Piero Carninci’s group at RIKEN published an article last year demonstrating that small RNAs can possess a large variety of 5′ cap structures including a 2,7 dimethylguanosine modification and found multiple cap structures (mGpppC, 7mGpppG, GpppG, GpppA, and 7mGpppA) in small RNA pools.

Source: DOI: 10.1371/journal.pone.0102895

This adds a further level of complication to small RNA sequencing library prep, as such protocols generally allow for sequencing only of RNAs with a 5′ monophosphate. In general, mature, functional microRNAs are thought to carry a simple 5′ monophosphate, and ligation kits such as the CleanTag™ Ligation Kit for Small RNA Library Preparation that use T4 RNA Ligase to fix oligonucleotides to the 5′ end of RNA will effectively oligocap this 5′-monophosphated RNA population. Carninci’s group used a variety of approaches including decapping with tobacco acid pyrophosphatase, CAGE, and immunoprecipitation with 2,2,7-trimethylguanosine to perform their study, but this isn’t the first time we have heard about cap structures on small RNAs.

Image Source: “MiRNA-biogenesis” by Narayanese, Wikipedia. 

We have known for quite some time that small RNAs can possess 5′ structures. The now discontinued ScriptMiner™ small-RNA–seq library prep kit from Epicentre® (an Illumina Company), for example, included protocols for removal of 5′ cap structures with Epicentre’s tobacco acid pyrophosphatase to allow capture of small 5′-capped and 5′-triphosphorylated RNAs in addition to 5′-monophosphate RNAs. Due to the discontinuation of tobacco acid pyrophosphatase, tebu-bio’s Decapping Pyrophosphohydrolase or CellScript’s Cap-Clip™ Acid Pyrophosphatase is now recommended to convert RNAs with diverse 5′ structures to 5′-monophosphate RNA for oligo-capping.

While next generation sequencing is quickly replacing array-based genomics/transcriptomics technologies, library prep for miRNA sequencing remains relatively complex. Due to the complexity of adapter-dimers and 5′ cap structures, array-based genome-wide miRNA expression analysis remains the first choice for many researchers.

The cap structures present on small RNAs appear to be either added during transcription or may be the result of a yet-to-be characterized mechanism for capping of RNA degradation products. An excellent resource for learning more about RNA 5′ structures is the Modomics website, which allows users to visualize various cap structures and gives the identify of the enzymes responsible for the modifications.

Source: http://modomics.genesilico.pl/pathways/pppN/

Here are some of tebu-bio’s top products and services for isolating and studying small RNAs:

• RNAzol® RT RNA Isolation Reagent – the most effective reagent for isolation of total RNA and small RNA from samples of various origins
• MasterPure™ RNA Purification Kits – for isolation of total RNA without columns and without dangerous solvents
• CleanTag™ Ligation Kit for Small RNA Library Prep –  includes chemically modified adapters that greatly reduce adapter dimers
• iSWAB™ RNA – collection kit for RNA from buccal swabs or blood drops stabilizes donor samples at room temperature
• miRNA solutions – qPCR primers, 3’UTR target clones, precursor miRNA expression clones, miRNA inhibitors
• tebu-bio’s 3D-Gene® miRNA profiling platform – a full service array-based miRNA profiling service
• custom synthetic miRNAs and 2’-O-methylated oligonucleotides – tebu-bio can provide the most complex chemically-modified oligonucleotides

We have been closely following the interesting case of the discontinuation of tobacco acid pyrophosphatase (TAP) and the efforts of the world’s RNA biologists to find a suitable alternative. Our previous post gives a bit of the background and discusses how protocols for 5′ RACE, transcriptional start site (TSS) mapping and GRO-Seq require a good decapping enzyme to remove cap structures from RNAs to allow oligocapping. Here we discuss two new commercial offerings to replace the discontinued TAP enzyme and show some user data comparing the 3 enzymes.

As detailed in our previous post, the discontinuation of the enzyme tobacco acid pyrophosphatase (TAP), has left many molecular biology researchers without a suitable enzyme for their protocols. Perhaps the most classic assay using this enzyme was 5′ RACE, which required TAP to remove the 5′ mRNA cap yielding a 5′-monophosphate RNA.