6MI Enhanced Fluorescence in a Specific DNA Pentamer Sequence

Abstract

The development of fluorescent nucleoside analogs, which hydrogen bond in the same fashion as their counterparts and minimally distort the structure of duplex DNA, has greatly improved the amount of information that can be obtained from both steady-state and time-resolved fluorescence experiments. Reduction in quantum yield observed when probes are incorporated into an oligomer or a duplex limits their potential application. 6-methylisoxanthopterin (6-MI) is a fluorescent guanosine analog which H-bonds with cytosine similar to guanosine. Investigating the photophysical properties of the nucleoside analog; we discovered a pentamer DNA sequence (ATFAA; where F=6-MI) that exhibits an enhancement of fluorescence upon formation of duplex DNA. The enhanced 6-MI fluorescence within a duplex broadens the potential applications by allowing binding and other experiments to occur at nanomolar concentrations. Within, the sequence context of ATFAA, time-resolved measurements reveal that the fluorescent populations shift from 0.4 to 7.2 ns upon formation of duplex and the relative quantum yield increases from 0.2 to 0.8. This implied the pentamer ATFAA fluorescence enhancement is due to 6MI adopting a single conformation that is either “flipped out” from the duplex or sterically constrained. To further investigate the enhancement of fluorescence upon duplex formation, we characterized oligonucleotides local and global structure. Temperature melt and iodide quenching experiments support a model in which enhancement of fluorescence is due to a solvent inaccessible geometry of 6MI remaining H-bonded to cytosine. An increase in solvent accessibility and reduction in the quantum yield were achieved through the introduction of a 3′ bulge or mismatch in the highly fluorescent duplex; suggesting limited dynamics of the 6MI is due to steric hiderance on the 3′ side. This information can now be used to generate other sequence contexts in which 6-MI will exhibit enhanced fluorescence upon duplex formation.