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Recognition and Selective Binding of DNA by Ionenes of Different Charge Density Elizabeth S. Trukhanova,† Vladimir A. Izumrudov,† Andrey A. Litmanovich,‡ and Alexander N. Zelikin*,† Department of Chemistry, M.V. Lomonosov Moscow State University, 119992 Moscow, Russia, and Moscow State Automobile & Road Technical University, Moscow, Russia Received July 31, 2005; Revised Manuscript Received September 14, 2005
The ability of aliphatic ionenes to recognize and bind DNA or poly(methacrylic acid) (PMA) in the equimolar mixture of these polyanions was studied by fluorescence quenching technique. Within a particular system, the selectivity of competitive interactions was shown to be determined by a component with the lowest degree of polymerization (DP). Ionene polycations with lowest DP values did not exhibit pronounced selectivity in binding DNA or PMA with higher values of DP. Increase in ionene DP resulted in a steady increase in selectivity of interaction and ultimately in almost exclusive binding of one of the two polyanions. The ability of the ionene to recognize and bind DNA in the mixture of polyanions was shown not to correlate with the affinity of the ionene to DNA in their binary mixture. Although ionenes with a higher charge density exhibited preferential binding to PMA, the ionenes with the lowest charge density selectively bound DNA. Introduction Selective recognition of DNA by nucleic acid binding proteins is of the utmost importance for the function of living organisms from single cell to complex organisms. Of the numerous reports on systems mimicking DNA-protein interactions, such as the interaction of DNA with synthetic polycations or interaction of other charged species, only a few studies focused on the dominant interaction of two polymeric components in multicomponent systems. Some notable examples are recognition of DNA topology by a linear polycation1 and polycationic gel,2 selective binding of DNA by a polycation in DNA/RNA mixtures,3 selection of A-T rich DNA in a mixture of different DNA molecules by polylysine,4 and the matching of polyelectrolytes per their chain lengths.5 Although these instances demonstrate the possibility of recognition in polyelectrolyte mixtures, a systematic study of factors underlying the phenomena is yet to be performed. Herein we report the recognition of nucleic acid or synthetic polyanion in an equimolar mixture of DNA and poly(methacrylic acid) (PMA) by an ionene polycation via judicious choice of ionene charge density. We then demonstrate that further control over selectivity of interaction is achieved via the proper choice of the degree of polymerization of the polycation. Together with our recent data on the role of ionic strength of solution and degree of polymerization of the synthetic polyanion in this process, the presented results demonstrate that within a particular * Corresponding author. Current address: Department of Chemical and Biomolecular Engineering, the University of Melbourne, Victoria, 3010 Australia. Phone: (+61 3) 8344 9833. Fax: (+613) 8344 4153. E-mail:
[email protected]. † M.V. Lomonosov Moscow State University. ‡ Moscow State Automobile & Road Technical University.
system, a mixture of DNA with a synthetic vinylic polyanion and aliphatic ionene, it is possible to achieve high recognition and almost selective binding of one of the two polyanions. These results can serve as a basis for creation of smart gene delivery vehicles which release the cargo DNA at desired external conditions. Experimental Section Ethidium bromide (EB), NaCl and, Tris (tris[hydroxymethyl]aminomethane) buffer were purchased from Sigma (U.S.A.) and used without purification. Aliphatic n,n-ionenes bromides were synthesized by Menshutkin reaction from N,N,N′,N′- tetramethylalkyldiamine and dibromoalkanes, subsequently modified by p-nitrotoluene bromide, fractionated by ion-exchange chromatography and characterized as described in details elsewhere.6 Calf thymus DNA (sodium salt, ∼1 × 104 base pairs) was purchased from Sigma (USA) and used without purification. Prior to experiments the purity of the DNA samples was verified by UV/vis spectroscopy and thermal denaturation profile of the sample. Poly(methacrylic acid) (PMA), DP 37, 55, 72, 128, 141, and 440 (Mw standards, PDI ∼ 1.14), was purchased from Fluka and used without purification. In each case, the concentration of the polymers is expressed in molarity of ionisable groups and the DP value signifies the number of ionizable groups per polymer chain. Spectrophotometric measurements were performed using a Hitachi 150-20 Spectrometer (Japan) in a waterthermostatic cell under permanent stirring. Fluorescence intensity was measured using a Jobin-Yvon-3CS Spectrofluorimeter (France) in a quartz fluorescence cell at permanent stirring in TRIS buffer, pH 9.0, at 25 °C in a waterthermostatic stirred cell holder. The excitation and emission
10.1021/bm050536i CCC: $30.25 © 2005 American Chemical Society Published on Web 10/07/2005
Recognition and Binding of DNA by Ionenes
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Scheme 1. Aliphatic n,n-Ionene
wavelengths were 535 and 595 nm, respectively. In all experiments, DNA‚EB complex of composition [EB]/[P] ) 1/20, where [EB] and [P] are molar concentrations of the dye and DNA phosphate groups, respectively, was used. This ratio of EB to DNA is within the linear range of change in ethidium fluorescence with its concentration.7 Solutions of polyelectrolyte complexes were prepared directly in the fluorimetric cell by mixing of aliquots of stock solutions of the polymers. In each case to ensure equilibration of the system, the final combination of polyelectrolytes was reached by at least two alternative routes via a change in the sequence of addition of reagents. The time required for equilibration was found to be a function of ionene charge density and degree of polymerization of the polyelectrolytes and typically varied from 5 to 10 min. Thus, the reading for each point was taken 10 min after mixing the polymers. Interaction of polycations with DNA charged with an intercalating dye leads to a decrease in fluorescence of the dye. This occurs primarily due to the displacement of the dye from the double helix into the bulk solution and can also occur due to the interaction of the fluorophore with fluorescence quenchers, if they are present on the polycation chain. In the case of the linear change in the fluorescence signal with the amount of polycation bound to DNA, fluorescence titration curves can be used to quantify the interaction of the polycation with DNA in their binary mixture or in the presence of other charged species. Thus, the share fraction of the ionene bound to DNA in its mixture with PMA was calculated from the data of titration curves according to8 Θ)
I0 - IC I0 - I P
where I0, IC, and IP are fluorescence intensity of solutions of DNA, a mixture of DNA, PMA, and ionene, and a mixture of DNA and ionene, respectively. Distribution of a polycation between two polyanions is a function of binding constants of the polycation to each of the polyanions and the degree of occupation of the polyanions with the polycation.9 Since the latter factor becomes more and more pronounced with an increase in polycation content, readings were taken within the initial part of the titration curves at [N]/[P] e 0.5. Results and Discussion Aliphatic ionenes (Scheme 1) are positively charged polymers with quaternary nitrogen atoms in the polymer backbone. These polycations have attracted interest due to their regular structure and the ability to predetermine their charge density solely by the choice of the proper monomers.10 Owing to this, they serve as appropriate model species to probe theories describing polyelectrolytes in solutions.11-13 Recently, we reported that the competitive interaction of highly polymerized 3,3-ionene with polyanions in mixtures
Figure 1. Titration curves of DNA (open circles) and equimolar mixture of DNA and PMA (closed circles) with 3,3-ionene monitored via fluorescence of ethidium bromide, intercalating into DNA double helix. DPionene ) 150, DPPMA ) 72. [DNA] ) 4 × 10-5 M, pH 9.0, [NaCl] ) 0.05 M.
of DNA and poly(acrylic acid) (PA) is controlled by the degree of polymerization of the synthetic polyanion and the ionic strength of the solution and can be varied from predominant binding with DNA to almost exclusive complexation with PA.14 In the framework of the ongoing research, we aimed to elucidate the role of molecular characteristics of the aliphatic ionenes in the selective binding. To this end, we synthesized and characterized a series of n,n-ionenes, n ) 3, 4, 6, and 10, differed by degree of polymerization. The interaction of ionenes with DNA in the presence or absence of poly(methacrylic acid) (PMA) was monitored by a fluorescence quenching technique using ethidium bromide (EB) as a fluorescence probe.7,8 The fluorescence intensity of EB intercalated in DNA decreased linearly upon introduction of the ionene15 to reach its minimum at the ratio of charged groups of ionene and DNA (φ) close to unity (Figure 1, curve 1). The linearity of I vs φ provides a basis to quantify the ionene-DNA interaction and determine the share fraction (Θ) of polycation bound to DNA in the multicomponent systems, 0 e Θ e 1, where Θ ) 1 and Θ ) 0 correspond to exclusive interaction of polycation with DNA and a competing polyanion, respectively. This approach was recently employed by us14,16 and others17 to monitor competitive interactions in solutions of DNA with synthetic polyelectrolytes. We determined Θ values in the systems containing an ionene and a mixture of DNA and PMA equimolar with regards to the negatively charged groups. The curve in Figure 2 was obtained for 4,4-ionene with DP ) 100 and typifies the dependence of Θ on the degree of polymerization of PMA. At DPPMA > DPionene, the fraction of polycation bound to DNA was constant regardless of DPPMA, whereas at DPPMA < DPionene a pronounced increase in Θ with a decrease in DPPMA occurred. This observation is a direct consequence of the fact that the binding energy of two sufficiently long oppositely charged polyions is primarily controlled by DP of the shorter component, which determines the number of ion pairs formed by the two polymers.9 In a system containing more than two polyelectrolytes, this holds true for each pair of the interacting polyelectrolytes and, other things
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Figure 2. Fraction of 4,4-ionene bound to DNA in its equimolar mixture with PMA as a function of DPPMA. DP4,4-ionene ) 100. [DNA] ) [PMA] ) 4 × 10-5 M, pH 9.0, [NaCl] ) 0.05 M. Line is a guide to the eye only.
Figure 3. Fraction of ionene bound to DNA in its equimolar mixture with PMA (unless stated otherwise DPPMA)140) as a function of DPionene for 3,3-ionenes (filled circles) and 10,10-ionenes (open circles). Experimental conditions are the same as in Figure 2.
being equal, the selectivity of interactions is controlled by DP of the shortest component of the mixture. This finding is not limited to the system investigated here, an analogous trend was observed with different mixtures of polyelectrolytes.14,16 To elucidate the influence of ionene DP and charge density on the competitive interaction, Θ values were determined in mixtures of different ionenes with an equimolar combination of DNA and PMA, DPPMA) 140 using ionene samples with DP e 100 to meet the requirement DPionene< DPPMA. Unlike the system described above (Figure 2) in which the DP of the shorter component (PMA) controlled the affinity between the polyelectrolytes in only one of the two polyelectrolyte pairs (ionene-PMA), for these systems, increase in ionene DP results in a growth of its affinity to both PMA and DNA. Hence, increase in ionene DP (DPionene< DPPMA) should result in a more pronounced selectivity of its interaction with DNA or PMA in the mixture of the polyanions,9 and the data presented in Figure 3 fully support this assumption. For 3,3-ionenes with the lowest tested DP (DP ) 20), Θ was close to 1/2 (Figure 3, filled circles). This implies that the system exhibits minor selectivity of interaction with an almost even distribution of the ionene between DNA and PMA. Still, Θ was slightly lower than 1/2; that is, interaction of 3,3-ionene with PMA is more
Trukhanova et al.
Figure 4. Fraction of ionene bound to DNA in its equimolar mixture with PMA. DPionene ) 100; DPPMA ) 140. Experimental conditions are the same as in Figure 2.
favorable than with DNA. In accordance with the reasoning mentioned above, an increase in the DP3,3-ionene was accompanied by a continuous decrease in Θ indicating the growing selectivity of the ionene binding to PMA. To probe the possibility of exclusive binding, highly polymerized samples of 3,3-ionene (DP ) 220) and PMA (DP ) 440) were used. As expected, the selectivity of the interaction was extremely high; the determined Θ value equalled zero within the experimental error (marked by arrow in Figure 3). For 10,10-ionene, the pronounced increase in the binding selectivity with an increase in the ionene DP also occurred (Figure 3, open circles). Yet the striking observation here is that, contrary to 3,3-ionene, 10,10-ionene bound preferentially to DNA. Θ deviated from values slightly higher than 1/2 at low DP10,10-ionene to subsequently higher values making the ionene-DNA interaction more and more favorable, and relatively long 10,10-ionene chains interacted with DNA almost exclusively. In other words, 3,3- and 10,10- ionenes which differ only by the charge spacing along the chain exhibited opposite selectivity in binding to DNA or PMA, and in both cases, the highly polymerized ionenes recognized and bound only one of the polyanions. The samples of 4,4-ionene and 6,6-ionene possessed intermediate selectivity with preferential binding to PMA and DNA, respectively (Figure 4). Thus, the ability of aliphatic ionenes to bind DNA in its mixture with PMA as a function of ionene charge spacing, as least within the studied array of the ionenes, increases with an increase in charge spacing of the polycation from 3 to 10 methylene groups in a monotonic fashion. This observation is rather unexpected in view of the affinity of integral type polyamines to DNA.18,19 Increase in the central methylene spacer from 4 to 10 carbon atoms in a series of spermine homologues was followed by a 2-fold decrease in the binding constant of the tetraamine to DNA with a maximal binding constant at five carbon methylene spacer.18 Comparing these data to the findings of our research reveals that the ability of the ionene to recognize and bind DNA in a mixture of polyanions does not correlate with its affinity to DNA in their binary mixture. The higher DNA-polyamine binding constant does not stipulate their preferential interaction in a multicomponent mixture in which case the ratio of
Recognition and Binding of DNA by Ionenes
the binding constants for each pair of interacting polyelectrolytes is a decisive parameter,14 and these are the ionenes with the lowest charge density which selectively bind DNA in its mixture with PMA. It is necessary to emphasize that the recognition of a higher charge density polyanion (supercoiled DNA) in its mixture with a lower charge density polyanion (linear DNA) by a polycation with high charge density has been described in the literature,1 and several factors were discussed to contribute to this phenomenon. To the best of our knowledge, this is the first example of recognition of DNA by a polycation with low charge density. The presented data imply that, although binding of natural polyamines (spermine and spermidine) and high charge density synthetic polycations to DNA can be greatly affected by other negatively charged species, the interaction between DNA and cationic species of lower charge density could be unaffected or affected to a much lesser extent. This finding can have an impact for the field of gene delivery since it is recognized that vector unpacking is an important step for successful gene delivery20 and the liberation of DNA within the cell is thought to proceed via competitive displacement of the carrier from nucleic acid with intracellular charged species.21 Our data show that charge density of the synthetic vector can be used as a means to tweak its ability to be successfully separated from DNA and allow for efficient gene transfer. In conclusion, the outlined data together with the results of our previous work14 show that within a particular system, a mixture of DNA with a synthetic vinylic polyanion and an aliphatic ionene, it is possible to achieve high recognition and almost selective binding of one of the two polyanions. The factors of effective control over the competitive interaction are the charge density of the polycation, the degree of polymerization of the interacting species, and the concentration of low molecular weight electrolyte. These findings can serve as groundwork for creation of smart gene delivery vehicles which liberate the cargo DNA upon a change in surrounding environment.
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References and Notes (1) Bronich, T. K.; Hong Khanh Nguyen; Eisenberg, A.; Kabanov, A. V. J. Am. Chem. Soc. 2000, 122, 8339. (2) Sergeyev, V. G.; Novoskoltseva, O. A.; Pyshkina, O. A.; Zinchenko, A. A.; Rogacheva, V. B.; Zezin, A. B.; Yoshikawa, K.; Kabanov, V. A. J. Am. Chem. Soc. 2002, 124, 11324. (3) Wahlund, P.-O.; Izumrudov, V. A.; Gustavsson, P.-E.; Larsson, P.O.; Galaev, I. Yu. Macromol. Biosci. 2003, 3, 404. (4) Shapiro, J. T.; Leng, M.; Felsenfeld, G. Biochemistry 1969, 8, 3219. (5) Harada, A.; Kataoka, K. Science 1999, 283, 65. (6) Zelikin, A. N.; Akritskaya, N. I.; Izumrudov, V. A. Macromol. Chem. Phys. 2001, 202, 3018. (7) Read, M. L.; Bettinger, T.; Oupicky, D. Methods for studying formation of polycation-DNA complexes and properties useful for gene delivery. In Methods in Molecular Medicine, V.65: NonViral Vectors for Gene Therapy; Findeis, M. A., Ed.; Humana Press Inc.: Totowa, NJ, 2001; pp 131-148. (8) Izumrudov, V. A.; Zhiryakova, M. V.; Akritskaya, N. I. Fluorescence quenching technique for study of DNA-containing polyelectrolyte complexes. In AdVanced macromolecular and supramolecular materials and processes; Guckeler, K., Ed.; Kluwer Academic/Plenum Publishers: Norwell, MA, 2003; pp 277-289 (9) Papisov, I. M.; Litmanovich, A. A. AdV. Polym. Sci. 1989, 90, 139. (10) Casson, D.; Rembaum, A. Macromolecules 1972, 5, 75. (11) Popov, A. M.; Hoagland, D. J. Polym. Sci. B 2004, 42, 3616. (12) Arh, K.; Pohar, C.; Vlachy, V. J. Phys. Chem. B 2002, 106, 9967. (13) Nagaya, J.; Minakata, A.; Tanioka, A. Colloids Surf. 1999, 148, 163. (14) Zelikin, A. N.; Trukhanova, E. S.; Izumrudov, V. A.; Litmanovich, A. A. Polym. Sci. Ser. B 2003, 9-10, 284. (15) The ionenes utilized in the current research possess terminal nitrobenzene groups, presence of which results in linearity in ethidium fluorescence decrease upon interaction of the ionene with DNA, a feature not observed on pristine aliphatic ionenes. (16) Zelikin, A. N.; Trukhanova, E. S.; Putnam, D.; Izumrudov, V. A.; Litmanovich, A. A. J. Am. Chem. Soc. 2003, 125, 13693. (17) Danielsen, S.; Strand, S.; de Lange Davies, C.; Stokke, B. T. Biochim. Biophys. Acta 2005, 1721, 44. (18) Vijayanathan, V.; Thomas, T.; Shirahata, A.; Thomas, T. J. Biochemistry 2001, 40, 13644. (19) Vijayanathan, V.; Lyall, J.; Thomas, T.; Shirahata, A.; Thomas T. J. Biomacromolecules 2005, 6, 1097. (20) Schaffer, D. V.; Fidelman, N. A.; Dan, N.; Lauffenburger, D. A. Biotechnol. Bioeng. 2000, 67, 598. (21) Erbacher, P.; Roche, A. C.; Monsigny, M.; Midoux, P. Bioconjugate Chem. 1995, 6, 401.
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