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Unexpected electrophoretic migration of RNA with different 3' termini causes a RNA sizing ambiguity that can be
J Cruz-Reyes1, K J Piller, L N Rusché
1Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Abstract:
It has been widely believed that the electrophoretic migration difference of otherwise identical RNAs with a P versus OH terminus would be the same as occurs for DNA, a fairly reproducible approximately 1/2 nucleotide (nt) offset. RNA with a 5'-OH indeed migrates =1 nt slower than if it had a 5'-P. Surprisingly, however, RNA with a 3'-OH terminus (generated by many cellular RNases of interest) migrates anywhere from approximately 1/4 to approximately 2 nts slower than the otherwise identical molecule with a 3'-P or 2', 3'-cyclic-P terminus (as present on standard RNase-generated sequencing ladders). This previously unrecognized variability in electrophoretic migration offset causes a 1-2 nt ambiguity in a commonly used method of RNA size determination. We also show two ways to overcome this problem and enable rigorous sizing of 3'-OH terminating RNAs. Most convenient is to use sequencing standards generated by nuclease P1, which is generally sequence-nonspecific but we show becomes G-specific or A-preferential under certain reaction conditions.