intermolecular ligation between ssRNA and ssDNA; 3. Synthesis of single-stranded oligoribonucleotides (ssOligo RNA); 4. Adapter ligation to the 5′ end of RNAs such as miRNA for cloning, library construction, or PCR detection, as well as cDNA library preparation.	14651ES
T4 RNA Ligase 2	1. Ligation of nicks in dsRNA; 2. Nick-joining between the 3′-hydroxyl group of RNA and the 5′-phosphate group of DNA within a duplex structure; 3. Facilitation of ssRNA end-joining with the assistance of an RNA splint.	14652ES

 

Extended Reading

1. Synthesis of siRNA and the Role of T4 RNA Ligase 2.

2. Overview of T4 DNA Ligase.

References

[1] Yin S, Ho C K, Shuman S. Structure-function analysis of T4 RNA ligase 2[J]. Journal of Biological Chemistry, 2003, 278(20): 17601-17608.

[2] Easey A. Synthetic Biology Methods to Optimise T4 RNA Ligase Activities[D]. University of East Anglia, 2018.

[3] Viollet S, Fuchs R T, Munafo D B, et al. T4 RNA ligase 2 truncated active site mutants: improved tools for RNA analysis[J]. BMC biotechnology, 2011, 11: 1-14.