TY - JOUR
T1 - Buffer-Dependent Photophysics of 2-Aminopurine
T2 - Insights into Fluorescence Quenching and Excited-State Interactions
AU - Poddar, Souvik
AU - Levitus, Marcia
N1 - Publisher Copyright:
© 2024 American Chemical Society
PY - 2024/3/21
Y1 - 2024/3/21
N2 - 2-Aminopurine (2AP) is the most widely used fluorescent nucleobase analogue in DNA and RNA research. Its unique photophysical properties and sensitivity to environmental changes make it a useful tool for understanding nucleic acid dynamics and DNA-protein interactions. We studied the effect of ions present in commonly used buffer solutions on the excited-state photophysical properties of 2AP. Fluorescence quenching was negligible for tris(hydroxymethyl)aminomethane (TRIS), but significant for phosphate, carbonate, 3-(N-morpholino) propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers. Results indicate that the two tautomers of 2AP (7H, 9H) are quenched by phosphate ions to different extents. Quenching by the H2PO4- ion is more pronounced for the 7H tautomer, while the opposite is true for the HPO42- ion. For phosphate ions, the results of the time-resolved fluorescence study cannot be explained using a simple collisional quenching mechanism. Instead, results are consistent with transient interactions between 2AP and the phosphate ions. We postulate that excited-state interactions between the 2AP tautomers and an H-bond acceptor (phosphate and carbonate) result in significant quenching of the singlet-excited state of 2AP. Such interactions manifest in biexponential fluorescence intensity decays with pre-exponential factors that vary with quencher concentration, and downward curvatures of the Stern-Volmer plots.
AB - 2-Aminopurine (2AP) is the most widely used fluorescent nucleobase analogue in DNA and RNA research. Its unique photophysical properties and sensitivity to environmental changes make it a useful tool for understanding nucleic acid dynamics and DNA-protein interactions. We studied the effect of ions present in commonly used buffer solutions on the excited-state photophysical properties of 2AP. Fluorescence quenching was negligible for tris(hydroxymethyl)aminomethane (TRIS), but significant for phosphate, carbonate, 3-(N-morpholino) propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers. Results indicate that the two tautomers of 2AP (7H, 9H) are quenched by phosphate ions to different extents. Quenching by the H2PO4- ion is more pronounced for the 7H tautomer, while the opposite is true for the HPO42- ion. For phosphate ions, the results of the time-resolved fluorescence study cannot be explained using a simple collisional quenching mechanism. Instead, results are consistent with transient interactions between 2AP and the phosphate ions. We postulate that excited-state interactions between the 2AP tautomers and an H-bond acceptor (phosphate and carbonate) result in significant quenching of the singlet-excited state of 2AP. Such interactions manifest in biexponential fluorescence intensity decays with pre-exponential factors that vary with quencher concentration, and downward curvatures of the Stern-Volmer plots.
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U2 - 10.1021/acs.jpcb.3c07269
DO - 10.1021/acs.jpcb.3c07269
M3 - Article
AN - SCOPUS:85187329482
SN - 1520-6106
VL - 128
SP - 2640
EP - 2651
JO - Journal of Physical Chemistry B
JF - Journal of Physical Chemistry B
IS - 11
ER -