UNEXPECTED ELECTROPHORETIC MIGRATION OF RNA WITH DIFFERENT 3' TERMINICAUSES A RNA SIZING AMBIGUITY THAT CAN BE RESOLVED USING NUCLEASE P1-GENERATED SEQUENCING LADDERS
J. Cruzreyes et al., UNEXPECTED ELECTROPHORETIC MIGRATION OF RNA WITH DIFFERENT 3' TERMINICAUSES A RNA SIZING AMBIGUITY THAT CAN BE RESOLVED USING NUCLEASE P1-GENERATED SEQUENCING LADDERS, Biochemistry, 37(17), 1998, pp. 6059-6064
It has been widely believed that the electrophoretic migration differe
nce of otherwise identical RNAs with a P versus OK terminus would be t
he same as occurs for DNA, a fairly reproducible similar to 1/2 nucleo
tide (nt) offset. RNA with a 5'-OH indeed migrates less than or equal
to 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) migra
tes anywhere from similar to 1/4 to similar to 2 nts slower than the o
therwise identical molecule with a 3'-P or 2',3'-cyclic-P terminus las
present on standard RNase-generated sequencing ladders). This previou
sly unrecognized variability in electrophoretic migration offset cause
s a 1-2 nt ambiguity in a commonly used method of RNA size determinati
on. We also show two ways to overcome this problem and enable rigorous
sizing of 3'-OH terminating RNAs. Most convenient is to use sequencin
g standards generated by nuclease P1, which is generally sequence-nons
pecific but we show becomes G-specific or A-preferential under certain
reaction conditions.