A simple fluorescence method for pK(a) determination in RNA and DNA reveals highly shifted pK(a)'s.

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.