ThermoSalt RNA Ligase 2 (70 µM) is a novel dsRNA ligase independently developed by Yeasen. In the presence of ATP, it efficiently catalyzes both intermolecular and intramolecular ligation of RNA strands. Compared with conventional T4 RNA Ligase 2, ThermoSalt RNA Ligase 2 offers the dual distinct advantages of high-temperature and high-salt tolerance.

Experimental data demonstrate that the enzyme retains over 90% of its ligation activity at 70°C, and maintains over 50% activity even in a 300 mM NaCl high-salt environment, fully satisfying the requirements of complex samples or special reaction conditions.

Features

High-temperature tolerance: Retains >50% activity at 70°C, facilitating high-temperature denaturation steps and improving ligation of structured RNA.

High-salt tolerance: Active in 300 mM NaCl, allowing direct ligation in saline buffers without the need for dilution or buffer exchange.

Flexible temperature compatibility: Functions across 25–70°C (validated at 25°C, 45°C, and 70°C) to accommodate diverse substrates and process conditions.

Substrate compatibility: Ligates nicks in dsRNA and DNA-splinted RNA; compatible with 2′-F, 2′-OMe, and 2′-F/OMe modified dsRNA.

Specifications

Application

RNA intermolecular and intramolecular ligation; dsRNA nick ligation; splint-dependent RNA ligation; 2′-F, 2′-OMe, and 2′-F/OMe modified dsRNA ligation; ligation under high-temperature and high-salt conditions.

Figures

1. Basic ligation activity

Figure 1. Comparison of ligation activity between ThermoSalt RNA Ligase 2 and T4 RNA Ligase 2

[Note]: Under the same enzyme input gradient (0.25 / 0.125 / 0.063 / 0.031 U), ThermoSalt RNA Ligase 2 exhibited superior ligation efficiency compared with T4 RNA Ligase 2. (The specific activity of ThermoSalt RNA Ligase 2 is approximately 30 U/µL, i.e., 70 pmol ≈ 30 U.)

2. Thermal stability

Figure 2. Comparison of thermal stability between ThermoSalt RNA Ligase 2 and T4 RNA Ligase 2 at different reaction temperatures

[Note]: The comparison was performed across a 25–90°C temperature gradient (enzyme input 0.25 U, 30 min reaction). T4 RNA Ligase 2 activity declined rapidly as the temperature increased; ThermoSalt RNA Ligase 2 retained over 50% ligation efficiency even after 30 min at 70°C, demonstrating markedly superior thermal stability.

3. Salt tolerance

Figure 3. Comparison of salt tolerance between ThermoSalt RNA Ligase 2 and T4 RNA Ligase 2 at different salt concentrations

[Note]: The comparison was performed across a 0–300 mM NaCl gradient (enzyme input 0.25 U). T4 RNA Ligase 2 activity dropped rapidly with increasing salt concentration, whereas ThermoSalt RNA Ligase 2 retained over 50% ligation efficiency even at 300 mM NaCl, demonstrating markedly superior salt tolerance.

4. Functional validation

(A). Splint-dependent RNA ligation

Figure 4. Splint DNA-mediated RNA ligation activity comparison

[Note]: ThermoSalt RNA Ligase 2 showed splint-dependent RNA ligation activity superior to that of T4 RNA Ligase 2.

(B). Modified dsRNA ligation

Figure 5. Ligation activity verification of 2′-F, 2′-OMe, and 2′-F/OMe modified dsRNA

[Note]: ThermoSalt RNA Ligase 2 efficiently ligates 2′-F, 2′-OMe, and 2′-F/OMe modified dsRNA.

Note: Reaction conditions: 0.3 U enzyme, 37°C, 1 h, 2 mM MgCl₂. A: 2′-F modified dsRNA; B: 2′-OMe modified dsRNA; C: 2′-F/OMe mixed modified dsRNA; D: unmodified dsRNA.

(C). Flexible temperature compatibility

Figure 6. Modified dsRNA ligation at different temperatures (25°C, 45°C, 70°C)

[Note]: A: 2′-F modified dsRNA; B: 2′-OMe modified dsRNA; C: 2′-F/OMe mixed modified dsRNA.

(D). Optimal Mg²⁺ concentration

Figure 7. Modified dsRNA ligation at different Mg²⁺ concentrations (2/5/10 mM)

[Note]: Reaction conditions: 0.3 U enzyme, 45°C, 1 h. A: 2′-F modified dsRNA; B: 2′-OMe modified dsRNA; C: 2′-F/OMe mixed modified dsRNA; NC: no-enzyme negative control; PC: unmodified substrate positive control.