Dependence of the internal relaxation time for DNA on salt type as inferred by quasielastic light scattering

Quasielastic light scattering methods were used to study calf thymus DNA in solutions of LiCl, NaCl, NH₄Ac, and NH₄Cl. Plots of the reciprocal relaxation time (1/τ) vs sin²(θ/2), where θ is the scattering angle, exhibit two linear regions, in accordance with theories for semiflexible polymers based on the t → 0 approximation. In these theories the slope of the linear region at low angles is associated with the translational diffusion coefficient (D_t), whereas the slope of the linear region at high angles is associated with the segmental diffusion coefficient (D_s = kT/?_s). The midpoint of the “transition” between these two linear regions is associated with the mean displacement between segments (b). Data presented here indicate that the Rouse-Zimm parameters b and ?_s are significantly different for DNA in 0.4M NH₄Cl relative to the other salts at comparable ionic strengths. It is suggested that this difference reflects local solvent structure and that both b and D_s are sensitive to the local water structure.     

Monitoring DNA/poly-L-lysine polyplex formation with time-resolved multiangle laser light scattering

Nonviral DNA complexes show promise as alternative and attractive gene delivery vectors for treating genetic diseases. Nonviral DNA complexes are typically formed by combining DNA with various condensing/complexing agents such as lipids, polyelectrolytes, polymers, polypeptides, and surfactants in solution. DNA/poly-L-lysine polyplex formation kinetics are probed by time-resolved multiangle laser light scattering (TR-MALLS), which yields the time evolution of the supramolecular complex mass and geometric size. Primary polyplexes whose geometric size is smaller than individual DNA molecules in solution are formed very rapidly upon mixing DNA and poly-L-lysine. Over time, these primary polyplexes aggregate into larger structures whose ultimate size is determined primarily by the relative concentrations of DNA and poly-L-lysine. This final polyplex size varies with the DNA/poly-L-lysine mass ratio in a non-monotonic fashion, with the maximum polyplex size occurring at a DNA/poly-L-lysine mass ratio of approximately two to three (charge ratio near unity). The utility of TR-MALLS for monitoring the temporal evolution of DNA loading and supramolecular complex size growth (mean square radius and molar mass) throughout the DNA/poly-L-lysine polyplex formation process is demonstrated. The polyplex DNA loading and size, both geometric and molar mass, are key to understanding the transfection process and for developing optimal gene therapy vectors.     

DNA condensation by polyamines: a laser light scattering study of structural effects.

Polyamines such as spermidine and spermine are abundant in living cells and are believed to aid in the dense packaging of cellular DNA. DNA condensation is a prerequisite for the transport of gene vectors in living cells. To elucidate the structural features of polyamines governing DNA condensation, we studied the collapse of lambda-DNA by spermine and a series of its homologues, H2N(CH2)3NH(CH2)n=2-12NH(CH2)3NH2 (n = 4 for spermine), using static and dynamic light scattering techniques. All polyamines provoked DNA condensation; however, their efficacy varied with the structural geometry of the polyamine. In 10 mM sodium cacodylate buffer, the EC50 values for DNA condensation were comparable (4 +/- 1 microM) for spermine homologues with n = 4-8, whereas the lower and higher homologues provoked DNA condensation at higher EC50 values. The EC50 values increased with an increase in the monovalent ion (Na+) concentration in the buffer. The slope of a plot of log [EC50(polyamine4+)] against log [Na+] was approximately 1.5 for polyamines with even number values of n, whereas the slope value was approximately 1 for compounds with odd number values of n. Dynamic light scattering measurements showed the presence of compact particles with hydrodynamic radii (Rh) of about 40-50 nm for compounds with n = 3-6. Rh increased with further increase in methylene chain length separating the secondary amino groups of the polyamines (Rh = 60-70 nm for n = 7-10 and >100 nm for n = 11 and 12). Determination of the relative binding affinity of polyamines to DNA using an ethidium bromide displacement assay showed that homologues with n = 2 and 3 as well as those with n > 7 had significantly lower DNA binding affinity compared to spermine and homologues with n = 5 and 6. These data suggest that the chemical structure of isovalent polyamines exerts a profound influence on their ability to recognize and condense DNA, and on the size of the DNA condensates formed in aqueous solution.     

Statistical length of DNA from light scattering

An analysis of hydrodynamic data of DNA by use of the wormlike coil model gave a statistical length of 1300 Å, in the absence of excluded volume corrections. The worm-like coil model and this hydrodynamically observed statistical length adequately describe the light scattering properties of DNA.     

Conformational studies of BSA using laser light scattering

The usefulness of laser light scattering as a technique for determining protein conformation has been investigated by studying the self-association and drug binding of bovine serum albumin (BSA). The diffusion coefficients of BSA monomers and dimers have been measured and the ratio of these two quantities indicates that in the dimer, the subunit separation is 2.2 times the monomeric hydrodynamic radius. The binding of salicylate to BSA causes an increase in its diffusion coefficient corresponding to a reduction in the frictional drag of the solvent on the protein molecules. It has been found that data obtained using laser light scattering may be interpreted confidently only when proper care has been taken in sample preparation and the scattered intensity autocorrelation function has been appropriately analyzed.     

A laser light scattering study of gellan gels

A study of gellan has been made using the technique of photon correlation spectroscopy. It has been confirmed that gellan gels are largely stationary at a molecular level like other polysaccharide gels and quite unlike the gels of flexible polymers such as polyacrylamide. Solution-gel transitions of deacetylated gellan in 0.025MNaCl have been studied both as a function of concentration and temperature, and the results compared with those of a parallel investigation of agarose. The interstitial spaces within gellan gels have also been studied by measuring the diffusion coefficients of dextran fractions within the gels. Since all gels are nonergodic systems, the theory of dynamic light scattering from such systems is discussed insofar as it affects the present work. It has been shown that the gellan and agarose aqueous systems are fundamentally different, in that agarose does not from a solution at very low concentrations, but splits up into macroscopic gel particles. At very low concentrations, gellan forms a solution in the presence of both gelleing and nongelling ions, the molecules of which shows little change in hydrodynamic diameter with temperature in the range 20–80°C. At higher concentrations where gels are formed, both gellan and agarose exhibit hystersis in their tempertature transitions from gel to solution and solution to gel, the solution being of large molecular aggregates. The transitions are sharp, but in both cases ther is a continous rearrangement in the structural morphology over the entire temperature range on heating, rendering the system more homogeneous prior to dissociation. In the case of gellan, however, there are two distincit phases in these structural changes—this is not true of agarose. The mean mass per unit length of the gellan fibre in the presence of 0.025M NaCl is 19 k daltons/nm at 0.7% concentration and varies with concentration to the power 0.15. The mass per unit length of the agarose fibre is much larger (ca. 110 k Daltons/nm), this difference being consistent with the difference in properties at very low concentrations. © 1994 John Wiley & Sons, Inc.     

Self-assembly of melanin studied by laser light scattering

The unknown molecular weight and chemical structure of melanin place the study of these pigments outside the range of the classical biochemical techniques; thus in this paper the problem of characterizing these heterogeneous biopolymers was approached by means of light scattering techniques, static and dynamic. The static technique allowed us to identify the macromolecular properties (MW and R(g)(2)(1/2)) of melanin extracted from sepia inksac and of two synthetic analogues: L-Dopa melanin obtained by autooxidation and by enzymatic oxidation by Tyrosinase. By dynamic light scattering (DLS), the hydrodynamic radius R(h) was measured to monitor the temporal behaviour of the polymerization and aggregation processes and R(h) variation by changing the chemical constraints of the polymerization medium, such as pH and ionic strength. The fractal dimension d of the aggregates of melanin, both natural and synthetic, in the past only recognized during the aggregation of the synthetic one by lowering the pH of the medium, was a useful parameter to further investigate and compare the structure of melanin granules of differing origins, revealing for the natural sample, a structure with clusters that are spherical, not largely hydrated and self-assembled, following a reaction limited aggregation kinetics (d=2.38).     

Characterization of acid-denatured DNA by low-angle light scattering

Low-angle light scattering results reported previously demonstrated that measurements on high molecular weight native DNA must be made at angles below 30° in order to obtain correct molecular weights. Earlier light-scattering data obtained on denaturated DNA at angles above 30° showed no change in molecular weight upon denaturation, even though other techniques clearly showed that strand separation occurred. This paper reports low-angle measurements on solutions of calf thymus and T7 DNA denatured under acidic conditions. The results demonstrate that a halving of molecular weight consistent with strand separation is detected by light scattering only when low-angle data are used to obtain correct molecular weights for native material. As expected from theoretical considerations, the scattering from denatured DNA is a linear function of sin²(θ/2), where θ is the angle of observation. This result shows that anticipated experimental artifacts have no significant effect on the low-angle measurements and demonstrates that the curvature in the scattering envelope observed for native DNA below 30° is an inherent property of the native molecule.     

Microscope laser light scattering spectroscopy of single biological cells

A microscope laser light scattering setup was developed, allowing us to do intensity autocorrelation spectroscopy on the light scattered from a volume as small as (2 micron)3. This non-invasive technique makes cytoplasmic studies possible inside single live biological cells. The effect of osmotic swelling and shrinking on the diffusion coefficient of hemoglobin inside intact red blood cells is shown as an illustrative example of the applicability and sensitivity of this new experimental method.     

Microscope laser light scattering spectroscopy of single biological cells

A microscope laser light scattering setup was developed, allowing us to do intensity autocorrelation spectroscopy on the light scattered from a volume as small as (2 μm)³. This non-invasive technique makes cytoplasmic studies possible inside single live biological cells. The effect of osmotic swelling and shrinking on the diffusion coefficient of hemoglobin inside intact red blood cells is shown as an illustrative example of the applicability and sensitivity of this new experimental method.     

Motility of bovine spermatozoa studied by laser light scattering

Laser light scattering has been employed to determine the swimming speed distribution and the fraction of motile cells in samples of bovine spermatozoa. As predicted from theory, average trajectory velocities determined by laser light scattering were approximately four times the average translational speed estimated using light microscopy. The proportion of motile spermatozoa decreased with time at the same rate when samples were prepared in either HEPES or phosphate buffers. However, whereas the mean swimming velocity declined slowly in HEPES buffer, it dropped rapidly when phosphate buffer was used. Dilution (in the range 40–0.4×10⁶ spermatozoa·ml^-1) in either of these two buffers reduced the fraction of motile spermatozoa in the sample, but the mean swimming velocity of the remaining active spermatozoa was unchanged. Lowering the temperature from 37° C to 15° C reduced the mean swimming speed by a factor of 2–3 and the fraction of motile cells by a factor of 4–5.     

Flow birefringence of T7 phage DNA: Dependence on salt concentration

We have determined extinction angles and flow birefringence of T7 bacteriophage DNA over a wide range of shear, polymer concentration, and solvent ionic strength. From these data, information on the simple salt dependence of coil permeability to solvent and on short-range intrachain interactions (persistence length) was obtained. At all ionic strengths, our results are consistent with a partially draining coil in the Gaussian subchain dynamical theory of Rouse-Zimm-Tschoegl-Bloomfield. Salt dependence of persistence length is comparable to, although somewhat less than, that obtained previously using similar methods with a fivefold higher-molecular-weight DNA (T2 bacteriophage DNA). Possible reasons for observed discrepancies are analyzed, and the results of this work are compared in detail to other current studies of solvent ionic strength dependence in persistence length and hydrodynamic properties of DNA.     

Continuous assay for DNA polymerization by light scattering

The time course of DNA polymerization, catalyzed by DNA polymerase I, can be monitored by following changes in light scattering that result from increases in molecular weight of the polymer. Increases in mass of 10% result in an easily measurable change in light scattering. It has been shown that the light scattering change is correlated with the incorporation of radioactive nucleotide, as measured by the filter binding method. Continuous monitoring of the time dependence of polymerization should prove useful in kinetic analysis of the polymerase reaction and related enzyme-DNA interactions.