T4 Ligase

As an expert in the field of gene therapy, Creative Biolabs has achieved great achievements in circular RNAs (circRNAs) synthesis. We focus on customizing circRNAs according to your specific needs. Our professional team has completed multiple circRNAs projects.

Introduction to CircRNAs

CircRNAs are a specialized class of RNAs characterized by their covalently closed structure. At present, more and more evidence indicated that circRNAs are gradually emerging as key new members of the gene regulatory environment. Moreover, circRNAs are emerging as potential regulators of cellular physiology and potential markers of disease progression. However, their mechanisms are not yet fully understood, thus further research is urgently needed to explore their functions and mechanisms. The T4 ligase-dependent circRNAs synthesis strategy has gradually become a popular method for circRNAs research.

Fig.1 Functions of circRNAs.¹

Overview of T4 Ligase

The promotion of circRNAs synthesis by T4 ligase mainly depends on three enzymes in phage T4, T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl 1) and T4 RNA ligase 2 (T4 Rnl 2).

• T4 Dnl

T4 Dnl is an efficient and accurate enzyme that is critical for ligating sticky-ended and blunt-ended DNA into plasmid vectors in a short time. T4 Dnl is suitable for joining ds duplexes such as DNA/RNA hybrids. Therefore, this approach requires a complementary DNA bridge to enable RNA ligation. Typically, DNA bridges have a dozen nucleotides on either side of the junction to ensure efficacious ligation. A significant feature of this way is that the accuracy of the connections is remarkably improved.

• T4 Rnl 1

Compared with T4 Dnl, T4 Rnl 1 contributes to the formation of phosphodiester bonds in single-stranded RNA molecules with a lower reaction specificity. T4 Rnl 1 is a powerful tool in many applications where short and long RNAs are used as substrates. In addition, the efficiency of this enzyme is closely linked to the structure of the linear precursor. Importantly, to facilitate intramolecular circularization, linear RNA should be pre-orientated to promote end proximity.

• T4 Rnl 2

T4 Rnl 2 has also been successfully used to circularize RNA. It is suitable for both single and double strand substrates. However, this RNA ligase is highly efficient at ligating gaps in dsRNA substrates compared to T4 Rnl 1. Furthermore, like T4 Rnl 1, T4 Rnl 2 is also accompanied by the disadvantages of low efficiency and side reactions towards large RNA molecules.

Comparison of T4 Ligases

In general, T4 Dnl and T4 Rnl 1 are very common in RNA junctions without complicated secondary structures while T4 Rnl 2 plays a crucial function in linear RNA precursors where the junction is in the double-stranded (ds) region. Thus, it is essential to select appropriate T4 ligases based on the RNA precursor structure. It is worth noting that these strategies are not ideal for the ligation of macromolecular RNAs, because intermolecular end-joining side reactions cannot be completely avoided. The advantages and disadvantages of various enzymes are listed in the table below:

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Reference

1. Shang, Qingfeng, et al. "The novel roles of circRNAs in human cancer." Molecular cancer 18.1 (2019): 1-10.

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