Temperature and base sequence dependence of 2-aminopurine fluorescence bands in single- and double-stranded oligodeoxynucleotides

Fluorescence excitation spectra of 2-aminopurine (2AP) incorporated into si ngle-stranded DNA di- and trinucleotides, as well as into single- and doubl e-stranded pentanucleotides, have been measured as a function of temperatur e from 5 to 65 degreesC. Spectral shifts have been precisely quantitated th rough difference spectroscopy and spectral fits. G(2AP)C and C(2AP)G oligon ucleotides have relatively blue-shifted excitation spectra (especially the former) compared to the 2AP free base. The position of the excitation peak of 2AP free base is temperature independent, those of (2P9)T, G(2AP)C, C(2A P)G and TT(2AP)TT shift about 0.4 nm to the blue from 5 to 65 degreesC, tho ugh the spectra of the G-C-containing oligomers also change shape. The temp erature dependence of the A(2AP)T spectral position is 2.5-times stronger, and just rises to that of the free base at high temperature. On the other h and, the decrease of yield with increasing temperature is smallest for A(2A P)T even compared to the free base. The dominant effect when A neighbors 2A P appears to be temperature-dependent stacking with accompanying energy tra nsfer, while in G- and C-containing trinucleotides a temperature-independen t interaction keeps the 2AP excitation spectrum blue-shifted. The effect of double strand formation appears to be small compared to stacking interacti ons. These spectra can be useful in identifying base neighbors and structur es of 2AP in unknown 2AP-labeled DNA.