T4 RNA Ligase 2, Truncated

• Specifically joins the pre-adenylated 5´ end of DNA or RNA to the 3´ end of RNA.
• In contrast to the full-length ligase, it is unable to join the phosphorylated 5´ end of RNA or DNA to the 3´ end of RNA.
• Ligation does not require ATP, but pre-adenylated substrate is needed. 
• Minimizes background ligation by utilizing only pre-adenylated linkers.

T4 RNA Ligase 2 Truncated catalyzes the phosphodiester bond formation between a pre-adenylated 5’ phosphate (DNA or RNA) and the 3’ hydroxyl of RNA. The truncated enzyme only contains the first 249 amino acids of the full-length T4 RNA Ligase 2, 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. Thus, this enzyme is an excellent choice for RNA library preparation for RNA sequencing (1-5).

Supplied in: 
10 mM Tris-HCl, 100 mM NaCl, 0.1 mM DTT, 0.1 mM EDTA, 50% glycerol (pH 7.5 at 25°C)

Supplied with: 
10X T4 RNA Ligase 2, Truncated Buffer (B6070): 500mM Tris-HCl, 100mM MgCl₂, 50mM DTT (pH 7.6 @ 25⁰C)

Properties:
Storage temperature: –25°C to –15°C

Source of Protein: Purified from a strain of E. coli that expresses the recombinant truncated T4 RNA Ligase 2 gene. 

Unit Definition: 1 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.

Molecular weight: 30,451 Daltons

Usage Instructions: Ligation of the 3’ OH of RNA to the 5’ pre-adenylated DNA 

1. Set up the following reaction mixture in a total volume of 20 µL:

   Components	Final Concentration	Volume
   Nuclease free water	N/A	X µL
   10X T4 RNA Ligase 2, Truncated  Reaction Buffer (B6070)	1X	2 µL
   3’ OH RNA	1 µM	X µL
   5’ pre-adenylated DNA or RNA	2 µM	X µL
   T4 RNA Ligase 2, Truncated  (L6070L)	5 U	1 µL
   	Total Volume =	20 µL
2. Incubate at 37°C for 30 minutes. 
3. Reaction can be stopped by adding EDTA or incubation at 75°C for 10 minutes.

Quality Control Analysis: 
 
Unit Activity is measured using a 2-fold 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 (5’pre-adenylated) 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 (OD₂₈₀) 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 protein of interest band 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 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 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 16S rDNA Contamination is evaluated using 5 µL replicate samples of enzyme solution 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 product is available for molecular biology applications such as:

• Library construction for RNA sequencing
• Increase ability to identify miRNAs

References: 

1. Ho, C.K. et al. (2004) Structure, 12, 327-339.
2. Ho, C.K. and Shuman, S. (2002) Proc. Natl. Acad. Sci. USA, 99, 12709-12714.
3. Nandakumar, J. et al. (2004) J. Biol. Chem, 279, 31337-31347.
4. Aravin, A. and Tusch, T. (2005) FEBS Letters, 579, 5830-5840.
5. Pfeffer, S. et al. (2005) Nat. Meth, 2, 269-276.