T4 RNA Ligase 2

• T4 RNA Ligase 2 ligates nicks on double- or single-stranded RNA
• T4 RNA Ligase 2 ligates 5ʹ phosphate and 3ʹ hydroxyl of RNA
• T4 RNA Ligase 2 Truncated facilitates linker ligations with pre-adenylated 5' DNA to 3' hydroxyl RNA
• T4 RNA Ligase 2 Truncated does not require ATP

T4 RNA Ligase 2 catalyzes phosphodiester bond formation between a 5ʹ phosphate and 3ʹ hydroxyl of RNA. The preferred substrate is nicked double-stranded RNA but single-stranded RNA can also serve as a substrate. Ligation of single-stranded RNA substrates generates either intramolecular or intermolecular products. Besides nicked double-stranded RNA substrates, other nicked nucleic acids hybrids can be sealed. The strand containing the 5ʹ phosphate can either be DNA or RNA. The non-ligated strand of the duplex can be either RNA or DNA. T4 RNA ligase 2 requires ATP for activity unless the substrate is preadenylated on the 5ʹ end. A truncated version of T4 RNA ligase 2 is a better enzyme for preadenylated substrates because it generates less side-reaction ligation products than the full-length enzyme.

T4 RNA Ligase 2 truncated catalyzes phosphodiester bond formation between a pre-adenylated 5′ phosphate (DNA or RNA) and the 3′ hydroxyl of RNA. The truncated enzyme contains the first 249 amino acids which makes the enzyme require a pre-adenylated 5′ terminal donor and eliminates the need for ATP. Because T4 RNA ligase 2 truncated cannot use the 5′ phosphate of RNA or DNA as a donor in the ligation reaction, it is useful for certain applications, such as linker ligations with pre-adenylated 5′ DNA to 3′ hydroxyl RNA. The desired specific ligation products are enhanced dramatically over unwanted background ligation products, making the truncated enzyme superior to the full-length enzyme for this use.

Our T4 RNA Ligase 2 Truncated demonstrates equivalent or superior volume/volume performance when compared to analogous T4 RNA Ligase 2 Truncated products on the market.

The full length and truncated T4 RNA Ligase 2 are supplied in 10 mM Tris-HCl, 100 mM NaCl, 0.1 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 7.5 at 25°C.

T4 RNA Ligase 2 is supplied with 10X Ligation Buffer (cat. no. B6030), which contains the following: 500 mM Tris-HCl, 100 mM MgCl₂, 50 mM DTT and 10 mM ATP; pH 7.6 at 25°C.

T4 RNA Ligase 2 Truncated is supplied with 10X T4 RNA Ligase 2, Truncated Buffer (cat. no. B6070), which contains the following: 500 mM Tris-HCl, 100 mM MgCl₂ and 50 mM DTT; pH 7.6 at 25°C.

Enzyme properties

• Storage temperature: –25°C to –15°C

References

1. Ho, C.K., et al. (2004) Structure 12:327.
2. Ho, C.K., and Shuman, S. (2002) Proc. Natl.Acad.Sci. USA 99:12709.
3. Nandakumar, J., et al. (2004) J. Biol. Chem. 279:31337.
4. Aravin, A., and Tusch, T. (2005) FEBS Letters 579:5830.
5. Pfeffer, S., et al. (2005) Nat. Meth. 2:269.

Source of recombinant enzyme protein
The T4 RNA 1 protien is produced by a recombinant E. coli strain carrying the T4 RNA Ligase gene from bacteriophage T4.
The T4 RNA Ligase 2 proteins are produced by a strain of E. coli that expresses the recombinant the full length or the truncated T4 RNA Ligase 2 gene.

Unit definition
One unit is defined as the amount of enzyme required to ligate 50% of 0.4 µg of an equimolar mix of a single-stranded 5′ FAM-labeled 17-mer RNA to the 5′ pre-adenylated end of a 18-mer DNA when both 17-mers are annealed to a complementary 35-mer DNA strand in 20 µl 1X reaction buffer following a 30 minute incubation at 37°C.

Quality control analysis

Specific activity of the full length and truncated T4 RNA Ligase 2 enzymes was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer and 2 µl of each enzyme dilution was added to 18 µl reactions in 1X reaction buffer containing 0.4 µg of an equimolar mix of one 17 base RNA oligonucleotide (5′ FAM-labeled) and one 18 base DNA oligonucleotide (5ʹ pre-adenylated for T4 RNA Ligase 2; 5ʹ phosphorylated for T4 RNA Ligase 2 Truncated) annealed to a complementary 35-mer DNA oligonucleotide. Reactions were incubated 30 minutes at 37°C, quenched, and analyzed on a 15% TBE-urea gel.

Protein concentration is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Non-specific RNAse contamination is assessed using the RNAse Alert kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Nick ligation in double-stranded RNA
• Ligate the 3' OH of RNA to the 5' phosphate of DNA in a double-stranded-format NGS RNA library construction (miRNA-seq or directional mRNA-seq)