We extended the measurable time scale of DNA dynamics to microsecond using $[Ru(phen)_2(dppz)]^{2+}$ (phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine) (RuPD), which displays a mean lifetime near 500 ns. To evaluate the usefulness of this luminophore (RuPD) for probing ...
We extended the measurable time scale of DNA dynamics to microsecond using $[Ru(phen)_2(dppz)]^{2+}$ (phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine) (RuPD), which displays a mean lifetime near 500 ns. To evaluate the usefulness of this luminophore (RuPD) for probing nucleic acid dynamics, its intensity and anisotropy decays when intercalated into supercoiled and linear pBluescript (pBS) II SK(+) phagemids were examined using frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source. The mean lifetime for the supercoiled phagemids (< $\tau$ > = 489.7 ns) was somewhat shorter than that for the linear phagemids (< $\tau$ > = 506.4 ns), suggesting a more efficient shielding from water by the linear phagemids. The anisotropy decay data also showed somewhat shorter slow rotational correlation times for supercoiled phagemids (997.2 ns) than for the linear phagemids (1175.6 ns). The slow and fast rotational correlation times appear to be consistent with the bending and torsional motions of the phagemids, respectively. These results indicate that RuPD can have applications in studies of both bending and torsional dynamics of nucleic acids.
We extended the measurable time scale of DNA dynamics to microsecond using $[Ru(phen)_2(dppz)]^{2+}$ (phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine) (RuPD), which displays a mean lifetime near 500 ns. To evaluate the usefulness of this luminophore (RuPD) for probing nucleic acid dynamics, its intensity and anisotropy decays when intercalated into supercoiled and linear pBluescript (pBS) II SK(+) phagemids were examined using frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source. The mean lifetime for the supercoiled phagemids (< $\tau$ > = 489.7 ns) was somewhat shorter than that for the linear phagemids (< $\tau$ > = 506.4 ns), suggesting a more efficient shielding from water by the linear phagemids. The anisotropy decay data also showed somewhat shorter slow rotational correlation times for supercoiled phagemids (997.2 ns) than for the linear phagemids (1175.6 ns). The slow and fast rotational correlation times appear to be consistent with the bending and torsional motions of the phagemids, respectively. These results indicate that RuPD can have applications in studies of both bending and torsional dynamics of nucleic acids.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
제안 방법
FD intensity and anisotropy decay measurements Measurements were performed with an ISS Koala instrument (ISS Inc., Champaign, USA) using a blue LED LNG992CFBW (Panasonic, Japan) as the excitation source. An LED driver LDX-3412 (ILX Lightwave, Boseman, USA) provided 30 mA of current at frequencies from 0.
In this study, we demonstrated the usefulness of RuPD, a long-lifetime MLC for probing the bending and torsional dynamics of supercoiled and linear pBS II SK(+) phagemids. The most important observation of this report is that the use of RuPD, which has a lifetime of about 500 ns (Table 1), enabled us to extend the measurable time scale of DNA dynamics to microsecond.
UVvisible absorption spectra were measured with a Hewlett-Packard 8453 diode array spectrophotometer. Steady-state intensity and anisotropy measurements were carried out using a Cary Eclipse fluorescence spectrophotometer (Varian Inc., Palo Alto, USA).
대상 데이터
Agarose gel electrophoresis pattern of supercoiled and linear pBluescript (pBS) II SK(+) phagemids. 1kb DNA ladder was used.
이론/모형
6), and the results are summarized in Table 2. The best fits of the anisotropy decay data were obtained using the two correlation time model. The slow and fast rotational correlation times appear to be consistent with the bending (flexure of the helix axis) and torsional (twisting of bp) motions of phagemids, respectively.
참고문헌 (27)
Badea, M. G. and Brand, L. (1979) Time-resolved fluorescence measurements. Methods Enzymol. 61, 378-425
Chirico, G. and Baldini, G. (1996) Rotational diffusion and internal motions of circular DNA. II. Depolarized photon correlation spectroscopy. J. Chem. Phys. 104, 6020-6026
DeGraff, B. A. and Demas, J. N. (1994) Direct measurement of rotational correlation times of luminescent ruthenium(II) molecular probes by differential polarized phase fluorometry. J. Phys. Chem. 98, 12478-12480
Friedman, A. E., Chambron, J. -C., Sauvage, J. -P., Turro, N. J. and Barton, J. K. (1990) Molecular light switch for DNA: $Ru(bpy)_2(dppz)^{2+}$ . J. Am. Chem. Soc. 112, 4960-4962
Gratton, E., Limkeman, M., Lakowicz, J. R., Maliwal, B. P., Cherek, H. and Laczko, G. (1984) Resolution of mixtures of fluorophores using variable-frequency phase and modulation data. Biophys. J. 46, 479-486
Haugen, G. R. and Lytle, F. E. (1981) Quantitation of fluorophores in solution by pulsed laser excitation of time-filtered detection. Anal. Chem. 53, 1554-1559
Holmin, R. E., Stemp, E. D. A. and Barton, J. K. (1998) $Ru(phen)_2dppz^+$ luminescence: dependence on DNA sequences and groove-binding agents. Inorg. Chem., 37, 29-34
Jenkin, Y., Friedman, A. E., Turro, N. J. and Barton, J. K. (1992) Characterization of dipyridophenazine complexes of ruthenium(II): The light switch effect as a function of nucleic acid sequence and conformation. Biochemistry 31, 10809- 10816
Kang, J. S., Abugo, O. O. and Lakowicz, J. R. (2002a) Dynamics of supercoiled and linear pTZ18U plasmids observed with a long-lifetime metal-ligand complex. Biopolymers 67, 121-128
Kang, J. S., Abugo, O. O. and Lakowicz, J. R. (2002b) Dynamics of supercoiled and relaxed pTZ18U plasmids probed with a long-lifetime metal-ligand complex. J. Biochem. Mol. Biol. 35, 389-394
Kang, J. S. and Lakowicz, J. R. (2001) Fluorescence resonance energy transfer in calf thymus DNA from a long-lifetime metal-ligand complex to nile blue. J. Biochem. Mol. Biol. 34, 551-558
Kang, J. S., Piszczek, G. and Lakowicz, J. R. (2002c) Highmolecular- weight protein hydrodynamics studied with a longlifetime metal-ligand complex. Biochim. Biophys. Acta 1597, 221-228
Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy, pp. 98-101, Kluwer Academic/Plenum Publishers, New York, USA
Lakowicz, J. R., Cherek, H., Kusba, J., Gryczinski, I. and Johnson, M. L. (1993) Review of fluorescence anisotropy decay analysis by frequency-domain fluorescence spectroscopy. J. Fluorescence 3, 103-116
Lakowicz, J. R. and Gryczinski, I. (1991) Frequency-domain fluorescence spectroscopy; in Topics in Fluorescence Spectroscopy, Volume 1, Techniques, Lakowicz, J. R. (ed.), pp. 293-355, Plenum Press, New York, USA
Lakowicz, J. R., Gryczinski, I., Piszczek, G., Tolosa, L., Nair, R., Johnson, M. L. and Nowaczyk, K. (2000) Microsecond dynamics of biological molecules. Methods Enzymol. 323, 473- 509
Lakowicz, J. R., Laczko, G., Cherek, H., Gratton, E. and Limkeman, M. (1984) Analysis of fluorescence decay kinetics from variable-frequency phase-shift and modulation data. Biophys. J. 46, 463-477
Lakowicz, J. R., Malak, H., Gryczynski, I., Castellano, F. N. and Meyer, G. J. (1995) DNA dynamics observed with long lifetime metal-ligand complexes. Biospectroscopy 1, 163-168
Lakowicz, J. R., Piszczek, G. and Kang, J. S. (2001) On the possibility of long-wavelength long-lifetime high-quantum yield luminophores. Anal. Biochem. 288, 62-75
Langowski, J. and Giesen, U. (1989) Configurational and dynamic properties of different length superhelical DNAs measured by dynamic light scattering. Biophys. Chem. 34, 9-18
Langowski, J., Kremer, W. and Kapp, U. (1992) Dynamic light scattering for study of solution conformation and dynamics of superhelical DNA. Methods Enzymol. 211, 430-448
Malak, H., Gryczinski, I., Lakowicz, J. R., Meyers, G. J. and Castellano, F. N. (1997) Long-lifetime metal-ligand complexes as luminescent probes for DNA. J. Fluorescence 7, 107-112
Schurr, J. M., Fujimoto, B. S., Wu, P. and Song, L. (1992) Fluorescence studies of nucleic acids: dynamics, rigidities, and structures; in Topics in Fluorescence Spectroscopy, Volume 3, Biochemical Applications, Lakowicz, J. R. (ed.), pp. 137-229, Plenum Press, New York, USA
Terpetschnig, E., Szmacinski, H. and Lakowicz, J. R. (1997) Long-lifetime metal-ligand complexes as probes in biophysics and clinical chemistry. Methods Enzymol. 278, 295-321
※ AI-Helper는 부적절한 답변을 할 수 있습니다.