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Methods Enzymol. 2008;447:83-98. doi: 10.1016/S0076-6879(08)02205-2.

PABLO analysis of RNA: 5'-phosphorylation state and 5'-end mapping.

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  • 1Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Microbiology, New York University School of Medicine, New York, USA


Recent studies have revealed that 5'-end-dependent RNA degradation in prokaryotes is triggered by pyrophosphate removal from the 5'-terminus to generate a monophosphorylated intermediate that is readily degraded. This chapter describes how to examine the 5'-phosphorylation state of any specific bacterial RNA by PABLO analysis. The method is based on the ability of monophosphorylated, but not triphosphorylated, RNA 5'-ends to undergo splinted ligation to a DNA oligonucleotide when juxtaposed by base pairing to a bridging oligonucleotide. PABLO analysis not only makes it possible to quantify the proportion of a particular RNA that is monophosphorylated in bacterial cells but also provides a more reliable method than primer extension for high-resolution mapping of RNA 5'-termini.

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