Fluorescence Studies of Nucleotides and Polynucleotides

Abstract

Formycin, 2-aminopurine ribonucleoside, and 2,6-diaminopurine ribonucleoside are analogues of adenosine in base pairing and in a wide variety of enzymatic reactions. These chromophores, in contrast to adenine and other naturally occurring bases, are fluorescent under physiological conditions. Another noteworthy feature of these analogues is that they can be selectively excited in the presence of normal bases because their absorption bands occur at longer wave lengths. The fluorescence properties of these compounds, and of their phosphorylated derivatives, over a wide range of temperature, pH, and ionic strength, are reported here. Incorporation of formycin or 2-aminopurine into a perfectly alternating ribocopolymer results in a 100-fold reduction of the quantum yield of fluorescence. The residual fluorescence comes from chromophores at the ends of the polymer. Thermal denaturation of the helical structure is accompanied by a 3- to 10-fold increase in quantum yield. The pH and temperature dependence of the fluorescence of transfer RNA with formycin residue in place of the terminal adenosine indicates that the terminal formycin residue interacts with other parts of the transfer RNA molecule. Some potential applications of these analogues in studies of the structure and function of nucleic acids and proteins are: analysis of protein-nucleic acid interaction, polynucleotide conformation and conformational transitions, enzyme-coenzyme interaction, and nuclease assay.