Cap-Clip™ Acid Pyrophosphatase

A: Cap-Clip™ Acid Pyrophosphatase is an improvement over Tobacco Acid Pyrophosphatase (TAP), a discontinued enzyme which was used to remove the 5’ Cap 0, Cap 1 and/or the β and γ phosphates of triphosphorylated nucleic acids. The enzyme uses the same buffer as the original TAP and has been shown in genome-wide studies to function as a direct replacement for TAP (see: Nat Protoc. 2016 Feb; 11(2): 359–376.)

A: Other enzymes may function at a slightly alkaline pH (7.5), while Cap-Clip™ Acid Pyrophosphatase works at a slightly acidic pH (6.0). Additionally, only Cap-Clip™ Acid Pyrophosphatase is compatible with EDTA, which will inhibit trace amounts of contaminating exonucleases inadvertently present in samples. We have found the decapping activity of Cap-Clip™ Acid Pyrophosphatase to be much more robust than other commercially-available mRNA decapping enzymes.

A: Yes, the efficiency of the enzyme is reduced when given reduced access to the 5’-end of the mRNA due to secondary structure that obscures the 5’-end. Structural interference can be overcome by use of more enzyme, higher incubation temperatures or longer incubation times.

A: No, the enzyme is metal-independent. As with TAP, the Cap-Clip™ Acid Pyrophosphatase Reaction Buffer contains EDTA to inhibit any potential Mg²⁺-dependent nuclease activity from incoming substrate sources.

A: Multiple applications make use of Cap-Clip™ Acid Pyrophosphatase (or TAP) to interrogate the 5’-end of RNAs, including the transcriptional start sites of mRNAs. These protocols have evolved over time in parallel to advancements in RNA sequencing technologies. These and other protocols can make use of Cap-Clip™ Acid Pyrophosphatase for the ligation of oligoribonucleotides to decapped cellular RNAs for cloning and mapping, 5’- and 3’-end mapping of RNA, mapping of transcription start sites for eukaryotic and prokaryotic RNA, labeling of RNA for sequencing and cloning or use as hybridization probes. Here are just some of the many protocols:

1. Reverse Ligation-mediated PCR (RLPCR): Nucleic Acids Res. 1993 Apr 11; 21(7): 1683–1684.
2. Oligo-capping: Gene. 1994 Jan 28;138(1-2):171-4.
3. 5’ and 3’ RACE (Rapid Amplification of cDNA Ends) Nature Methods volume 6, pages i–ii (2009)
4. CIP-TAP: Nature. 2009 Feb 19; 457(7232): 1028–1032.
5. DEEP-RACE: Biotechniques. 2009 Feb;46(2):130-2.
6. 5p’Seq: Nat Protoc. 2016 Feb;11(2):359-76.
7. STAP-Seq: Nat Biotechnol. 2017 Feb; 35(2): 136–144.
8. 5′ End Analysis of Terminator-Resistant 18S and 25S rRNA. BMC Mol Cell Biol. 2020; 21: 59.
9. PRO-Seq: Biorxiv, 2020.
10. Thin Layer Chromatography (TLC) Detection of N1 2′-O-Me: Cell Rep. 2020 Oct 20; 33(3): 108269.
11. CPA-Seq: Cell Discov. 2021 Apr 19;7(1):25.
12. CapTag Seq, TSS-Seq and CLAM-Cap-seq: Nature. 2023 Feb;614(7947):358-366.
13. NAP-Seq: Nat Commun. 2024 Mar 18;15(1):2425.