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 pK_a 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 ³¹P 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.