Mark-free analysis of the efficiency of adding caps to the RNA using rnasa h, probes and liquid chromatography / mass spectrometry

摘要

A radiolabel-free method for identifying a 5 'end cap on a target ribonucleic acid (RNA), comprising the steps of: (a) hybridizing a non-radiolabeled labeled probe to the target RNA, where the nucleotide sequence of the probe labeled non-radiolabeled is complementary to the 5 'end of the target RNA, thereby forming a duplex polynucleotide; (b) treating the duplex polynucleotide with RNase H, thereby cleaving the 5 'end of the target RNA and forming a duplex polynucleotide containing the 5' end of the target RNA; (c) isolating the duplex polynucleotide, using a surface-coated substrate that is coated with a reagent that binds to the non-radiolabeled labeled probe; (d) removing the duplex polynucleotide from the coated surface substrate; (e) denaturing the duplex polynucleotide, thereby producing a single-stranded fragment of the 5 'end of the target RNA and the non-radiolabeled labeled probe; (f) isolating the single stranded fragment from the 5 'end of the target RNA; (g) analyzing the single stranded fragment from the 5 'end of the target RNA by liquid chromatography / mass spectrometry (LC-MS); and (h) identifying the 5 'end cap.