Unexpected electrophoretic migration of RNA with different 3' termini causes a RNA sizing ambiguity that can be resolved using nuclease P1-generated sequencing ladders

Biochemistry
Jorge Cruz-ReyesBarbara Sollner-Webb

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.

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Citations

Mar 29, 2003·Journal of Molecular Biology·Bruno LamontagneSherif Abou Elela
May 18, 2010·Nature Structural & Molecular Biology·Anthony GobertPhilippe Giegé
Feb 5, 2009·Nucleic Acids Research·Moritz NiemannH Ulrich Göringer
Aug 27, 2010·Nucleic Acids Research·Julia Starega-RoslanWlodzimierz J Krzyzosiak
Jan 12, 2001·Molecular and Cellular Biology·J Cruz-ReyesB Sollner-Webb
Jun 8, 2002·Molecular and Cellular Biology·Jorge Cruz-ReyesBarbara Sollner-Webb
Mar 16, 2005·Molecular and Cellular Biology·Julie A LawBarbara Sollner-Webb
Jan 29, 2013·RNA·Man-Gen SongMegerditch Kiledjian
Feb 24, 2006·RNA·Alevtina G ZhelonkinaBarbara Sollner-Webb
Sep 12, 2006·Proceedings of the National Academy of Sciences of the United States of America·Xuedong KangLarry Simpson
Aug 12, 2015·Nucleic Acids Research·Tatsuya MaehigashiChristine M Dunham
Dec 17, 2003·Virology·Yuanzheng ZhangJames L Van Etten
Sep 11, 2019·Nucleic Acids Research·Ian J PavelichChristine M Dunham

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