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Cellular uptake of modified oligonucleotides: fluorescence approach
Journal of Molecular Structure 744–747 (2005) 151–153 www.elsevier.com/locate/molstruc
Cellular uptake of modified oligonucleotides: fluorescence approach Eva Kocˇisˇova´a,*, Petr Prausa, Ivan Rosenbergb, Olivier Seksekc, Franck Sureauc, Josef Sˇteˇpa´neka, Pierre-Yves Turpinc a
Faculty of Mathematics and Physics, Division of Biomolecular Physics, Institute of Physics, Charles University, Ke Karlovu 5, CZ-12116 Prague 2, Czech Republic b Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo sq. 2, 16610 Prague 6, Czech Republic c Laboratoire de Physicochimie Biomole´culaire et Cellulaire (CNRS ESA 7033), Universite´ P. et M. Curie, Case 138, 4 Place Jussieu, F-75252 Paris Cedex 05, France Received 31 October 2004; accepted 18 November 2004 Available online 11 January 2005
Abstract Cellular uptake and intracellular distribution of the synthetic antisense analogue of dT15 oligonucleotide (homogenously containing 3 0 -O– P–CH2–O-5 0 internucleotide linkages and labeled with tetramethylrhodamine dye) was studied on B16 melanoma cell line by fluorescence micro-imaging and time-resolved microspectrofluorimetry. By using amphotericin B 3-dimethylaminopropyl amide as an enhancer molecule for the uptake process, homogenous staining of the cells with rather distinct nucleoli staining was achieved after 4 h of incubation. Two spectral components of 2.7 and 1.3 ns lifetime, respectively, were resolved in the emission collected from the cell nucleus. The way of staining and the long-lived component differed from our previous experiments demonstrating complexity of the intracellular oligonucleotide distribution and in particular of the binding inside the nucleus. q 2004 Elsevier B.V. All rights reserved. Keywords: Antisense oligonucleotide; Cellular uptake; Fluorescence microimaging; Phase resolved fluorescence spectroscopy
1. Introduction Advancement in research of novel synthetic modified oligonucleotides for the application in antisense, antigene and aptamer technologies forged ahead recently. These molecules (single-stranded sequences of modified deoxyribo- or ribonucleotides) are significantly effective in downregulation and/or blocking of the gene expression (transcription arrest—antigene technology, translation arrest—antisense technology and arrest of the function of the target protein—aptamer technology) in the case of various viral or malignant diseases. It can also be of great help in testing and could contribute to better insight into the biological function of any gene [1–7]. The cellular membrane passage with required proper intracellular distribution and stability of prepared * Corresponding author. Tel.: C420 221 911 471; fax: C420 224 922 797. E-mail address: [email protected] (E. Kocˇisˇova´). 0022-2860/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2004.11.057
oligonucleotide analogues must always be guaranteed besides minimizing their negative side effects. The search for appropriate delivery system is more or less common problem of the above-mentioned strategies. Majority of transfected oligonucleotide molecules in cellular uptake experiments tends to pass the membrane by endocytosis mechanism with subsequent sequestration into endosome/ lysosomal compartments which is characterized by punctuate cytoplasmic distribution. Compartmentalized oligonucleotides are considered to be degraded and so they cannot accomplish ‘antisense’ and/or ‘antigene’ function [8]. In our studies we apply two fluorescence techniques (fluorescence microimaging and multifrequency phase/modulation microspectrofluorimetry) to monitor the modified oligonucleotide penetration inside the cell, its distribution in the cellular environment and subsequent interaction with the subcellular structures [9–11]. Recently we used successfully amphotericin B 3-dimethylaminopropyl amide as an enhancer molecule for the uptake process.
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E. Kocˇisˇova´ et al. / Journal of Molecular Structure 744–747 (2005) 151–153
The 3T3 and B16 cell lines incubated with dT15 oligonucleotide analog, homogenously modified by phosphonatebased 3 0 -O–P–CH2–O-5 0 linkage and labeled by tetramethylrhodamine marker, exhibited regular intensive staining of the cell nucleus connected with two-component peculiar fluorescence signal [12]. In this work we proceeded with studies of the oligonucleotide penetration into B16 cells and analyzed time-resolved fluorescence signal from the nucleus in the case of another type of the intracellular staining process.
2. Experimental 2.1. Chemicals and the cell culture The modified oligonucleotide—oligothymidylate (15-mer)—with the 3 0 -O–P–CH2–O-5 0 internucleotide linkage labeled by tetramethylrhodamine (TMR) at the 3 0 end was prepared in two-steps: synthesis of modified oligomer and coupling of the oligomer with the rhodamine marker. The used chemical binding of TMR had been found as very stable in the cellular environment [11]. Amphotericin B 3-dimethylaminopropyl amide (AmA) for oligonucleotide uptake enhancement was kindly provided by Prof. Borowski (Technical University of Gdansk, Poland). Mouse melanoma cell line (B16) used in the study was cultured as monolayers in 25 cm2 flask at 37 8C in a humidified 5% CO2 atmosphere, in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, streptomycin (0.1 mg/ml) and penicillin (100 U/ml), all from Biomedia. Cells were subcultured in 35 mm diameter Petri dishes for 48 h before time resolved microspectrofluorimetric and fluorescence imaging experiments. Cells were treated by AmA/oligonucleotide mixture 4 h before the experiment. AmA/oligonucleotide mixture was prepared of 120 ml of AmA stock solution (10K3 M) gently mixed with 15 ml of the oligonucleotide solution (stock solution 1.6!10K4 M). After 15 min the mixture was added into prepared Petri dishes with cells and incubated at 37 8C in a standard humidified atmosphere.
The microspectrofluorimeter is configured for a multifrequency heterodyne measurement mode. Its detailed optical and electronic scheme was described elsewhere [13]. Laser excitation beam intensity is sine modulated from 1 to 200 MHz (set of eight frequencies 30, 45, 50, 90, 100, 135, 154 and 175 MHz is used in our experiment). Elastic scattering taken as a reference and fluorescence signal are collected in a back-scattering geometry and focused on the input slit of a spectrograph. A high frequency gain modulated MCP image intensifier is placed in front of an optical multichannel analyzer. Its gain is sine modulated in precise phase-lock to the excitation laser beam with a 1 Hz frequency offset (cross-correlation detection). Phase shift and demodulation due to the fluorescence lifetime are preserved in a low frequency modulation (1 Hz) of the received signal, detected for each detector pixel by means of a synchronizing unit. The determined spectral dependence of the phase shift and modulation is treated by a fitting procedure succeeded by Global analysis that is able to decompose multi-component fluorescence spectra to particular spectral components characterized by their specific lifetimes [13].
3. Results and discussion AmA makes possible to cross directly the oligonucleotide through membrane inside the cell (not by the endocytosis) and to achieve its regular distribution in the subcellular compartments [14], although its mechanism of action is still not understood completely. The fluorescence image (Fig. 1) shows intracellular distribution of modified oligonucleotide TMR-dT15 in mouse melanoma cells (line B16) observed after approx. 4 h incubation with AmA/oligonucleotide mixture. The cells are stained
2.2. Measuring methods Fluorescence micro-imaging experiments were performed on an Optiphot-2 epifluorescence microscope equipped with a Nipkow wheel coaxial-confocal attachment (Technical Instrument). Confocal fluorescence images were collected by aqueous-immersion objective (Zeiss, Neofluar X63—numerical aperture 1.2) and imaged by TE cooled CCD camera (Micromax, Princeton Instruments). TMR emission was visualized with a standard rhodamine filter set (BP 530G15, LP 600) using 4 s exposure time. Camera images were processed by IPLab software (Scanalytics).
Fig. 1. Fluorescence image of the mouse melanoma B16 cells after 4 h incubation with AmA/TMR-dT15 mixture. The circle indicates the region from which the emission for time-resolved microspectrofluorimetric measurements was collected.
E. Kocˇisˇova´ et al. / Journal of Molecular Structure 744–747 (2005) 151–153
Fig. 2. Decomposition of the emission spectrum (solid line) collected from the nucleus region into two spectral components. Dashed-line with circles: 2.7 ns component, dashed-line with triangles: 1.3 ns component.
almost homogenously with only more distinct staining of the nucleoli and part of the nucleus membrane. This result differs though radically from those obtained in our previous experiments carried out at equal conditions [12] when after 3 h incubation almost exclusive staining of the nucleus was observed and just after 24 h incubations the subsequent oligonucleotide redistribution throughout the cellular volume occurred. The differences in the staining development demonstrate that the distribution of the modified oligonucleotide may proceed in various ways depending (probably) very sensitively on the state of the cells and the experiment conditions. Although we still do not know the reason for the different staining process, it provided us with a new type of intracellular oligonucleotide distribution for time-resolved microfluorimetric measurements. Fig. 2 displays fluorescence time-resolved spectra coming from the region of the cell nucleus. After decomposition into two fluorescence components we obtained the major component (92% of integral intensity) with lifetime of 2.7 ns and spectral maximum at 586 nm and the minor component with 1.3 ns lifetime and emission maximum at about 570 nm. This result confirms existence of the exceptional short-lived component (1.4 ns lifetime in our previous experiment [12]) without any analogue in our reference experiments [11]. The 2.7 ns lifetime corresponds to the lifetime of TMR in interaction with DNA [11]. In the previous experiment [12] the second component was however of substantially longer lifetime (4.84 ns), lower intensity, and slightly shifted maximum to higher wavelengths. This noticable difference we interpret as a proof of several types of possible binding partners for the modified oligonucleotide inside the nucleus and/or nucleolus. The complex formations lead to various
153
emission characteristics. Due to unequal thermodynamic stabilities, varied level of the staining influences the proportions of particular spectral components. Decomposition into only two components is not able to characterize the emission completely, but describes only the dominant components whose characteristics are somewhat disturbed by the unresolved ones. Our method is unfortunately not able to provide reliable decomposition into more than two components because of limited current precision of the experimental data. Further progress we expect after enlarging our set of reference data (emission characteristics of the oligonucleotide complexes with various model biomolecules) and improvement of the microspectrofluorimeter to carry out experiments at higher modulation frequencies (increased precision of the phase shift and demodulation data).
Acknowledgements Special thanks of authors are addressed to Prof. J. Bok (Institute of Physics) for writing the procedure for the spectra fitting. The financial support awarded by the Grant Agency of the Czech Republic (project No. 202/03/D118) and by the Czech Ministry of Education (MSM 113200001) is gratefully acknowledged.
References [1] [2] [3] [4]
[5] [6] [7] [8] [9]
[10] [11] [12] [13] [14]
E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990) 543. C. Helene, Eur. J. Cancer 27 (1991) 1466. J. Kurreck, Eur. J. Biochem. 270 (2003) 1628. V.V. Vlassov, I.E. Vlassova, L.V. Pautova, in: K. Moldave (Ed.), Progress in Nucleic Acid Research and Molecular Biology, Oligonucleotides and Polynucleotides as Biologically Active Compounds, Academic Press, San Diego, 1997, pp. 95–143. A.M. Gewirtz, C.A. Stein, P.M. Glazer, Proc. Natl Acad. Sci. USA 93 (1996) 3161. G. Wang, M.M. Seidman, P.M. Glazer, Science 271 (1996) 802. M.D. Matteucci, R.W. Wagner, Nature 384 (1996) 20. A.J. Hudson, W. Lee, J. Porter, J. Akhtar, R. Duncan, S. Akhtar, Int. J. Pharm. 133 (1996) 257. P. Praus, D. Gaskova, E. Kocisova, R. Chaloupka, J. Stepanek, J. Bok, D. Rejman, I. Rosenberg, P.-Y. Turpin, F. Sureau, Biopolymers 67 (2002) 339. P. Praus, F. Sureau, E. Kocisova, I. Rosenberg, J. Stepanek, P.-Y. Turpin, Spectrosc. Int. J. 17 (2003) 429. E. Kocisova, F. Sureau, P. Praus, I. Rosenberg, J. Stepanek, P.-Y. Turpin, J. Mol. Struct. 115 (2003) 651. E. Kocisova, P. Praus, I. Rosenberg, O. Seksek, F. Sureau, J. Stepanek, P.-Y. Turpin, Biopolymers 74 (2004) 110. P. Praus, F. Sureau, J. Fluorescence 10 (2000) 361. Ch. Garcia-Chaumont, O. Seksek, B. Jolles, J. Bolard, Antisense Nucleic Acid Drug Dev. 10 (2000) 177.
Cellular uptake of modified oligonucleotides: fluorescence approach Eva Kocˇisˇova´a,*, Petr Prausa, Ivan Rosenbergb, Olivier Seksekc, Franck Sureauc, Josef Sˇteˇpa´neka, Pierre-Yves Turpinc a
Faculty of Mathematics and Physics, Division of Biomolecular Physics, Institute of Physics, Charles University, Ke Karlovu 5, CZ-12116 Prague 2, Czech Republic b Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo sq. 2, 16610 Prague 6, Czech Republic c Laboratoire de Physicochimie Biomole´culaire et Cellulaire (CNRS ESA 7033), Universite´ P. et M. Curie, Case 138, 4 Place Jussieu, F-75252 Paris Cedex 05, France Received 31 October 2004; accepted 18 November 2004 Available online 11 January 2005
Abstract Cellular uptake and intracellular distribution of the synthetic antisense analogue of dT15 oligonucleotide (homogenously containing 3 0 -O– P–CH2–O-5 0 internucleotide linkages and labeled with tetramethylrhodamine dye) was studied on B16 melanoma cell line by fluorescence micro-imaging and time-resolved microspectrofluorimetry. By using amphotericin B 3-dimethylaminopropyl amide as an enhancer molecule for the uptake process, homogenous staining of the cells with rather distinct nucleoli staining was achieved after 4 h of incubation. Two spectral components of 2.7 and 1.3 ns lifetime, respectively, were resolved in the emission collected from the cell nucleus. The way of staining and the long-lived component differed from our previous experiments demonstrating complexity of the intracellular oligonucleotide distribution and in particular of the binding inside the nucleus. q 2004 Elsevier B.V. All rights reserved. Keywords: Antisense oligonucleotide; Cellular uptake; Fluorescence microimaging; Phase resolved fluorescence spectroscopy
1. Introduction Advancement in research of novel synthetic modified oligonucleotides for the application in antisense, antigene and aptamer technologies forged ahead recently. These molecules (single-stranded sequences of modified deoxyribo- or ribonucleotides) are significantly effective in downregulation and/or blocking of the gene expression (transcription arrest—antigene technology, translation arrest—antisense technology and arrest of the function of the target protein—aptamer technology) in the case of various viral or malignant diseases. It can also be of great help in testing and could contribute to better insight into the biological function of any gene [1–7]. The cellular membrane passage with required proper intracellular distribution and stability of prepared * Corresponding author. Tel.: C420 221 911 471; fax: C420 224 922 797. E-mail address: [email protected] (E. Kocˇisˇova´). 0022-2860/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2004.11.057
oligonucleotide analogues must always be guaranteed besides minimizing their negative side effects. The search for appropriate delivery system is more or less common problem of the above-mentioned strategies. Majority of transfected oligonucleotide molecules in cellular uptake experiments tends to pass the membrane by endocytosis mechanism with subsequent sequestration into endosome/ lysosomal compartments which is characterized by punctuate cytoplasmic distribution. Compartmentalized oligonucleotides are considered to be degraded and so they cannot accomplish ‘antisense’ and/or ‘antigene’ function [8]. In our studies we apply two fluorescence techniques (fluorescence microimaging and multifrequency phase/modulation microspectrofluorimetry) to monitor the modified oligonucleotide penetration inside the cell, its distribution in the cellular environment and subsequent interaction with the subcellular structures [9–11]. Recently we used successfully amphotericin B 3-dimethylaminopropyl amide as an enhancer molecule for the uptake process.
152
E. Kocˇisˇova´ et al. / Journal of Molecular Structure 744–747 (2005) 151–153
The 3T3 and B16 cell lines incubated with dT15 oligonucleotide analog, homogenously modified by phosphonatebased 3 0 -O–P–CH2–O-5 0 linkage and labeled by tetramethylrhodamine marker, exhibited regular intensive staining of the cell nucleus connected with two-component peculiar fluorescence signal [12]. In this work we proceeded with studies of the oligonucleotide penetration into B16 cells and analyzed time-resolved fluorescence signal from the nucleus in the case of another type of the intracellular staining process.
2. Experimental 2.1. Chemicals and the cell culture The modified oligonucleotide—oligothymidylate (15-mer)—with the 3 0 -O–P–CH2–O-5 0 internucleotide linkage labeled by tetramethylrhodamine (TMR) at the 3 0 end was prepared in two-steps: synthesis of modified oligomer and coupling of the oligomer with the rhodamine marker. The used chemical binding of TMR had been found as very stable in the cellular environment [11]. Amphotericin B 3-dimethylaminopropyl amide (AmA) for oligonucleotide uptake enhancement was kindly provided by Prof. Borowski (Technical University of Gdansk, Poland). Mouse melanoma cell line (B16) used in the study was cultured as monolayers in 25 cm2 flask at 37 8C in a humidified 5% CO2 atmosphere, in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, streptomycin (0.1 mg/ml) and penicillin (100 U/ml), all from Biomedia. Cells were subcultured in 35 mm diameter Petri dishes for 48 h before time resolved microspectrofluorimetric and fluorescence imaging experiments. Cells were treated by AmA/oligonucleotide mixture 4 h before the experiment. AmA/oligonucleotide mixture was prepared of 120 ml of AmA stock solution (10K3 M) gently mixed with 15 ml of the oligonucleotide solution (stock solution 1.6!10K4 M). After 15 min the mixture was added into prepared Petri dishes with cells and incubated at 37 8C in a standard humidified atmosphere.
The microspectrofluorimeter is configured for a multifrequency heterodyne measurement mode. Its detailed optical and electronic scheme was described elsewhere [13]. Laser excitation beam intensity is sine modulated from 1 to 200 MHz (set of eight frequencies 30, 45, 50, 90, 100, 135, 154 and 175 MHz is used in our experiment). Elastic scattering taken as a reference and fluorescence signal are collected in a back-scattering geometry and focused on the input slit of a spectrograph. A high frequency gain modulated MCP image intensifier is placed in front of an optical multichannel analyzer. Its gain is sine modulated in precise phase-lock to the excitation laser beam with a 1 Hz frequency offset (cross-correlation detection). Phase shift and demodulation due to the fluorescence lifetime are preserved in a low frequency modulation (1 Hz) of the received signal, detected for each detector pixel by means of a synchronizing unit. The determined spectral dependence of the phase shift and modulation is treated by a fitting procedure succeeded by Global analysis that is able to decompose multi-component fluorescence spectra to particular spectral components characterized by their specific lifetimes [13].
3. Results and discussion AmA makes possible to cross directly the oligonucleotide through membrane inside the cell (not by the endocytosis) and to achieve its regular distribution in the subcellular compartments [14], although its mechanism of action is still not understood completely. The fluorescence image (Fig. 1) shows intracellular distribution of modified oligonucleotide TMR-dT15 in mouse melanoma cells (line B16) observed after approx. 4 h incubation with AmA/oligonucleotide mixture. The cells are stained
2.2. Measuring methods Fluorescence micro-imaging experiments were performed on an Optiphot-2 epifluorescence microscope equipped with a Nipkow wheel coaxial-confocal attachment (Technical Instrument). Confocal fluorescence images were collected by aqueous-immersion objective (Zeiss, Neofluar X63—numerical aperture 1.2) and imaged by TE cooled CCD camera (Micromax, Princeton Instruments). TMR emission was visualized with a standard rhodamine filter set (BP 530G15, LP 600) using 4 s exposure time. Camera images were processed by IPLab software (Scanalytics).
Fig. 1. Fluorescence image of the mouse melanoma B16 cells after 4 h incubation with AmA/TMR-dT15 mixture. The circle indicates the region from which the emission for time-resolved microspectrofluorimetric measurements was collected.
E. Kocˇisˇova´ et al. / Journal of Molecular Structure 744–747 (2005) 151–153
Fig. 2. Decomposition of the emission spectrum (solid line) collected from the nucleus region into two spectral components. Dashed-line with circles: 2.7 ns component, dashed-line with triangles: 1.3 ns component.
almost homogenously with only more distinct staining of the nucleoli and part of the nucleus membrane. This result differs though radically from those obtained in our previous experiments carried out at equal conditions [12] when after 3 h incubation almost exclusive staining of the nucleus was observed and just after 24 h incubations the subsequent oligonucleotide redistribution throughout the cellular volume occurred. The differences in the staining development demonstrate that the distribution of the modified oligonucleotide may proceed in various ways depending (probably) very sensitively on the state of the cells and the experiment conditions. Although we still do not know the reason for the different staining process, it provided us with a new type of intracellular oligonucleotide distribution for time-resolved microfluorimetric measurements. Fig. 2 displays fluorescence time-resolved spectra coming from the region of the cell nucleus. After decomposition into two fluorescence components we obtained the major component (92% of integral intensity) with lifetime of 2.7 ns and spectral maximum at 586 nm and the minor component with 1.3 ns lifetime and emission maximum at about 570 nm. This result confirms existence of the exceptional short-lived component (1.4 ns lifetime in our previous experiment [12]) without any analogue in our reference experiments [11]. The 2.7 ns lifetime corresponds to the lifetime of TMR in interaction with DNA [11]. In the previous experiment [12] the second component was however of substantially longer lifetime (4.84 ns), lower intensity, and slightly shifted maximum to higher wavelengths. This noticable difference we interpret as a proof of several types of possible binding partners for the modified oligonucleotide inside the nucleus and/or nucleolus. The complex formations lead to various
153
emission characteristics. Due to unequal thermodynamic stabilities, varied level of the staining influences the proportions of particular spectral components. Decomposition into only two components is not able to characterize the emission completely, but describes only the dominant components whose characteristics are somewhat disturbed by the unresolved ones. Our method is unfortunately not able to provide reliable decomposition into more than two components because of limited current precision of the experimental data. Further progress we expect after enlarging our set of reference data (emission characteristics of the oligonucleotide complexes with various model biomolecules) and improvement of the microspectrofluorimeter to carry out experiments at higher modulation frequencies (increased precision of the phase shift and demodulation data).
Acknowledgements Special thanks of authors are addressed to Prof. J. Bok (Institute of Physics) for writing the procedure for the spectra fitting. The financial support awarded by the Grant Agency of the Czech Republic (project No. 202/03/D118) and by the Czech Ministry of Education (MSM 113200001) is gratefully acknowledged.
References [1] [2] [3] [4]
[5] [6] [7] [8] [9]
[10] [11] [12] [13] [14]
E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990) 543. C. Helene, Eur. J. Cancer 27 (1991) 1466. J. Kurreck, Eur. J. Biochem. 270 (2003) 1628. V.V. Vlassov, I.E. Vlassova, L.V. Pautova, in: K. Moldave (Ed.), Progress in Nucleic Acid Research and Molecular Biology, Oligonucleotides and Polynucleotides as Biologically Active Compounds, Academic Press, San Diego, 1997, pp. 95–143. A.M. Gewirtz, C.A. Stein, P.M. Glazer, Proc. Natl Acad. Sci. USA 93 (1996) 3161. G. Wang, M.M. Seidman, P.M. Glazer, Science 271 (1996) 802. M.D. Matteucci, R.W. Wagner, Nature 384 (1996) 20. A.J. Hudson, W. Lee, J. Porter, J. Akhtar, R. Duncan, S. Akhtar, Int. J. Pharm. 133 (1996) 257. P. Praus, D. Gaskova, E. Kocisova, R. Chaloupka, J. Stepanek, J. Bok, D. Rejman, I. Rosenberg, P.-Y. Turpin, F. Sureau, Biopolymers 67 (2002) 339. P. Praus, F. Sureau, E. Kocisova, I. Rosenberg, J. Stepanek, P.-Y. Turpin, Spectrosc. Int. J. 17 (2003) 429. E. Kocisova, F. Sureau, P. Praus, I. Rosenberg, J. Stepanek, P.-Y. Turpin, J. Mol. Struct. 115 (2003) 651. E. Kocisova, P. Praus, I. Rosenberg, O. Seksek, F. Sureau, J. Stepanek, P.-Y. Turpin, Biopolymers 74 (2004) 110. P. Praus, F. Sureau, J. Fluorescence 10 (2000) 361. Ch. Garcia-Chaumont, O. Seksek, B. Jolles, J. Bolard, Antisense Nucleic Acid Drug Dev. 10 (2000) 177.