RNA Ligation

New England Biolabs provides a large selection of highly characterized RNA ligases which are recommended for common research and development applications. Our RNA Ligase Selection Chart lists all of the ligases available for RNA, along with their recommended applications. These products are useful for the ligation of single-stranded or double-stranded RNA to either RNA or DNA.

• T4 RNA ligase 1 (NEB #M0204) is a ssRNA ligase that catalyzes the ATP-dependent covalent joining of single-stranded 5´-phosphoryl termini of DNA or RNA to single-stranded 3´-hydroxyl termini of DNA or RNA.
• T4 RNA ligase 2 (NEB #M0239) is a dsRNA ligase that also catalyzes the joining of a 3´-hydroxyl terminus of RNA to a 5´-phosphorylated RNA or DNA; unlike T4 RNA ligase 1, this enzyme prefers double-stranded substrates.
• A truncated form of T4 RNA ligase 2, T4 RNA Ligase 2, truncated KQ (NEB #M0373) requires a pre-adenylated substrate for ligation.
  
  

Reported ligation activities of various ligases for RNA and DNA

The RtcB Ligase is provided through NEB’s Enzymes for Innovation Project which aims to offer unique enzymes with interesting properties to enable the discovery of innovative applications. In contrast to most of the RNA and DNA ligases that require a 5´ phosphate for ligation, the RtcB ligase (NEB# M0458), ligates RNA with a 3´ phosphate to a 5´OH acceptor.

Guidance for RNA labeling, RNA circularization and a unique approach for detection

NEB provides specific reaction conditions, as well as protocols for radioactive labeling of the 3´ termini of RNA, circularizing oligodeoxyribonucleotides and oligoribonucleotides, ligating oligomers and nicks, creating hybrid and chimeric DNA/RNA molecules, and miRNA cloning.

RNA Ligase enzymes are also commonly used to join linkers to the ends of RNA for amplification and next generation sequencing and are incorporated some of our NEBNext products for RNA and Small RNA Library Preparation.

Additionally, RNA ligation can be used for nucleic acid detection. A common method for miRNA detection involves the extension of a complementary DNA probe with a reverse transcriptase. An alternative method, developed by New England Biolabs uses ligation. Specifically, two miRNA specific DNA probes are joined by an RNA splint and ligated using SplintR Ligase (NEB #M0375). The ligated product can then be amplified and detected by qPCR. This approach is sensitive and able to detect miRNAs that differ by a single nucleotide ^[1].  

References

1.    Jin et al (2016), Sensitive and specific miRNA detection method using SplintR Ligase, Nucleic Acids Research 44(13):e116, doi: 10.1093/nar/gkw399