title: A simple fluorescence method for pK(a) determination in RNA and DNA reveals highly shifted pK(a)'s.
abstract: Charged nucleobases exist in RNA and DNA at neutral pH owing to pK(a) shifting. These bases can affect polymerase fidelity and participate in ribozyme general acid-base catalysis. Protonated RNA bases further influence miRNA processing and viral frameshifting. It is therefore important to have a simple and rapid method for determining the pKa of nucleobases in RNA and DNA. Here we describe the application of 2-aminopurine (2AP), a fluorescent isomer of adenine, to report on the pK(a) of a nearby ionizing base both in DNA secondary structure and RNA tertiary structure. We observe large, up to 5-fold quenching in fluorescence upon protonation of a nearby base. Using this method, we identify highly shifted pK(a)'s of 7.6 for adenine in a DNA oligonucleotide and 8.15 for cytidine in a tertiary structure element from beet western yellows virus (BWYV) RNA. These pK(a) values, which were corroborated by (31)P NMR measurements and comparison to literature, are shifted over 4 units from their standard values. This fluorescence method can be used to determine pK(a)'s for ionization of both A and C and reveals that shifted pK(a)'s are prevalent in DNA and RNA secondary and tertiary structures.
authors: Department of Chemistry and Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
authors: Jennifer L
authors: JL
authors: Wilcox
authors: Philip C
authors: PC
authors: Bevilacqua
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D005453:Fluorescence; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D017793:Luteovirus / Q000737:chemistry; D009690:Nucleic Acid Conformation; D011522:Protons; D012367:RNA, Viral / Q000737:chemistry*
chemical_list: D011522:Protons; D012367:RNA, Viral; D015075:2-Aminopurine; D004247:DNA