Fluorescence probing of metal-ion-mediated hybridization of oligonucleotides

Abstract

The sensitivity of fluorescence of pyrrolocytosine-containing oligonucleotides to changes in their secondary structure has been harnessed to monitor the hybridization of modified metal-ion-chelating oligonucleotides with their unmodified counterparts. With short double-helical oligonucleotides, quenching of fluorescence correlated well with the length and, hence, T_m of the duplex provided that the pyrrolo-dC residue was incorporated within the duplex. Furthermore, hybridization of the metal-ion-chelating oligonucleotides was greatly enhanced on addition of 1 eq. of Cu²⁺, as evidenced by both T_m and fluorometric measurements. As an example of a case where interpretation of a conventional UV-melting profile is challenging, application of the fluorometric method was extended to probing hybridization of short metal-ion-carrying oligonucleotides with the trinucleotide bulge motif of TAR RNA models. In this case the results were more ambiguous, presumably due to weak hybridization with the short target sequence.