DNA condensation

The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N 4 ,N 9 -dioleoylspermine and N 1 -cholesteryl spermine carbamate. Methods. Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen \ (PG) to allow fluorescence intensity fluctuation measurement and analysis. Results. N 4 ,N 9 -Dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 Â 10 j12 m 2 /s and particle number (PN) = 0.7 [at ammonium/phosphate (N/P) charge ratio=1.0Y1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N 1 -Cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5Y2.5) with D = 1.3 Â 10 j12 m 2 /s and PN value of 5.2. N 4 ,N 9 -Dioleoylspermine is a more efficient DNA-condensing agent than N 1 -cholesteryl spermine carbamate. Conclusions. FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.

The spermine-induced DNA condensation is a first-order phase transition. Here, we apply a novel technique fluorescence lifetime correlation spectroscopy to analyze this transition in a greater detail. We show that the method allows for the observation of the condensed and uncondensed molecules simultaneously based solely on different fluorescence lifetimes of the intercalating fluorophore PicoGreen in the folded und unfolded domains of DNA. The auto-and cross-correlation functions reveal that a small fraction of the DNA molecules is involved in the dynamic intramolecular equilibrium. Careful inspection of the cross-correlation curves suggests that folding occurs gradually within milliseconds.

The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N 4 ,N 9 -dioleoylspermine and N 1 -cholesteryl spermine carbamate. Methods. Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen \ (PG) to allow fluorescence intensity fluctuation measurement and analysis. Results. N 4 ,N 9 -Dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 Â 10 j12 m 2 /s and particle number (PN) = 0.7 [at ammonium/phosphate (N/P) charge ratio=1.0Y1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N 1 -Cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5Y2.5) with D = 1.3 Â 10 j12 m 2 /s and PN value of 5.2. N 4 ,N 9 -Dioleoylspermine is a more efficient DNA-condensing agent than N 1 -cholesteryl spermine carbamate. Conclusions. FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.

The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N 4 ,N 9 -dioleoylspermine and N 1 -cholesteryl spermine carbamate. Methods. Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen \ (PG) to allow fluorescence intensity fluctuation measurement and analysis. Results. N 4 ,N 9 -Dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 Â 10 j12 m 2 /s and particle number (PN) = 0.7 [at ammonium/phosphate (N/P) charge ratio=1.0Y1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N 1 -Cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5Y2.5) with D = 1.3 Â 10 j12 m 2 /s and PN value of 5.2. N 4 ,N 9 -Dioleoylspermine is a more efficient DNA-condensing agent than N 1 -cholesteryl spermine carbamate. Conclusions. FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.

The spermine-induced DNA condensation is a first-order phase transition. Here, we apply a novel technique fluorescence lifetime correlation spectroscopy to analyze this transition in a greater detail. We show that the method allows for the observation of the condensed and uncondensed molecules simultaneously based solely on different fluorescence lifetimes of the intercalating fluorophore PicoGreen in the folded und unfolded domains of DNA. The auto-and cross-correlation functions reveal that a small fraction of the DNA molecules is involved in the dynamic intramolecular equilibrium. Careful inspection of the cross-correlation curves suggests that folding occurs gradually within milliseconds.

Cationic dendrimers are promising vectors for non-viral gene due to their well-defined size and chemistry. We have synthesized a series of succinylated fourth generation (G4) PAMAM dendrimers to control the DNA packaging in dendriplexes, allowing us to probe the role of charge on DNA packaging. The self-assembly of DNA induced by these zwitterionic PAMAM (zPAMAM) was investigated using small-angle x-ray scattering (SAXS). We demonstrate that changing the degree of modification in zPAMAM-DNA significantly alters the packing density of the resulting dendriplexes. Salt sensitivities and pH dependence on the inter-DNA spacing were also examined. The swelling and stability to salt is reduced with increasing degree of PAMAM modification. Lowering the pH leads to significantly tighter hexagonal DNA packaging. In combination, these results show zPAMAM is an effective means to modulate nucleic acid packaging in a deterministic manner.

Understanding the strength and specificity of interactions among biologically important macromolecules that control cellular functions requires quantitative knowledge of intermolecular forces. Controlled DNA condensation and assembly are particularly critical for biology, with separate repulsive and attractive intermolecular forces determining the extent of DNA compaction. How these forces depend on the charge of the condensing ion has not been determined, but such knowledge is fundamental for understanding the basis of DNA-DNA interactions. Here, we measure DNA force-distance curves for a homologous set of arginine peptides. All forces are well fit as the sum of two exponentials with 2.4-and 4.8-A ˚decay lengths. The shorter-decay-length force is always repulsive, with an amplitude that varies slightly with length or charge. The longerdecay-length force varies strongly with cation charge, changing from repulsion with Arg 1 to attraction with Arg 2 . Force curves for a series of homologous polyamines and the heterogeneous protein protamine are quite similar, demonstrating the universality of these forces for DNA assembly. Repulsive amplitudes of the shorter-decay-length force are species-dependent but nearly independent of charge within each species. A striking observation was that the attractive force amplitudes for all samples collapse to a single curve, varying linearly with the inverse of the cation charge.

Protamines are small, highly positively charged peptides used to package DNA to very high densities in sperm nuclei. Tight DNA packing is considered essential to minimize DNA damage by mutagens and reactive oxidizing species. A striking and general feature of protamines is the almost exclusive use of arginine over lysine for the positive charge to neutralize DNA. We have investigated whether this preference for arginine might arise from a difference in DNA condensation by arginine and lysine peptides. The forces underlying DNA compaction by arginine, lysine, and ornithine peptides are measured using the osmotic stress technique coupled with x-ray scattering. The equilibrium spacings between DNA helices condensed by lysine and ornithine peptides are significantly larger than the interhelical distances with comparable arginine peptides. The DNA surface-to-surface separation, for example, is some 50% larger with polylysine compared to poly-arginine. DNA packing by lysine rich peptides in sperm nuclei would allow much greater accessibility to small molecules that could damage DNA. The larger spacing with lysine peptides is due to both a weaker attraction and a stronger short ranged repulsion relative to the arginine peptides. A previously proposed model for poly-arginine and protamine binding to DNA provides a convenient framework for understanding the differences between the ability of lysine and arginine peptides to assemble DNA. The compaction of DNA is a common theme in cell biology. The most tightly packed DNA is found in bacteriophages (1, 2) and vertebrate sperm nuclei (3, 4). In both cases, highly negatively charged DNA is found to be hexagonally organized with interhelical spacings of only 27-30 Å (or surface-to-surface separations of ~ 7-10 Å), corresponding to packing densities of 400 -500 mg/ml. In these two cases, packing is accomplished quite differently. The DNA is actively transported into the bacteriophage capsid head using very strong molecular motors. Once in the head, the DNA is under tremendous pressure that is used to help eject the DNA into bacterial cells. In vertebrate sperm nuclei, DNA is spontaneously compacted, typically to 28-30 Å spacings by small, highly positively charged, arginine-rich