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.
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