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Keywords:

  • DNA;
  • energy transfer;
  • fluorescence;
  • fluorescent probes;
  • G-quadruplexes;
  • RNA

Abstract

Because of their environment-sensitive fluorescence quantum yields, base analogues such as 2-aminopurine (2AP), 6-methylisoxanthopterin (6-MI), and 3-methylisoxanthopterin (3-MI) are widely used in nucleic-acid folding and catalysis assays. Emissions from these guanine mimics are quenched by base-stacking interactions and collisions with purine residues. Fluorescent base analogues that remain highly emissive in folded nucleic acids can provide sensitive means to differentiate DNA/RNA structures by participating in energy transfer from proximal ensembles of unmodified nucleobases. The development of new, highly emissive guanine mimics capable of proper base stacking and base-pairing interactions is an important prerequisite to this approach. Here we report a comparison of the most commonly used probe, 2-aminopurine (2AP), to 8-(2-pyridyl)-2′-deoxyguanosine (2PyG). The photophysical properties of these purine derivatives are very different. 2PyG exhibits enhanced fluorescence quantum yields upon its incorporation into folded nucleic acids—approximately 50-fold brighter fluorescence intensity than 2AP in the context of duplex DNA. Due to its bright fluorescence and compatibility with proper DNA folding, 2PyG can be used to accurately quantify energy-transfer efficiencies, whereas 2AP is much less sensitive to structure-specific trends in energy transfer. When using nucleoside monomers, Stern–Volmer plots of 2AP fluorescence revealed upward curvature of F0/F upon titration of guanosine monophoshate (GMP), whereas 2PyG exhibited unusual downward curvature of F0/F that resulted in a recovery of fluorescence at high GMP concentrations. These results are consistent with the trends observed for 2PyG- and 2AP-containing oligonucleotides, and furthermore suggest that solutions containing high concentrations of GMP can, in some ways, mimic the high local nucleobase densities of folded nucleic acids.