Unusual behavior exhibited by multistranded guanine-rich DNA complexes

The structural properties of oligonucleotides containing two different types of G-rich sequences at the 3′-ends were compared. It is shown that oligonucleotides with uninterrupted runs of guanine residues at the 3′-end, e.g., d(T₁₅G₁₂), form multistranded structures stabilized by guanine-guanine interactions. The chemical and physical properties of these complexes differ from those of the complexes formed by oligonucleotides with telomere-like sequences, e.g., d(T₁₅G₄T₂G₄). In methylation protection and methylation interference experiments, we found all the guanines in complexes formed by d(T₁₅G₁₅) and d(T₁₅G₁₂) to be accessible to methylation. Furthermore, the methylated monomers retain the ability to polymerize. This contrasts with the inaccessibility of the guanines in d(T₁₅G₄T₂G₄) to methylation and the inability of the methylated monomer to form supramolecular structures. The stoichiometry of the complexes arising from the two types of oligonucleotides also differs. The complexes formed by d(T₁₅G₁₅) consist of consecutive integer numbers of DNA strands, whereas complexes formed by telomere-like oligonucleotides contain 1, 2, 4, or multiples of four strands. Magnesium ions favor formation of high molecular weight complexes by d(T₁₅G₁₅) and d(T₁₅G₁₂), but not by d(T₁₅G₄T₂G₄). The d(T₁₅G₁₅) and d(T₁₅G₁₂) complexes have very high thermal stability compared with telomeric complexes. However, at low temperatures, the thymine bases within the telomeric motif, TTGGGGTTGGGG, appear to allow for the formation of stable high-molecular weight species with a longer nonguanine portion. © 1998 John Wiley & Sons, Inc. Biopoly 45: 427–434, 1998     

Conformation of intercalated DNA plasmids investigated by circular dichroism and dynamic light scattering

Two DNA plasmids, pEGF and pACL29, intercalated with ethidium bromide (EB), have been examined by circular dichroism (CD) and dynamic light scattering (DLS). CD and DLS data show significant changes when the EB/DNA (phosphates) ratio reaches a value of r approximately equal to 0.13. The translational and rotational diffusion coefficients, predicted assuming that plasmids can be described by a string of beads, and the CD spectrum, suggest that a transition from an interwound to a toroidal conformation is likely to occur.     

Measurement of inherent particle properties by dynamic light scattering: introducing electrorotational light scattering.

Common dynamic light scattering (DLS) methods determine the size and zeta-potential of particles by analyzing the motion resulting from thermal noise or electrophoretic force. Dielectric particle spectroscopy by common microscopic electrorotation (ER) measures the frequency dependence of field-induced rotation of single particles to analyze their inherent dielectric structure. We propose a new technique, electrorotational light scattering (ERLS). It measures ER in a particle ensemble by a homodyne DLS setup. ER-induced particle rotation is extracted from the initial decorrelation of the intensity autocorrelation function (ACF) by a simple optical particle model. Human red blood cells were used as test particles, and changes of the characteristic frequency of membrane dispersion induced by the ionophore nystatin were monitored by ERLS. For untreated control cells, a rotation frequency of 2 s-1 was induced at the membrane peak frequency of 150 kHz and a field strength of 12 kV/m. This rotation led to a decorrelation of the ACF about 10 times steeper than that of the field free control. For deduction of ERLS frequency spectra, different criteria are discussed. Particle shape and additional field-induced motions like dielectrophoresis and particle-particle attraction do not significantly influence the criteria. For nystatin-treated cells, recalculation of dielectric cell properties revealed an ionophore-induced decrease in the internal conductivity. Although the absolute rotation speed and the rotation sense are not yet directly accessible, ERLS eliminates the tedious microscopic measurements. It offers computerized, statistically significant measurements of dielectric particle properties that are especially suitable for nonbiological applications, e.g., the study of colloidal particles.     

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.     

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.     

Vesicle sizing: Number distributions by dynamic light scattering

A procedure is described which optimizes nonnegative least squares and exponential sampling fitting methods for analysis of dynamic light scattering (DLS) data from aqueous suspensions of vesicle/liposome systems. This approach utilizes a Rayleigh-Gans-Debye form factor for a coated sphere and yields number distributions which can be compared directly to distributions obtained by freeze-fracture electron microscopy (EM). Excellent agreement between the DLS and EM results are obtained for vesicle size distributions in the 100-200-nm range.     

Rotational relaxation of macromolecules determined by dynamic light scattering. II. Temperature dependence for DNA

Correlation functions have been determined for the fluctuating intensity of the depolarized component of forward-scattered laser light from solutions of DNA. The molecular correlation function of calf thymus DNA (mol wt ～15 × 10⁶) appears to exhibit a longest relaxation time (τ_25,w, ～ 18 msec) close to what one would predict from the flowdichroism measurements of Callis and Davidson and, in addition, manifests a spectrum of faster times down to tenths of milliseconds. Furthermore, a major fraction of the amplitude of fluctuations in the angular distribution of segment axes is relaxed on a very much shorter time scale (of the order of 20 microseconds) that appears to be relatively insensitive to molecular weight of the DNA, or to near-melting temperatures. The temperature profile of the longest relaxation time has been obtained and found to exhibit a peculiar spike near T_m, which, together with the absence of a corresponding spike in the (high shear) viscosity, has been interpreted as indicative of an increase in the molecular weight of the DNA in a narrow temperature region near T_m. Correlation functions for polarized light scattered at finite angles were obtained in an attempt to determine the temperature dependence of the translational diffusion coefficient. Although the data contain an extremely slow component that does not admit a simple interpretation, there is some indication of a decrease in the translational diffusion coefficient near T_m, thus supporting the notion of an aggregation occurring near T_m. Finally, a “counterion escape” mechanisn is proposed for the apparent aggregation.     

Gelatin-alpha olefin sulfonate interactions studied by dynamic light scattering

Dynamic light scattering (DLS) measurements were performed to study the binding of anionic surfactant alpha olefin sulfonate (AOS) to gelatin chains at various NaCl concentrations at 30 degrees C in aqueous sodium phosphate buffer (pH = 6.8) solutions. The surfactant concentration was varied from 0 to 80 mM and the NaCl concentrations chosen were 0.025, 0.05, and 0.1 M. AOS exhibited electrostatic binding to the positively charged sites of the polypeptide chain resulting in considerable reduction in its hydrodynamic radius up to critical micellar concentration (cmc = 8 mM for no salt, 0.01 and 0.025 M, and 5 mM for 0.05 M and 2 mM for 0.1 M solutions). The correlation function revealed the presence of two types of structures above cmc; namely the micelles of AOS and gelatin-AOS micelle complexes. The micellar radii (Rm), the effective gelatin-surfactant complex radii (Rc), have been determined as a function of salt concentration. No critical aggregation concentration (cac) was observed. The inter-gelatin-surfactant complex (kD1) and inter-micellar interactions (kD2), were determined by fitting the concentration dependence of Rm and Rc to a virial expansion in reduced concentration (c - cmc), which are compared. While kD1 showed strong ionic strength dependence, kD2 remained invariant of the same. The protein to surfactant binding ratio was found to be smaller than normal. Results have been discussed within the framework of the necklace-bead model of polymer-surfactant interactions.     

Solution dimensions of the gramicidin dimer by dynamic light scattering

Gramicidin is thought to form dimeric helical rods in alcohol solutions. In addition, there is evidence that the rod dimensions change upon addition of potassium ions. The present work reports values for the translational and rotational diffusion coefficients of gramicidin in methanol and 95% ethanol and in these same solvents with added KSCN. Solution dimensions are calculated from the diffusion coefficients. The results suggest that gramicidin exists primarily as dimers in these solutions and that the gramicidin rod does indeed become shorter upon addition of potassium ion. These results are consistent with those obtained from X-ray studies on single crystals grown from alcohol solutions.     

Interaction of YOYO-1 with guanine-rich DNA

The oxazole homodimer YOYO-1 has served as a valuable tool for the detection and quantification of nucleic acids. While the base specificity and selectivity of binding of YOYO-1 has been researched to some extent, the effect of unorthodox nucleic acid conformations on dye binding has received relatively less attention. In this work, we attempt to correlate the quadruplex-forming ability of G-rich sequences with binding of YOYO-1. Oligonucleotides differing in the number of tandem G repeats, total length, and length of loop sequence were evaluated for their ability to form quadruplexes in presence of sodium (Na⁺) or potassium (K⁺) ions. The fluorescence behavior of YOYO-1 upon binding such G-rich sequences was also ascertained. A distinct correlation was observed between the strength and propensity of quadruplex formation, and the affinity of YOYO-1 to bind such sequences. Specifically, as exemplified by the oligonucleotides 5′-G4T2G4-3′ and 5′-G3TG3TG3-3′, sequences possessing longer G-rich regions and shorter loop sequences formed stronger quadruplexes in presence of K⁺ which translated to weaker binding of YOYO-1. The dependence of binding of YOYO-1 on sequence and structural features of G-rich DNA has not been explored previously and such studies are expected to aid in more effective interpretation of applications involving the fluorophore.     

Characterization of precrystallization aggregation of canavalin by dynamic light scattering.

The aggregation processes leading to crystallization and precipitation of canavalin have been investigated by dynamic light scattering (DLS) in photon correlation spectroscopy (PCS) mode. The sizes of aggregates formed under various conditions of pH, salt concentration, and protein concentrations were deduced from the correlation functions generated by the fluctuating intensity of light scattered by the solutions of the protein. Results obtained indicate that the barrier to crystallization of canavalin is the formation of the trimer, a species that has been characterized by x-ray crystallographic studies (McPherson, A. 1980. J. Biol. Chem. 255:10472-10480). The dimensions of the trimer in solution are in good agreement with those obtained both from the crystal (McPherson, A. 1980. J. Biol. Chem. 255:10472-10480) and from a low angle x-ray scattering study in solution (Plietz, P., P. Damaschun, J. J. Müller, and B. Schlener. 1983. FEBS [Fed. Eur. Biochem. Soc.] Lett. 162:43-46). Furthermore, under conditions known to lead to the formation of rhombohedral crystals of canavalin, a limiting size is reached at high concentrations of canavalin. The size measured corresponds to an aggregate of trimers making a unit rhombohedral cell consistent with x-ray crystallographic data (McPherson, A. 1980. J. Biol. Chem. 255:10472-10480). Presumably, such aggregates are the nuclei from which crystal growth proceeds. The present study was undertaken primarily to test the potential of DLS (PCS) as a tool for rapid, routine screening to determine the ultimate fate of protein solutions (i.e., crystallization or amorphous precipitation) at an early stage, therefore eliminating the need for long-term visual observation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Configurational and dynamic properties of different length superhelical DNAs measured by dynamic light scattering

The solution conformation and internal motions of five superhelical DNAs between 2100 and 10200 base-pairs in length have been characterized by dynamic light scattering (DLS). Variations in the diffusion coefficients and rotational relaxation times with molecular weight are both indicative of an anisotropic extended structure of these DNAs; we therefore conclude that under our conditions the interwound superhelical structure prevails. The internal dynamics can be described by a superposition of rotational diffusion and internal relaxation. The latter process is characterized by the internal diffusion of persistence length size segments within the DNA chain and faster bending motions within these segments.     

Aggregation of amphiphilic pullulan derivatives evidenced by on-line flow field flow fractionation/multi-angle laser light scattering

Size-exclusion chromatography (SEC) is a useful steric separation technique for the analysis of water-soluble polysaccharides in aqueous solution. However, in the case of amphiphilic derivatives, the usefulness is limited because of interactions between hydrophobic segments and the stationary phase. Alkyl-bearing pullulans differing from the extent and the length of alkyl groups were characterized using flow-field flow fractionation with on-line coupling multi-angle laser light scattering (F⁴/MALLS). Comparison of SEC and F⁴ is presented and the interest of F⁴ in the field of amphiphilic derivatives is demonstrated.     

The onset of amelogenin nanosphere aggregation studied by small-angle X-ray scattering and dynamic light scattering

Proteins with predominantly hydrophobic character called amelogenins play a key role in the formation of the highly organized enamel tissue by forming nanospheres that interact with hydroxyapatite crystals. In the present investigation, we have studied the temperature and pH-dependent self-assembly of two recombinant mouse amelogenins, rM179 and rM166, the latter being an engineered version of the protein that lacks a 13 amino acid hydrophilic C-terminus. It has been postulated that this hydrophilic domain plays an important role in controlling the self-assembly behavior of rM179. By small-angle X-ray and neutron scattering, as well as by dynamic light scattering, we observed the onset of an aggregation of the rM179 protein nanospheres at pH 8. This behavior of the full-length recombinant protein is best explained by a core-shell model for the nanospheres, where hydrophilic and negatively charged side chains prevent the agglomeration of hydrophobic cores of the protein nanospheres at lower temperatures, while clusters consisting of several nanospheres start to form at elevated temperatures. In contrast, while capable of forming nanospheres, rM166 shows a very different aggregation behavior resulting in the formation of larger precipitates just above room temperature. These results, together with recent observations that rM179, unlike rM166, can regulate mineral organization in vitro, suggest that the aggregation of nanospheres of the full-length amelogenin rM179 is an important step in the self-assembly of the enamel matrix.     

Quasi-elastic light scattering by calf thymus DNA and lamdaDNA.

The quasi-elastic light-scattering (homodyne) time-correlation functions of calf thymus and λDNA are shown to contain contributions from at least two relaxation processes. A method of asymptotic analysis is described and used to obtain an estimate of the longest relaxation time as well as the “average” relaxation time and the mean-squared dispersion in this average. Most theories of scattering from macromolecules in the limit of inifinite dilution predict that the longest relaxation time is due to translational self-diffusion. The data obtained, however, indicate that the longest time is not simply related to the translational self-diffusion coefficient of unaggregated macromolecules. It is also shown that the longest relaxation time of λDNA decreases in the later stages of the denaturation transition region. Some possible mechanisms for the origin of this long time are discussed, including a model of restricted motion of a molecule by its neighbors.     

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.