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. Author manuscript; available in PMC: 2017 Sep 13.
Published in final edited form as: Methods Enzymol. 2014;549:189–219. doi: 10.1016/B978-0-12-801122-5.00009-X

Figure 6.

Figure 6

Fluorescence-based determination of pKa using 2AP-substituted oligonucleotides. A) 2AP is structurally similar to adenine and is able to form a two-hydrogen base pair with thymine. B) DNA constructs containing the A+•C wobble (2AP5) and the control construct containing an AT Watson-Crick (2AP-c) base pair. The signal change for the 2AP substitution was optimal when 2AP was positioned directly above the wobble base pair. C) Fluorescence spectra at different pH for 2AP5 construct, with pink corresponding to low pH and blue to high pH. D) Plot of observed fluorescence as a function of pH for 2AP5 (solid circles) and 2AP5-c (hollow circles). 2AP5-c shows no change in fluorescence with pH, whereas the 2AP5 shows a 4-fold change in fluorescence. Adapted with permission from Wilcox et al. 2013. Copyright (2013) American Chemical Society.