Observation of Oligonucleotide Dynamics by means of Fluorescent Nucleoside analog 6MI

Abstract

To improve current understanding of the structural recognition mechanism of architectural DNA binding proteins such as HU and IHF, we are investigating the structure and dynamics of different DNA substrates. We are able to make these observations on both global and local levels by incorporating the fluorescent guanosine nucleoside analog 6-methylisoxanthopterin (6-MI), with H-bonds with cytosine similar to guanosine. We have previously shown this probe does not significantly perturb the global structures of duplex DNA molecules. 6-MI was systematically incorporated into a 34 base oligonucleotide. Initial characterization of local DNA environment included time resolved fluorescence and rotational correlation measurements of the duplex oligomers relative to 6-MI monomer and single stranded DNA. Analysis of time-resolved fluorescence decay yields 3 lifetime components of 0.4 ns, 4 ns and 6.5 ns. The largest lived component is similar to that of 6-MI monomer, 7 ns. The position of the probe shifts the fluorescent populations from 0.4 ns to 6.5ns upon formation of duplex, which implies that 6-MI local environment in these positions resembles that of the solvent exposed monomer. However, no direct correlation between adjacent base sequence and the fluorescent properties of 6MI was observed. To further investigate the increase in fluorescence upon duplex formation, we characterized the local and global structure of several oligonucleotides through temperature melts, quantum yield calculations, quenching assays, and Raman spectroscopy. The results suggest that, the position of 6-MI in the duplex sequence, helical turn, and surrounding base sequence determines the dynamics of 6-MI. This potentially leads to the formation of a fixed geometry of 6-MI which stacks poorly with adjacent bases. The lack of stacking interactions causes 6-MI to exhibit fluorescent properties of the monomer.