RtcB Ligase from E. coli joins single stranded RNA with a 3´-phospate or 2´,3´-cyclic phosphate to another RNA with a 5´ -hydroxyl (1,2). Ligation requires both GTP and MnCl₂ and proceeds through a 3´-guanylate intermediate (3). With substrates having a 2´,3´-cyclic phosphate end, hydrolysis to a 3´-phosphate precedes 3´ end activation with GMP and ligation.

Product Source

RtcB Ligase is expressed as a His-tagged fusion in E. coli.

Reagents Supplied

The following reagents are supplied with this product:

Applications

• Ligate ssRNA or ssDNA with a 3´-phosphate or a 2´,3´-cyclic phosphate to the 5´-OH of ssRNA
• Circularization of ssRNA with compatible ends, as above
• Detection of 3´-phosphorylated or 5´ -OH RNA using labeled probes

Reaction Conditions

1X RtcB Reaction Buffer
Supplement with 0.1 mM GTP and 1 mM MnCl₂
Incubate at 37°C

1X RtcB Reaction Buffer:
50 mM Tris-HCl
75 mM KCl
3 mM MgCl₂
10 mM DTT
pH 8.3 @ 25°C

Storage Temperature

-20°C

Storage Conditions

50 mM NaCl
10 mM Tris-HCl
0.1 mM EDTA
1 mM DTT
50% Glycerol
pH 7.4 @ 25°C

For difficult substrates, addition of PEG 8000 up to 15% can improve ligation yields.

1.Naoko, T., Shuman, S. (2011). J. Biol. Chem. 286, 7727-7731.

2.Naoko, T. et al. (2011). J. Biol. Chem. 286, 43134-43143.

3.Chakravarty, A. K. et al (2012). Proc. Natl. Acad. Sci. USA. 109, 6072-6077.