Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides

Abstract

We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5′ end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5′-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5′ end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5′ end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).