Dynamics of supercoiled and linear pTZ18U plasmids observed with a long-lifetime metal-ligand complex.
The metal-ligand complex, [Ru(bpy)2(dppz)]2+ (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) (Ru-BD), was used as a spectroscopic probe for studying nucleic acid dynamics. The Ru-BD complex displays a long lifetime of over 100 ns and a molecular light switch property upon DNA binding due to shielding of its dppz ligand from water. To further show the usefulness of this luminophore (Ru-BD) for probing DNA dynamics, we examined its intensity and anisotropy decays when intercalated into supercoiled and linear pTZ18U plasmids using frequency-domain fluorometry with a light-emitting diode (LED) as the modulated light source. Compared to the supercoiled plasmids with an average intensity decay time of 120.8 ns at 25 degrees C, we obtained somewhat longer lifetimes for the linear plasmids ((tau) = 141.4 ns at 25 degrees C), suggesting a more efficient shielding from water by the linear plasmids. The anisotropy decay data also showed longer rotational correlation times for the linear plasmids (495 and 35 ns at 25 degrees C) as compared to the supercoiled plasmids (412 and 27 ns at 25 degrees C). The slow and fast rotational correlation times appear to be consistent with the bending and torsional motions of the plasmids, respectively. The anisotropy values were quite similar, although the values of the supercoiled plasmids were slightly higher in both the steady-state and anisotropy decay measurements. These results indicate that Ru-BD can be applied in the study of both bending and torsional dynamics of nucleic acids.
Kang
Jung Sook
JS
Department of Oral Biochemistry and Molecular Biology, College of Dentistry, Pusan National University, Korea.
Abugo
Omoefe O
OO
Lakowicz
Joseph R
JR
D004278:DNA, Superhelical / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D005454:Fluorescence Polarization; D007700:Kinetics; D008024:Ligands; D008670:Metals; D009690:Nucleic Acid Conformation; D009942:Organometallic Compounds / Q000737:chemistry*; D010619:Phenazines / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry*; D012428:Ruthenium
D004278:DNA, Superhelical; D008024:Ligands; D008670:Metals; D009942:Organometallic Compounds; D010619:Phenazines; D012428:Ruthenium; C077849:bis(2,2'-bipyridyl)(dipyrido(3,2-alpha-2',3'-c)phenazine)ruthenium (II)