The conformation of a bulged DNA base, whether looped-out of the DNA helix or stacked-in between the flanking bases, can be distinguished using fluorescence spectroscopy of an inserted fluorescent base. If 2-aminopurine, a structural analog of adenine and guanine, is placed in duplex DNA as the bulged base replacing an adenine or guanine, it loops out of the DNA helix into solution. This is determined by the decrease or increase of 2-aminopurine fluorescence during DNA thermomelting: if the 2-aminopurine base stacks into the helix, its fluorescence increases or remains about the same during DNA duplex melting, but if the 2-aminopurine base loops out of the helix, its fluorescence decreases upon melting of the DNA duplex.

The fluorescence spectroscopy of 7-azaindole ( 7a In) incorporated in DNA oligonucleotides is investigated. Incorporation of 7a In into DNA oligonucleotides is accomplished through standard solid-phase phosphoramidite chemistry. Fluorescence emission of the 7a In chromophore shifts slightly to the red (from 386 nm to 388 nm) upon glycosylation at the N − 1 position, but its relative fluorescence quantum yield increases 23 times, from 0.023 to 0.53. Upon incorporation into DNA, the fluorescence emission of 7a In is greatly quenched with fluorescence quantum yields of 0.020 and 0.016 in single and double strand DNA, respectively. The fluorescence emission for 7a In in DNA oligonucleotides shifts to the blue with an emission maximum at 379 nm. Both the strong fluorescence quenching and the blue shift of the emission spectrum signify that 7a In is stacked with neighboring DNA bases in both single and double strand DNA. As the duplex DNA melts due to temperature increase, the fluorescence of the 7a In chromophore increases, indicating the transition from the less fluorescent duplex DNA to the more fluorescent single strand DNA. Since this fluorescent 7a In is a structural analog of purine, its fluorescence property may be utilized as a probe for studying nucleic acid structure and dynamics.