Influence of Monovalent Salt on the Molecular ... - Semantic Scholar
MEETING ABSTRACT
Single Mol. 3 (2002) 5-6, 315-316
Single 315 Molecules
Influence of Monovalent Salt on the Molecular Structure of Single DNA Complexes with Positively Charged Dendronized Polymers Illdiko Gössl A), Lijin Shu B), A. Dieter Schlüter B) and Jürgen P. Rabe A)
A)
Department of Physics, Humboldt University Berlin, 10099 Berlin, Germany.
B)
Institute of Chemistry, Free University Berlin, Takustr. 3, 14195 Berlin, Germany
Correspondence to Jürgen P. Rabe Department of Physics Humboldt University Berlin 10099 Berlin, Germany phone *49-30-2093-7621 fax *49-30-2093-7632 email [email protected]
submitted 15 Aug 2002 accepted 30 Oct 2002 published 15 Nov 2002
keywords: DNA complex, salt concentration, single molecules, scanning force microscopy
Abstract In cells and viruses as well as non-viral gene delivery systems, DNA is complexed with different molecules to form highly condensed structures. A wide range of conditions that cause DNA to collapse into compact structures has been discovered [1]. However, in most of these cases an exact description of these structures cannot be given. Since the complex stability
is largely due to electrostatic forces, it can be modulated by varying the salt concentration. Apart from the biological aspects the study of the molecular structure of polyelectrolyte complexes may be used to improve our general understanding of polyelectrolyte interactions. Theoretical models reveal the structure of complexes formed between a stiff charged cylinder and an oppositely charged flexible or semiflexible polymer [2], [3]. Here we determine the influence of salt on the structure of linearized pUC19 plasmid DNA and positively charged dendronized polymers of generation two (PG2). The repeat units of the polymers are styrenes functionalized with dendrons carrying protonated amine groups at the periphery. Starting with the amino-terminated dendronized polystyrene of generation one (PG1), higher generations were obtained by the so called mixed ”attach-to” approach [4]. For the analysis of the structure of the polyelectrolyte complexes scanning force microscopy (SFM) was used. The molecules were allowed to adsorb from solution onto mica or poly-L-ornithin coated mica, rinsed three times with water and finally dried under a stream of N2. Further details of sample preparation are given elsewhere [5]. Complexes of DNA and dendronized polymers of generation two, deposited from different NaCl solutions (10, 50, 100 and 300 mM) onto poly-L-ornithin coated mica, were visualized via SFM (Fig. 1). For the analysis of the complexes the heights and the contour lengths of both the complex (LC) and of the DNA that belonged to the complex (LDNA-C) were determined. While for DNA/PG2 complexes in 0 mM NaCl the average height was (4.0 ± 0.3) nm [5], within the errors the same heights were obtained for the complex using different NaCl solutions (4.2 ± 0.4) nm. The underestimation of the height of molecules in SFM images due to tip-sample interactions (deformation of the sample) is a well known feature. To evaluate the contour lengths of the complex and DNA, their contour was divided into straight segments of 2-5 nm. For their analysis only those complexes were chosen which
2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 69469 1438-5163/02/5-611-0315 $17.50+.50/0
Single Molecules
316
Single Mol. 3 (2002) 5-6
MEETING ABSTRACTS
8 nm
+ + + + + Na
0 nm
+
_ _ _ _ _
2 nm
0 nm
Cl-
10 nm
0 nm
Fig. 1. Schema of complex formation: positively charged PG2 and negatively charged DNA form well structured complexes in the present of NaCl. SFM images of PG2 ( upper left deposited onto mica) , negatively charged DNA (upper right deposited onto poly-L-ornithine coated micaand DNA/PG2 complexes (middle deposited onto poly-L-ornithine coated mica ). The scale bar represents 250 nm.
exhibited a constant height along their contour, and where single DNA strands that came out of this complex belonged clearly to the complex (see [5]). The length of the DNA that contributes to the complex (LDNA-C) can be obtained by subtracting the measured contour length of the DNA molecule out of the complex (Lout) from the length of the monodisperse DNA (L0) by LDNA-C = L0-Lout. In Fig. 2 the contour lengths of the complexes (Lc) are plotted versus the DNA that contributes to the complex (LDNA-C) [5]. In the presence of elevated salt concentration, the obtained data for each NaCl concentration exhibit an overall linear dependence where the slope (m) decreased for increasing concentration of NaCl. Using the estimated radius for PG2 (1.6 ± 10 %) [5] and the theoretical diameter for DNA (2 nm), we calculate the DNA length required for one turn around the dendronized polymers (U) to be (16.3 ± 1.0). With Xi=miU (i stands for the different salt concentrations), the pitch (X) of the wrapped DNA can be calculated. Comparing the results to the case of 0 mM NaCl obtained previously [5], the increase in NaCl concentration
Fig. 2. Linear dependence of the complex length (LC) and DNA length used for complexes (LDNA-C). Complexes formed with 0 mM (,), 10 mM ("), 50 mM (8), 100 mM (x) and 300 mM (-) NaCl.
lead to a decrease in the pitch separation of DNA which is consistent with the theory [3]. We propose a molecular level structural model for a DNA/dendronized polymer complex, according to which the polyelectrolyte with the smaller linear charge density (DNA) is wrapped around the more highly charged dendronized polymer (PG2) and the pitch (X) depends upon the salt concentration. The dendronized polymers together with DNA are a useful model system to test theories on the interaction of oppositely charged polyelectrolytes.
References [1] Bloomfield, V. A. Biopolymers 31 (1991) 1471. [2] Park, S. Y., Bruinsma, R. F., and Gelbart, W. M. Europhys. Lett. 46 (1999) 454. [3] Kunze, K.-K., and Netz, R. R. Europhys. Lett. 58 (2002) 299. [4] Shu, L., Schäfer, A., and Schlüter, A. D. Macromolecules 33 (2000) 4321. [5] Gössl, I., Shu, L., Schlüter, A. D., and Rabe, J. P. J. Am. Chem. Soc. 124 (2002) 6860.
Single Mol. 3 (2002) 5-6, 315-316
Single 315 Molecules
Influence of Monovalent Salt on the Molecular Structure of Single DNA Complexes with Positively Charged Dendronized Polymers Illdiko Gössl A), Lijin Shu B), A. Dieter Schlüter B) and Jürgen P. Rabe A)
A)
Department of Physics, Humboldt University Berlin, 10099 Berlin, Germany.
B)
Institute of Chemistry, Free University Berlin, Takustr. 3, 14195 Berlin, Germany
Correspondence to Jürgen P. Rabe Department of Physics Humboldt University Berlin 10099 Berlin, Germany phone *49-30-2093-7621 fax *49-30-2093-7632 email [email protected]
submitted 15 Aug 2002 accepted 30 Oct 2002 published 15 Nov 2002
keywords: DNA complex, salt concentration, single molecules, scanning force microscopy
Abstract In cells and viruses as well as non-viral gene delivery systems, DNA is complexed with different molecules to form highly condensed structures. A wide range of conditions that cause DNA to collapse into compact structures has been discovered [1]. However, in most of these cases an exact description of these structures cannot be given. Since the complex stability
is largely due to electrostatic forces, it can be modulated by varying the salt concentration. Apart from the biological aspects the study of the molecular structure of polyelectrolyte complexes may be used to improve our general understanding of polyelectrolyte interactions. Theoretical models reveal the structure of complexes formed between a stiff charged cylinder and an oppositely charged flexible or semiflexible polymer [2], [3]. Here we determine the influence of salt on the structure of linearized pUC19 plasmid DNA and positively charged dendronized polymers of generation two (PG2). The repeat units of the polymers are styrenes functionalized with dendrons carrying protonated amine groups at the periphery. Starting with the amino-terminated dendronized polystyrene of generation one (PG1), higher generations were obtained by the so called mixed ”attach-to” approach [4]. For the analysis of the structure of the polyelectrolyte complexes scanning force microscopy (SFM) was used. The molecules were allowed to adsorb from solution onto mica or poly-L-ornithin coated mica, rinsed three times with water and finally dried under a stream of N2. Further details of sample preparation are given elsewhere [5]. Complexes of DNA and dendronized polymers of generation two, deposited from different NaCl solutions (10, 50, 100 and 300 mM) onto poly-L-ornithin coated mica, were visualized via SFM (Fig. 1). For the analysis of the complexes the heights and the contour lengths of both the complex (LC) and of the DNA that belonged to the complex (LDNA-C) were determined. While for DNA/PG2 complexes in 0 mM NaCl the average height was (4.0 ± 0.3) nm [5], within the errors the same heights were obtained for the complex using different NaCl solutions (4.2 ± 0.4) nm. The underestimation of the height of molecules in SFM images due to tip-sample interactions (deformation of the sample) is a well known feature. To evaluate the contour lengths of the complex and DNA, their contour was divided into straight segments of 2-5 nm. For their analysis only those complexes were chosen which
2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 69469 1438-5163/02/5-611-0315 $17.50+.50/0
Single Molecules
316
Single Mol. 3 (2002) 5-6
MEETING ABSTRACTS
8 nm
+ + + + + Na
0 nm
+
_ _ _ _ _
2 nm
0 nm
Cl-
10 nm
0 nm
Fig. 1. Schema of complex formation: positively charged PG2 and negatively charged DNA form well structured complexes in the present of NaCl. SFM images of PG2 ( upper left deposited onto mica) , negatively charged DNA (upper right deposited onto poly-L-ornithine coated micaand DNA/PG2 complexes (middle deposited onto poly-L-ornithine coated mica ). The scale bar represents 250 nm.
exhibited a constant height along their contour, and where single DNA strands that came out of this complex belonged clearly to the complex (see [5]). The length of the DNA that contributes to the complex (LDNA-C) can be obtained by subtracting the measured contour length of the DNA molecule out of the complex (Lout) from the length of the monodisperse DNA (L0) by LDNA-C = L0-Lout. In Fig. 2 the contour lengths of the complexes (Lc) are plotted versus the DNA that contributes to the complex (LDNA-C) [5]. In the presence of elevated salt concentration, the obtained data for each NaCl concentration exhibit an overall linear dependence where the slope (m) decreased for increasing concentration of NaCl. Using the estimated radius for PG2 (1.6 ± 10 %) [5] and the theoretical diameter for DNA (2 nm), we calculate the DNA length required for one turn around the dendronized polymers (U) to be (16.3 ± 1.0). With Xi=miU (i stands for the different salt concentrations), the pitch (X) of the wrapped DNA can be calculated. Comparing the results to the case of 0 mM NaCl obtained previously [5], the increase in NaCl concentration
Fig. 2. Linear dependence of the complex length (LC) and DNA length used for complexes (LDNA-C). Complexes formed with 0 mM (,), 10 mM ("), 50 mM (8), 100 mM (x) and 300 mM (-) NaCl.
lead to a decrease in the pitch separation of DNA which is consistent with the theory [3]. We propose a molecular level structural model for a DNA/dendronized polymer complex, according to which the polyelectrolyte with the smaller linear charge density (DNA) is wrapped around the more highly charged dendronized polymer (PG2) and the pitch (X) depends upon the salt concentration. The dendronized polymers together with DNA are a useful model system to test theories on the interaction of oppositely charged polyelectrolytes.
References [1] Bloomfield, V. A. Biopolymers 31 (1991) 1471. [2] Park, S. Y., Bruinsma, R. F., and Gelbart, W. M. Europhys. Lett. 46 (1999) 454. [3] Kunze, K.-K., and Netz, R. R. Europhys. Lett. 58 (2002) 299. [4] Shu, L., Schäfer, A., and Schlüter, A. D. Macromolecules 33 (2000) 4321. [5] Gössl, I., Shu, L., Schlüter, A. D., and Rabe, J. P. J. Am. Chem. Soc. 124 (2002) 6860.
