T4 RNA Ligase 2 is an ATP-dependent double-strand RNA ligase (dsRNA Ligase), also called T4 Rnl2 (gp24.1). It can be used for intramolecular circular ligation and intermolecular linear ligation of double-stranded RNA.Different from T4 RNA Ligase 1, T4 RNA Ligase 2 has much higher ligation activity for nicks in double-stranded RNA than for the termini of single-stranded RNA. T4 RNA Ligase 2 can also be used for intramolecular or intermolecular ligation between the 3'-hydroxyl group of RNA strands and the 5'-phosphate group of DNA strands in duplex nucleic acids (double-stranded RNA, RNA/DNA heteroduplexes and/or double-stranded DNA).The ligation reaction of T4 RNA Ligase 2 requires the presence of 5'-phosphate and 3'-hydroxyl groups; the ligation reaction can occur between the 5'-phosphate group of an RNA or DNA strand and the 3'-hydroxyl group of an RNA strand.The reaction process of dsRNA cohesive-end ligation catalyzed by T4 RNA Ligase 2 is as follows. Firstly, T4 RNA Ligase 2 directly consumes ATP to form the intermediate T4 RNA Ligase 2-AMP and release pyrophosphate; secondly, T4 RNA Ligase 2-AMP binds to the nick site of dsRNA, and transfers AMP from the intermediate T4 RNA Ligase 2-AMP to the 5'-phosphate terminus at the dsRNA nick to form an adenylated nicked dsRNA intermediate; finally, under the catalysis of T4 RNA Ligase 2, the 3'-hydroxyl group at the dsRNA nick attacks the 5'-phosphate group at the same nick, forming a 3'-5' phosphodiester bond and releasing AMP.
| Item | Content |
|---|
| Source | Recombinantly expressed in Escherichia coli | | Appearance | Sterile liquid | | Storage Buffer | 10 mM Tris-HCl, 50 mM KCl, 35 mM (NH₄)₂SO₄, 0.1 mM EDTA, 0.1 mM DTT, 50% (v/v) Glycerol, pH7.5.
| | Enzyme Concentration | 10 U/μL | | Purity | Free of DNA endonuclease, DNA exonuclease, RNase and phosphatase | | Activity Definition | One unit is defined as the amount of enzyme required to ligate 0.4 μg of an equimolar mix of a 23-mer and 17-mer RNAs in a total reaction volume of 20 μL within 30 minutes at 37 °C. |
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Components List
T750873
| Components
| Appearance
| 1000U | 5×1000U
| Storage
| | T750873A | T4 RNA Ligase 2 (10U/µl) | Liquid | 100ul | 5×100ul
| -20℃. Avoid freeze/thaw cycle. | | T750873B | 10× T4 Rnl2 Reaction Buffer
| Liquid
| 250ul | 5×250ul | -20℃. Avoid freeze/thaw cycle. |
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Applications
It is mainly used for ligation of nicks in double-stranded RNA (i.e., cohesive-end ligation of dsRNA). It can also catalyze nick ligation between the 3'-hydroxyl of RNA and the 5'-phosphate of DNA within duplex structures.
Product Advantages
This product features high enzymatic activity and superior ligation efficiency.
Instructions for Use
1. Annealing of double-stranded RNA or DNA/RNA heteroduplexes. Mix two single-stranded RNAs at equimolar ratios; the recommended stock concentration is 20 μM (10–50 μM is acceptable). Incubate at 90 °C for 1 min, then perform gradient cooling down to 25 °C to form dsRNA. It is recommended to use 5× Annealing Buffer for RNA oligos and carry out annealing reactions following the manufacturer’s instructions of this buffer. Annealing conditions for DNA/RNA heteroduplexes can refer to those for double-stranded RNA. The annealed duplexes are recommended to be stored at -80 °C.
2. For ligation of nicks in double-stranded RNA, prepare the following reaction system on ice according to the table below:
| Reagent | Volume | Final Concentration |
|---|
| DEPC-treated Water | 16 μL | - | | Nicked dsRNA Substrate (20 μM) | 1 μL | 1 μM | | 10× T4 Rnl2 Reaction Buffer | 2 μL | 1× | | T4 RNA Ligase 2 (10 U/μL) | 1 μL | 0.5 U/μL | | Total Volume | 20 μL | - |
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Notes:a. Since RNA manipulation is involved, standard RNA handling protocols must be strictly followed to prevent RNase contamination. All relevant reagents and consumables shall be treated with DEPC to remove RNase or certified RNase-free. This reaction system contains double-stranded RNA, which is resistant to RNase A, RNase T1 and other ribonucleases. If single-stranded RNA is present, including partially single-stranded RNA regions formed after duplex annealing, an appropriate amount of RNase Inhibitor is recommended to be supplemented.
b. When setting up multiple ligation reactions at the same time, pre-mix all solutions and enzymes listed in the above table except the Nicked dsRNA Substrate in advance, then aliquot the mixture into separate reaction tubes.
c. The final concentration of Nicked dsRNA Substrate in the reaction system can reach 1 µM, a concentration sufficient to guarantee complete ligation. In practical applications, the dosage of Nicked dsRNA Substrate can be appropriately reduced if substrate is limited; for instance, the final concentration can be adjusted to 0.5 µM or 0.2 µM.
3. For nick ligation between the 3'-hydroxyl group of RNA and the 5'-phosphate group of DNA within RNA/DNA heteroduplexes, prepare the reaction system on ice as specified in the table below:
| Reagent | Volume | Final Concentration |
|---|
| DEPC-treated Water | 10.4μl | - | | Nicked DNA/RNA Substrate (20μM) | 1μl | 1μM | | 10X T4 Rnl2 Reaction Buffer | 2μl | 1X | | PEG8000 (50%, RNase Free) | 4μl | 0.1 | | MgCl₂ (100mM, DEPC-treated) | 1.6μl | 8mM | | T4 RNA Ligase 2 (10U/μl) | 1μl | 0.5U/μl | | Total Volume | 20μl | - |
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Notes:
a. Since RNA handling is involved, standard RNA operation specifications must be strictly followed to avoid RNase contamination. All relevant reagents and consumables shall be treated with DEPC to eliminate RNase or be verified RNase-free. This reaction system contains double-stranded RNA, which can resist RNase A, RNase T1 and other ribonucleases. If single-stranded RNA exists, including partially single-stranded RNA regions formed after duplex annealing, appropriate amount of RNase Inhibitor is recommended to be added.b. When setting up multiple ligation reactions simultaneously, pre-mix all solutions and enzymes listed in the above table except the Nicked DNA/RNA Substrate in advance, then aliquot the mixture into individual reaction tubes.c. The final concentration of Nicked dsRNA Substrate in the reaction system can reach 1 µM, which can ensure complete ligation. In practical use, if the substrate is insufficient, the dosage of Nicked dsRNA Substrate can be reduced appropriately, for example, adjusting the final concentration to 0.5 µM or 0.2 µM.
4. Ligation reaction: Incubate at 37 °C for 30 min. If unsatisfactory ligation efficiency is observed, incubation at 25 °C for 2 h can be attempted. The incubation time can be extended appropriately to achieve more sufficient ligation.
5. Termination of reaction: The reaction can be terminated by adding Proteinase K or EDTA after incubation. T4 RNA Ligase 2 requires heating at 85 °C for 5 min for thermal inactivation, which may denature dsRNA or DNA/RNA heteroduplexes. Therefore, thermal inactivation of T4 RNA Ligase 2 is generally not recommended. If maintenance of duplex structure is not required for subsequent experiments, heating at 85 °C for 5 min is suggested to inactivate T4 RNA Ligase 2.
Precautions:
(1) If the ligation substrate is ssRNA, products such as T4 RNA Ligase 1 are recommended.
(2) Appropriate operating procedures can be selected according to specific applications, and extra reagents such as RNase Inhibitor and DEPC-treated Water may be required.
(3) The supplied 10× T4 Rnl2 Reaction Buffer is suitable for nick ligation within double-stranded RNA. For nick ligation between the 3'-hydroxyl of RNA and the 5'-phosphate of DNA in RNA/DNA heteroduplexes, the final concentration of MgCl₂ in the reaction system shall be increased to 10 mM, and PEG8000 shall be supplemented to a final concentration of 10–15%. This modification can markedly enhance enzymatic activity without altering reaction characteristics.
(4) This product is only for scientific research use by professional personnel. It shall not be used for clinical diagnosis or treatment, food or pharmaceutical manufacturing, nor stored in residential premises.
(5) Please wear lab coat and disposable gloves during operation for your safety and health.
