T4 RNA Ligase 2

T4 RNA ligase catalyses the formation of a phosphodiester bond between a 5′ phosphate and 3′ hydroxyl of RNA. It is most active in sealing nicked double-stranded RNA but it will also join the ends of single-stranded RNA and can ligate the 3′ hydroxyl of RNA to the 5´ phosphate of DNA. The enzyme requires ATP unless the 5’ end of the substrate is preadenylated.

Key Features:

• ligates ds-RNA
• Ligates RNA to DNA creating RNA:DNA hybrids
• ATP-dependent

Applications:

• RNA Library construction, e.g. connecting short RNA fragments or adapters for NGS applications
• Modified RNA Synthesis - Ligation of modified or therapeutic RNA molecules 
• mRNA manufacturing - repair or circularisation of RNA molecules
• RNA repair and editing

Bulk quantities of this enzyme are available, animal free origin, and meeting high regulatory specifications. Please contact us for more information ([email protected]. uk or on our Contact Us page)

Unit Definition:

One cohesive end ligation unit (CELU) is the amount of enzyme which gives 50% ligation of 10 pmol of a ds RNA-DNA substrate at 37°C for 30 minutes.

Ligation Assay       

ds RNA-DNA substrate

5’-Flu-GGC CAG UG-AAT TCG AGC TCG-3’
      3’- CCG  GTC AC TTA AGC TCG AGC-5’ 

10 pmols of a double stranded 20-mer with a nick in one strand, 5’ end-labelled on the nicked strand, is incubated with ligase and 1x Assay Buffer supplemented with 1 mM ATP, at 37°C for 30 minutes, run on a 15 % (w/v) acrylamide/8M urea gel and visualised.