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Bioconjugate Chem. 1999, 10, 624−628
Caged DNA Does Not Aggregate in High Ionic Strength Solutions Vladimir S. Trubetskoy,*,† Aaron Loomis,† Paul M. Slattum,† James E. Hagstrom,† Vladimir G. Budker,‡ and Jon A. Wolff‡ Mirus Corporation, 545 Science Drive, Madison, Wisconsin 53711, and Departments of Pediatrics and Medical Genetics, Waisman Center, University of WisconsinsMadison, Room 361, 1500 Highland Avenue, Madison, Wisconsin 53705. Received December 28, 1998; Revised Manuscript Received March 22, 1999
The assembly of DNA into compact particles that do not aggregate in physiologic salt solution occurs naturally in chromatin and viral particles but has been challenging to duplicate using artificial constructs. Cross-linking amino-containing polycations in the presence of DNA with bisimidoester cross-linker leads to the formation of caged DNA particles that are stable in salt solutions. This first demonstration of caged DNA provides insight into how natural condensation processes avoid aggregation and a promising avenue for developing nonviral gene therapy vectors.
INTRODUCTION
The condensation of DNA is an integral process of chromatin and viral assembly (1-4). These processes have been modeled by use of low molecular weight cations such as spermine and polycations such as polylysine which compact DNA into toroid and rod structures when the DNA’s negative charge is more than 90% neutralized (5). These practices are also important for the preparation of nonviral vectors for gene therapy (6-8). In contrast to chromatin and viral particles, these artificial DNA particles aggregate in semidilute DNA solutions containing physiologic concentrations of NaCl (9, 10). The aggregation of large polycation/DNA complexes is not well understood and hampers the use of such condensates for gene therapy because they are rapidly cleared from the circulation (11). We now show that covalent crosslinking of polyamines in complexes with DNA increases the stability of the condensed DNA particles in salt. MATERIALS AND METHODS
Reagents. Plasmid pCILuc encoding the firefly luciferase was used as DNA throughout the study (12). Sodium chloride, poly-L-lysine (PLL, molecular mass 34 and 210 kDa), histone H1 (type III-S) and dextran sulfate (DS, MW 500 kDa) were obtained from Sigma. Polyallylamine, (molecular mass 10 kDa) and fluorescein isothiocyanate (FITC) were purchased from Aldrich. Crosslinking reagent, dimethyl-3,3′-dithiobispropionimidate (DTBP), was a product of Pierce (Rockford, IL). Reagents for covalent labeling of DNA (LabelIT Fluorescein and LabelIT Rhodamine) were products of Mirus Corp. Labeling of Polyions with Fluorescent Moieties. DNA was labeled using LabelIT Rhodamine reagent (13) at 1:1 reagent/DNA w/w ratio to yield covalent rhodamine-DNA conjugate (Rh-DNA). DNA was incubated with the reagent at 1 mg/mL in Hepes buffer for 1 h at 37 °C and was purified by two ethanol precipitations and immediately redissolved in water at concentration 1 mg/ * To whom correspondence should be addressed. Phone: (608) 238-4400. Fax: (608) 233-3007. E-mail: vladimirt@ genetransfer.com. † Mirus Corporation. ‡ University of WisconsinsMadison.
mL. PLL was labeled with FITC at 1:100 fluorophore/ lysine initial ratio to yield fluorescein-PLL (Fl-PLL). PLL (10 mg in 1 mL of 0.1 M sodium tetraborate, pH 9.0) was mixed with 0.18 mg of FITC dissolved in 50 µL of anhydrous methyl sulfoxide (Aldrich). After 1 h of incubation at room temperature, the polymer was precipitated with 2-propanol and redissolved in the same buffer supplemented with 0.5 M NaCl. Preparation of DNA/Polycation Complexes and Their Cross-Linking. The complexes were prepared at different amine/phosphate charge ratios in 25 mM Hepes solution adjusted to pH 8.0 with 4N NaOH (Hepes buffer) by adding stock solution of polycation (typically 10 mg/ mL in 6-30 µL of water) into 1 mL of Hepes solution of DNA (50 µg/mL) and vortexing for 30 s. Cross-linking of DNA/polycation DTBP occurred at a 2:1 molar ratio of DTBP to primary amine (e.g., lysine monomer for polylysine and histone). Reaction was conducted for 3 h at room temperature. Cleavage of DTBP was performed by treating cross-linked complexes with a final concentration of 20 mM dithiothreitol (DTT) (Sigma) for 30 min at 37 °C. To premodify PLL with DTBP, PLL (10 mg in 1 mL of Hepes buffer) and DTBP (15 mg as dry powder) were incubated for 3 h at room temperature. The reaction mixture was used without further purification. Particle Sizing and ζ-Potential Measurements. Particle sizing and ζ-potential measurements were performed at 25 °C using a Zeta Plus Particle Analyzer (Brookhaven Instruments Corp., Holtsville, NY) with a laser wavelength of 532 nm. Dialysis of DNA/Polycation Complexes. For dialysis experiments, Rh-DNA/Fl-PLL complexes were formed at 100 µg/mL DNA concentration with a charge ratio of DNA to PLL of 1:6. DTBP modification was then done for 3 h. Samples were loaded into Spectra/Por CE (Cellulose Ester) Membrane MWCO 300 000 (Spectrum Medical Industries, Houston TX) and then dialyzed against Hepes buffer for 48 h with two buffer exchanges. Ultracentrifugation of Rh-DNA/Fl-PLL Complexes. For ultracentrifuge precipitation experiments, Rh-DNA/ Fl-PLL complexes were prepared at [DNA] ) 100 µg/mL in Hepes buffer at 1:2, 1:4, and 1:6 charge ratios. Within 1 h after mixing, the complexes (1 mL) were centrifuged in Beckman TX-100 benchtop ultracentrifuge at 60 000 rpm for 1 h using a Ti 100 rotor. Spectra of the complexes
10.1021/bc9801530 CCC: $18.00 © 1999 American Chemical Society Published on Web 05/29/1999
Stabilization of DNA/Polycation Complexes
were registered before and after centrifugation in a range of 400-700 nm using Beckman DU-7 spectrophotometer. Difference spectra were derived using the instrument’s software to obtain spectra of precipitated complexes. Spectra of Rh-DNA and Fl-PLL were also registered separately in order to measure absorbance at 590 and 495 nm respectively for individual components. Polycation exchange experiments. For polycation exchange experiments, Fl-PLL was used as a reporter molecule. Its fluorescence within DNA/Fl-PLL complex is decreased due to polyelectrolyte condensation. The addition of excess of nonlabeled PLL displaces Fl-PLL from the complex and leads to fluorescence increase. Complex was formed at an initial charge ratio (3:1) and at a DNA concentration of 20 µg/mL in Hepes buffer (total volume 0.5 mL). NaCl was added up to 50 mM in order to achieve the equilibrium quicker. After the signal was stable, nonlabeled PLL (20× molar excess) was added to displace Fl-PLL from the complex. The result was expressed as the percentage of nonexchangeable Fl-PLL in the complex: (F0 - F)/(F0 - Fmin) × 100% where F0 is the initial fluorescence of Fl-PLL, Fmin is the fluorescence of Fl-PLL/DNA complex, and F is the fluorescence after addition of 20× excess of nonlabeled PLL. Release of Fl-DNA from Fl-DNA/PLL Complexes in Decondensation Conditions. Release of Fl-DNA from Fl-DNA/PLL complexes in decondensation conditions was assessed by monitoring the increase in Fl-DNA fluorescence (13) after interaction with dextran sulfate (DS, molecular mass 500 kDa, Sigma). Fl-DNA/PLL complexes (cross-linked and non-cross-linked) were mixed in 450 µL of Hepes buffer at [Fl-DNA] ) 50 µg/mL with 50 µL of increasing amount of DS for 5 min at room temperature (PLL/DS ratio was varied from 0.15 to 16). Sample fluorescence (0.5 mL of total volume) was measured using a spectrofluorometer (λem ) 490, λex ) 530) and expressed as % of uncondensed Fl-DNA fluorescence. Electron Microscopy of Condensed DNA Samples. DNA complexes were placed onto glow-discharged Formvar-coated 200-mesh grids for 1 min followed by staining with 1% uranyl acetate for 30 s. The grids were blotted dry with filter paper and examined using a JEOL JEM 100S electron microscope. Transfection with DTBP-Treated Plasmid DNA. A luciferase expression plasmid DNA/histone H1 complex was cross-linked with DTBP. After DTT treatment, the plasmid DNA was transfected into 3T3 cells using TransIT LT-1 reagent (Mirus Corp.) and luciferase expression was similar to that of non-cross-linked plasmid DNA preparation. RESULTS AND DISCUSSION
The salt stability of polycation complexes with plasmid DNA was assessed using dynamic light scattering (Figure 1) and further confirmed using static light scattering. For PLL complexes, raising the charge ratio of PLL/DNA from 2:1 to 6:1 increased salt stability but the complexes still aggregated in 50 mM NaCl (Figure 1a). The addition of DTBP, which cross-links the amines on the PLL, substantially increased the salt stability of the complexes. DTBP was chosen because its product with primary amino groups preserves positive charge upon coupling. The salt stabilization is reversed upon addition of dithiotreithol which cleaves DTBP, indicating that it is not due to the conversion of the amines to amidines and is dependent upon the cross-linking. DTBP also stabilized complexes of DNA and polyallylamine (Figure 1b). Remarkably, cross-linking of histone H1/DNA complexes
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Figure 1. Relationship between particle size and NaCl concentration for polycation/DNA cross-linked complexes (filled symbols) and non-cross-linked ones (open symbols). (a) PLL (molecular mass ) 34 kDa) and DNA were mixed at 2:1 and 6:1 charge ratios in low salt Hepes buffer following cross-linking of the complexes with DTBP. The 6:1 cross-linked complex was treated with 20 mM DTT. (b) PAA/DNA complex formation and cross-linking. (c) Histone H1/DNA complex formation and crosslinking. Charge ratios of polycation to DNA are indicated in the legends. The average size of all the PLL, PAA, and histone DNA particles under 100 nm with and without DTBP were ∼50, 60, and 75 nm in diameter, respectively.
created particles that were stable even in 800 mM NaCl (Figure 1c). Similar effects of cross-linking by DTBP on salt stabilization were evident when aggregation was assessed using static light scattering. The cross-linked particles did not aggregate for at least 7 days after the addition of salt. Taken together, these results suggest that the cross-linking of many polycations would stabilize their DNA complexes in salt. The addition of the cross-linker DTBP could induce salt stabilization by either modifying the dispersion medium or the DNA-containing particles. If the DTBP was added to a concentrated solution of PLL first and this mixture is then added to DNA, no salt stabilization was observed. In addition, when the 6:1 PLL/DNA complexes (crosslinked and non-cross-linked) were dialyzed so as to reduce the net charge ratio of PLL/DNA to ∼1.7, the cross-linked
626 Bioconjugate Chem., Vol. 10, No. 4, 1999
Figure 2. Transmission electron microscopy image of crosslinked PLL/DNA particles. The cross-linking reaction was carried at 6:1 PLL/DNA charge ratio. Bar length is 90 nm.
particles still had greater salt stability than the noncross-linked complexes (data not shown). These results indicate that cross-linking increased salt stability by modifying the polyamine/DNA complex. It is noteworthy that the cross-linked DNA particles also did not aggregate in physiologic salt solutions that did not contain large amounts of excess polycation. One possible mechanism by which cross-linking increases salt stability is by recruiting polycation to the particle thus providing electrosteric stabilization (14, 15). Following cross-linking, neither ζ-potential nor particle size was significantly altered as compared to non-crosslinked DNA/PLL complexes. For example, particle size and ζ-potential for PLL/DNA 6:1 complex were found to be 61.6 nm and +59 mV for non-cross-linked vs 62.1 nm and + 53 mV for cross-linked complex, respectively. Similarly, electron microscopic analysis indicated that the
Trubetskoy et al.
cross-linked particles were toroids and rods that were similar in size and shape to non-cross-linked particles (Figure 2). To directly determine the ratio of PLL to DNA in the complexes, the particles formed with fluorescently labeled Rh-DNA and Fl-PLL were pelleted by ultracentrifugation. The difference between the amount of PLL in solution before and after centrifugation indicates the amount of PLL in complexation with DNA. The noncross-linked particles contained approximately a 1:1 ratio of amine/phosphate regardless of the initial ratios in the mixture (Figure 3). This is contrary to a common assumption in the polycation/DNA field, but is consistent with 1:1 charge ratios observed in some complexes of synthetic polyelectrolytes (16, 17). Furthermore, crosslinking did not modify the ratio of PLL/DNA in the complexes (Figure 3). The ratio of PLL/DNA found in the precipitate was ∼1:1 independent of both the initial PLL/ DNA ratio and the addition of cross-linker. Similar results were obtained using analytical ultracentrifugation (data not shown). These studies indicate that crosslinking does not stabilize the DNA particles by recruiting additional polycations. One hypothesis for the mechanism by which salt induces the aggregation of polycation/DNA complexes is by enabling the polycation on one particle to become bound to another particle by interparticle cross-bridging as it was proposed for both DNA condensates (18, 19) and polyelectrolyte complexes in general (20, 21). Crosslinking markedly decreases the exchange of unlabeled PLL in solution with Fl-PLL in 3:1 PLL/DNA complex. The percentage of nonexchangeable PLL in 3:1 complexes was found to decrease 3 times upon cross-linking (data not shown). Furthermore, cross-linking prevents high concentrations of NaCl and dextran sulfate (DS), a strong polyanion, from displacing the DNA in PLL complexes (Figure 4). DNA complexes containing larger PLL molecules of molecular mass 210 kDa were destabilized by DS (Figure 4b) and high salt (data not shown), which argues that the stabilization effect of DTBP is not merely due to increasing the size of PLL on the DNA. Taken together, these results suggest that cross-linking in fact “cages” condensed DNA inside the complexes. Even in the presence of high salt and DS, the “caged” DNA appears to be partially condensed. The cross-linked particles are more resistant to salt-induced aggregation
Figure 3. Difference spectra for precipitated portion of chromophore-labeled PLL/DNA cross-linked and non-cross-linked complexes with 2:1, 4:1, and 6:1 charge ratios. PLL and DNA were labeled with fluorescein isothiocyanate (λmax ) 495 nm) and rhodamine LabelIT (λmax ) 590 nm) reagent respectively (15). For stoichiometry measurements, the spectra were compared with the spectra of standard Fl-PLL and Rh-DNA solutions.
Stabilization of DNA/Polycation Complexes
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Figure 4. Decondensation of Fl-DNA in cross-linked and non-cross-linked PLL/DNA 6:1 complexes with different dissociative media. DNA covalently labeled with Fluorescein LabelIT reagent (3 fluorophore moieties per 100 base pairs) sustains considerable fluorescence quenching upon complexing with polycations. (A) Relative fluorescence of Fl-DNA and its cross-linked and non-cross-linked complexes with PLL upon decondensation in 2.5 M NaCl (20). (B) Decondensation of Fl-DNA with dextran sulfate. PLL (210 kDa) and precross-linked PLL (34 kDa pre DTBP) were added as additional controls.
Cross-linking of protein macromonomers accompanies the assembly of viral particles (24, 25). Cross-linking of polyamine/DNA complexes mimics some aspects of viral particle assembly and provides a rationale for why it occurs during virus formation. The lack of aggregation of caged DNA particles in physiologic salt solutions presents an important advance in the development of nonviral vectors. ACKNOWLEDGMENT Figure 5. The model illustrates that salt induces the aggregation of polycation/DNA complexes by lowering the threshold for interparticle crossbridging with exchangeable polycations. Crosslinking of the polycation prevents this from occurring. Black, curving lines (+) indicate the polycations, and the gray, straight lines indicate the cross-links in the caged DNA.
because part of a polyamine molecule on one particle is less able to cross-bridge to another particle (Figure 5). In essence, cross-linking “freezes” the polycations on the particle. Preliminary experiments indicated that caged complexes are inactive in gene transfer in vitro. On the other hand, experiments with cross-linked DNA/polycation (histone H1) complexes showed that DNA remains intact and expressable after reversing the cross-linking procedure with DTT treatment (data not shown). Considering intracellular reducing conditions (22, 23), more labile cross-linkers may result in transfection-competent caged complexes.
We would like to thank Dr. Grayson Scott for his assistance in obtaining electron microscopy images of caged DNA complexes and Dr. Darrell McCaslin for his expert advice in analytical ultracentrifugation experiments. LITERATURE CITED (1) Furlong, D., Swift, H., and Roizman, B. (1972) Arrangement of Herpesvirus Deoxyribonucleic Acid in the Core. J. Virol. 10, 1071-1074. (2) Chattoraj, D., Gosule, L. C., and Schellman, J. A. (1978) DNA Condensation with Polyamines. II. Electron Microscopic Studies. J. Mol. Biol. 121, 327-337. (3) Lepault, J., Dubochet, J., Baschong, W., and Kellenberger, E. (1987) Organization of double-stranded DNA in bacteriophages: a study by cryo-electron microscopy of vitrified samples. EMBO J. 6, 1507-1512. (4) Hud, N. V. (1995) Double-Stranded DNA Organization in Bacteriophage Heads: An Alternative Toroid-Based Model. Biophys. J. 69, 1355-1362.
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BC9801530
