A Simple Fluorescence Method for pK(a) Determination in RNA and DNA Reveals Highly Shifted pK(a)'s

作者：Wilcox Jennifer L; Bevilacqua Philip C^*

来源：Journal of the American Chemical Society, 2013, 135(20): 7390-7393.

DOI：10.1021/ja3125299

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 plc of nucleobases in RNA and DNA. Here we describe the application of 2-aminopurine (ZAP), 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 P-31 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.

• 出版日期2013-5-22