title: Photophysical Characterization of Enhanced 6-Methylisoxanthopterin Fluorescence in Duplex DNA.
abstract: The structure and dynamic motions of bases in DNA duplexes and other constructs are important for understanding mechanisms of selectivity and recognition of DNA-binding proteins. The fluorescent guanine analogue, 6-methylisoxanthopterin 6-MI, is well suited to this purpose as it exhibits an unexpected 3- to 4-fold increase in relative quantum yield upon duplex formation when incorporated into the following sequences: ATFAA, AAFTA, or ATFTA (where F represents 6-MI). To better understand some of the factors leading to the 6-MI fluorescence increase upon duplex formation, we characterized the effect of local sequence and structural perturbations on 6-MI photophysics through temperature melts, quantum yield measurements, fluorescence quenching assays, and fluorescence lifetime measurements. By examining 21 sequences we have determined that the duplex-enhanced fluorescence (DEF) depends on the composition of bases adjacent to 6-MI and the presence of adenines at locations n ± 2 from the probe. Investigation of duplex stability and local solvent accessibility measurements support a model in which the DEF arises from a constrained geometry of 6-MI in the duplex, which remains H-bonded to cytosine, stacked with adjacent bases and inaccessible to quenchers. Perturbation of DNA structure through the introduction of an unpaired base 3' to 6-MI or a mismatched basepair increases 6-MI dynamic motion leading to fluorescence quenching and a reduction in quantum yield. Molecular dynamics simulations suggest the enhanced fluorescence results from a greater degree of twist at the X-F step relative to the quenched duplexes examined. These results point to a model where adenine residues located at n ± 2 from 6-MI induce a structural geometry with greater twist in the duplex that hinders local motion reducing dynamic quenching and producing an increase in 6-MI fluorescence.
authors.affiliation: Departments of Chemistry and ‡Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University , 52 Lawn Ave, Middletown, Connecticut 06459, United States.
authors.forename: Andrew
authors.initials: A
authors.lastname: Moreno
authors.affiliation: Departments of Chemistry and ‡Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University , 52 Lawn Ave, Middletown, Connecticut 06459, United States.
authors.forename: J L
authors.initials: JL
authors.lastname: Knee
authors.affiliation: Departments of Chemistry and ‡Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University , 52 Lawn Ave, Middletown, Connecticut 06459, United States.
authors.forename: Ishita
authors.initials: I
authors.lastname: Mukerji
mesh_terms: D004247:DNA / Q000737:chemistry*; D005453:Fluorescence; D055668:Photochemical Processes; D013816:Thermodynamics; D014976:Xanthopterin / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: C483770:6-methylisoxanthopterin; D004247:DNA; D014976:Xanthopterin