No evidence of pathogenic involvement of cathelicidins in patient cohorts and mouse models of lupus and arthritis.

Apart from their role in the immune defence against pathogens evidence of a role of antimicrobial peptides (AMPs) in autoimmune diseases has accumulated in the past years. The aim of this project was to examine the functional impact of the human cathelicidin LL-37 and the mouse cathelicidin-related AMP (CRAMP) on the pathogenesis of lupus and arthritis. Serum LL-37 and anti-LL-37 levels were measured by ELISA in healthy donors and patients with Systemic Lupus Erythematosus (SLE) and Rheumatoid arthritis (RA). Pristane-induced lupus was induced in female wild type (WT) and cathelicidin-deficient (CRAMP-/-) mice. Serum levels of anti-Sm/RNP, anti-dsDNA, and anti-histone were determined via ELISA, cytokines in sera and peritoneal lavages were measured via Multiplex. Expression of Interferon I stimulated genes (ISG) was determined by real-time PCR. Collagen-induced arthritis (CIA) was induced in male WT and CRAMP-/- mice and arthritis severity was visually scored and analysed histomorphometrically by OsteoMeasure software. Serum levels of anti-LL-37 were higher in SLE-patients compared to healthy donors or patients with RA. However, no correlation to markers of disease activity or organ involvement was observed. No significant differences of autoantibody or cytokine/chemokine levels, or of expression of ISGs were observed between WT and CRAMP-/- mice after pristane-injection. Furthermore, lung and kidney pathology did not differ in the absence of CRAMP. Incidence and severity of CIA and histological parameters (inflammation, cartilage degradation, and bone erosion) were not different in WT and CRAMP-/- mice. Although cathelicidins are upregulated in mouse models of lupus and arthritis, cathelicidin-deficiency did not persistently affect the diseases. Also in patients with SLE, autoantibodies against cathelicidins did not correlate with disease manifestation. Reactivity against cathelicidins in lupus and arthritis could thus be an epiphenomenon caused by extensive overexpression in blood and affected tissues. In addition, other cationic AMPs could functionally compensate for the deficiency of cathelicidins.

Vipericidins: a novel family of cathelicidin-related peptides from the venom gland of South American pit vipers.

Cathelicidins are phylogenetically ancient, pleiotropic host defense peptides-also called antimicrobial peptides (AMPs)-expressed in numerous life forms for innate immunity. Since even the jawless hagfish expresses cathelicidins, these genetically encoded host defense peptides are at least 400 million years old. More recently, cathelicidins with varying antipathogenic activities and cytotoxicities were discovered in the venoms of poisonous snakes; for these creatures, cathelicidins may also serve as weapons against prey and predators, as well as for innate immunity. We report herein the expression of orthologous cathelicidin genes in the venoms of four different South American pit vipers (Bothrops atrox, Bothrops lutzi, Crotalus durissus terrificus, and Lachesis muta rhombeata)-distant relatives of Asian cobras and kraits, previously shown to express cathelicidins-and an elapid, Pseudonaja textilis. We identified six novel, genetically encoded peptides: four from pit vipers, collectively named vipericidins, and two from the elapid. These new venom-derived cathelicidins exhibited potent killing activity against a number of bacterial strains (S. pyogenes, A. baumannii, E. faecalis, S. aureus, E. coli, K. pneumoniae, and P. aeruginosa), mostly with relatively less potent hemolysis, indicating their possible usefulness as lead structures for the development of new anti-infective agents. It is worth noting that these South American snake venom peptides are comparable in cytotoxicity (e.g., hemolysis) to human cathelicidin LL-37, and much lower than other membrane-active peptides such as mastoparan 7 and melittin from bee venom. Overall, the excellent bactericidal profile of vipericidins suggests they are a promising template for the development of broad-spectrum peptide antibiotics.

Electric and hydrodynamic stretching of DNA-polymer conjugates in free-solution electrophoresis.

The conjugation of an uncharged polymer to DNA fragments makes it possible to separate DNA by free-solution electrophoresis. This end-labeled free-solution electrophoresis method has been shown to successfully separate ssDNA with single monomer resolution up to about 110 bases. It is the aim of this paper to investigate in more detail the coupled hydrodynamic and electrophoretic deformation of the ssDNA-label conjugate at fields below 400 V/cm. Our model is an extension of the theoretical approach originally developed by Stigter and Bustamante [Biophys. J. 75, 1197 (1998)] to investigate the problems of a tethered chain stretching in a hydrodynamic flow and of the electrophoretic stretch of a tethered polyelectrolyte. These two separate models are now used together since the charged DNA is "tethered" to the uncharged polymer (and vice versa), and the resulting self-consistent model is used to predict the deformation and the electrophoretic velocity for the hybrid molecule. Our theoretical and experimental results are in good qualitative agreement.

Capillary electrophoretic separation of uncharged polymers using polyelectrolyte engines. Theoretical model.

We recently demonstrated that the molecular mass distribution of an uncharged polymer sample can be analyzed using free-solution capillary electrophoresis of DNA-polymer conjugates. In these conjugates, the DNA is providing the electromotive force while the uncharged polydisperse polymer chains of the sample retard the DNA engine with different amounts of hydrodynamic drag. Here we present a theoretical model of this new analytical method. We show that for the most favourable, diffusion-limited electrophoresis conditions, there is actually an optimal DNA size to achieve the separation of a given polymer sample. Moreover, we demonstrate that the effective friction coefficient of the polymer chains is related to the stiffness of the two polymers of the conjugate, thus offering a method to estimate the persistence length of the uncharged polymer through mobility measurements. Finally, we compare some of our predictions with available experimental results.

Peptoid oligomers with alpha-chiral, aromatic side chains: sequence requirements for the formation of stable peptoid helices.

The achiral backbone of oligo-N-substituted glycines or "peptoids" lacks hydrogen-bond donors, effectively preventing formation of the regular, intrachain hydrogen bonds that stabilize peptide alpha-helical structures. Yet, when peptoids are N-substituted with alpha-chiral, aromatic side chains, oligomers with as few as five residues form stable, chiral, polyproline-like helices in either organic or aqueous solution. The adoption of chiral secondary structure in peptoid oligomers is primarily driven by the steric influence of these bulky, chiral side chains. Interestingly, peptoid helices of this class exhibit intense circular dichroism (CD) spectra that closely resemble those of peptide alpha-helices. Here, we have taken advantage of this distinctive spectroscopic signature to investigate sequence-related factors that favor and disfavor stable formation of peptoid helices of this class, through a comparison of more than 30 different heterooligomers with mixed chiral and achiral side chains. For this family of peptoids, we observe that a composition of at least 50% alpha-chiral, aromatic residues is necessary for the formation of stable helical structure in hexameric sequences. Moreover, both CD and 1H-13C HSQC NMR studies reveal that these short peptoid helices are stabilized by the placement of an alpha-chiral, aromatic residue on the carboxy terminus. Additional stabilization can be provided by the presence of an "aromatic face" on the helix, which can be patterned by positioning aromatic residues with three-fold periodicity in the sequence. Extending heterooligomer chain length beyond 12-15 residues minimizes the impact of the placement, but not the percentage, of alpha-chiral aromatic side chains on overall helical stability. In light of these new data, we discuss implications for the design of helical, biomimetic peptoids based on this structural motif.

Peptoid oligomers with alpha-chiral, aromatic side chains: effects of chain length on secondary structure.

Oligomeric N-substituted glycines or "peptoids" with alpha-chiral, aromatic side chains can adopt stable helices in organic or aqueous solution, despite their lack of backbone chirality and their inability to form intrachain hydrogen bonds. Helical ordering appears to be stabilized by avoidance of steric clash as well as by electrostatic repulsion between backbone carbonyls and pi clouds of aromatic rings in the side chains. Interestingly, these peptoid helices exhibit intense circular dichroism (CD) spectra that closely resemble those of peptide alpha-helices. Here, we have utilized CD to systematically study the effects of oligomer length, concentration, and temperature on the chiral secondary structure of organosoluble peptoid homooligomers ranging from 3 to 20 (R)-N-(1-phenylethyl)glycine (Nrpe) monomers in length. We find that a striking evolution in CD spectral features occurs for Nrpe oligomers between 4 and 12 residues in length, which we attribute to a chain length-dependent population of alternate structured conformers having cis versus trans amide bonds. No significant changes are observed in CD spectra of oligomers between 13 and 20 monomers in length, suggesting a minimal chain length of about 13 residues for the formation of stable poly(Nrpe) helices. Moreover, no dependence of circular dichroism on concentration is observed for an Nrpe hexamer, providing evidence that these helices remain monomeric in solution. In light of these new data, we discuss chain length-related factors that stabilize organosoluble peptoid helices of this class, which are important for the design of helical, biomimetic peptoids sharing this structural motif.

Molar mass profiling of synthetic polymers by free-solution capillary electrophoresis of DNA-polymer conjugates.

The molar mass distribution of a polymer sample is a critical determinant of its material properties and is generally analyzed by gel permeation chromatography or more recently, by MALDI-TOF mass spectrometry. We describe here a novel method for the determination of the degree of polymerization of polydisperse, uncharged, water-soluble polymers (e.g., poly(ethylene glycol) (PEG)), based upon single-monomer resolution of DNA-polymer conjugates by free-solution capillary electrophoresis. This is accomplished by end-on covalent conjugation of a polydisperse, uncharged polymer sample (PEG) to a monodisperse, fluorescently labeled DNA oligomer, followed by electrophoretic analysis. The monodisperse, charged DNA "engine" confers to each conjugate an equal amount of electromotive force, while the varying contour lengths of the uncharged, polydisperse polymers engender different amounts of hydrodynamic drag. The balance of electromotive and hydrodynamic forces enables rapid, high-resolution separation of the DNA-polymer conjugates as a function of the size of the uncharged PEG tail. This provides a profile of the molar mass distribution of the original polymer sample that can be detected by laser-induced fluorescence through excitation of the dye-labeled DNA. We call this method free solution conjugate electrophoresis (FSCE). Theory-based analysis of the resulting electrophoresis data allows precise calculation of the degree of polymerization of the PEG portion of each conjugate molecule. Knowledge of the molecular mass of the uncharged polymer's repeat unit allows for direct calculation of the molar mass averages as well as sample polydispersity index. The results of these analyses are strikingly reminiscent of MALDI-TOF spectra taken of the same PEG samples. PEG samples of 3.4-, 5-, and 20-kDa nominal average molar mass were analyzed by FSCE and MALDI-TOF; the values of the molar mass averages, Mw and Mn, typically agree to within 5%. Measurements and molar mass calculations are performed without any internal standards or calibration. Moreover, when DNA-polymer conjugate analysis is performed in a chip-based electrophoresis system, separation is complete in less than 13 min. FSCE offers an alternative to MALDI-TOF for the characterization of uncharged, water-soluble polymers that can be uniquely conjugated to DNA.

Impact of polymer hydrophobicity on the properties and performance of DNA sequencing matrices for capillary electrophoresis.

To elucidate the impact of matrix chemical and physical properties on DNA sequencing separations by capillary electrophoresis (CE), we have synthesized, characterized and tested a controlled set of different polymer formulations for this application. Homopolymers of acrylamide and N,N-dimethylacrylamide (DMA) and copolymers of DMA and N,N-diethylacrylamide (DEA) were synthesized by free radical polymerization and purified. Polymer molar mass distributions were characterized by tandem gel permeation chromatography - laser light scattering. Polymers with different chemical compositions and similar molar mass distributions were selected and employed at the same concentration so that the variables of comparison between them were hydrophobicity and average coil size in aqueous solution. We find that the low-shear viscosities of 7% w/v polymer solutions decrease by orders of magnitude with increasing polymer hydrophobicity, while hydrophilic polymers exhibit more pronounced reductions in viscosity with increased shear. The performance of the different matrices for DNA sequencing was compared with the same sample under identical CE conditions. The longest read length was produced with linear polyacrylamide (LPA) while linear poly-N,N-dimethylacrylamide (PDMA) gave approximately 100 fewer readable bases. Read lengths with DMA/DEA copolymers were lower, and decreased with increasing DEA content. This study highlights the importance of polymer hydrophilicity for high-performance DNA sequencing matrices, through the formation of robust, highly-entangled polymer networks and the minimization of hydrophobic interactions between polymers and fluorescently-labeled DNA molecules. However, the results also show that more hydrophobic matrices offer much lower viscosities, enabling easier microchannel loading at low applied pressures.

Microchannel DNA sequencing matrices with a thermally controlled "viscosity switch".

Polymers and hydrogels that swell or shrink in response to environmental stimuli such as changes in temperature, pH, or ionic strength are of interest as switchable materials for applications in biotechnology. In this paper, we show that thermoresponsive polymers offer some particular advantages as entangled matrices for DNA sequencing by capillary and microchip electrophoresis. Matrices based on conventional water-soluble polymers demand a compromise in their design for microchannel electrophoresis: whereas highly entangled solutions of high molar mass polymers provide optimal sequencing performance, their highly viscous solutions require application of high pressures to be loaded into electrophoresis microchannels. Here, we demonstrate the reproducible synthesis, precise characterization, and excellent DNA sequencing performance of high molar mass, thermoresponsive polymer matrices that exhibit a reversible, temperature-controlled "viscosity switch" from high-viscosity solutions at 25 degrees C to low-viscosity, microphase-separated colloidal dispersions at a chosen, elevated temperature. The viscosity switch decouples matrix loading and sieving properties, enabling acceleration of microchannel flow by 3 orders of magnitude. DNA sequencing separations yielding read lengths of 463 bases of contiguous sequence in 78 min with 97% base-calling accuracy can be achieved in these matrices. Switchable matrices will be particularly applicable to microfluidic devices with dynamic temperature control, which are likely to provide the next major leap in the efficiency of high-throughput DNA analysis.

The use of light scattering for precise characterization of polymers for DNA sequencing by capillary electrophoresis.

The ability of a polymer matrix to separate DNA by capillary electrophoresis (CE) is strongly dependent upon polymer physical properties. In particular, recent results have shown that DNA sequencing performance is very sensitive to both the average molar mass and the average coil radius of the separation matrix polymers, which are affected by both polymer structure and polymer-solvent affinity. Large polymers with high average molar mass provide the best DNA sequencing separations for CE, but are also the most challenging to characterize with accuracy. The methods most commonly used for the characterization of water-soluble polymers with application in microchannel electrophoresis have been gel permeation chromatography (GPC) and intrinsic viscosity measurements, but the limitations and potential inaccuracies of these approaches, particularly for large or novel polymers and copolymers, press the need for a more universally accurate method of polymer molar mass profiling for advanced DNA separation matrices. Here, we show that multi-angle laser light scattering (MALLS) measurements, carried out either alone or in tandem with prior on-line sample fractionation by GPC, can provide accurate molar mass and coil radius information for polymer samples that are useful for DNA sequencing by CE. Wider employment of MALLS for characterization of novel polymers designed as DNA separation matrices for microchannel electrophoresis should enable more rapid optimization of matrix properties and formulation, and assist in the development of novel classes of polymer matrices.

DNA sequencing up to 1300 bases in two hours by capillary electrophoresis with mixed replaceable linear polyacrylamide solutions.

This paper presents results on ultralong read DNA sequencing with relatively short separation times using capillary electrophoresis with replaceable polymer matrixes. In previous work, the effectiveness of mixed replaceable solutions of linear polyacrylamide (LPA) was demonstrated, and 1000 bases were routinely obtained in less than 1 h. Substantially longer read lengths have now been achieved by a combination of improved formulation of LPA mixtures, optimization of temperature and electric field, adjustment of the sequencing reaction, and refinement of the base-caller. The average molar masses of LPA used as DNA separation matrixes were measured by gel permeation chromatography and multiangle laser light scattering. Newly formulated matrixes comprising 0.5% (w/w) 270 kDa and 2% (w/w) 10 or 17 MDa LPA raised the optimum column temperature from 60 to 70 degrees C, increasing the selectivity for large DNA fragments, while maintaining high selectivity for small fragments as well. This improved resolution was further enhanced by reducing the electric field strength from 200 to 125 V/cm. In addition, because sequencing accuracy beyond 1000 bases was diminished by the low signal from G-terminated fragments when the standard reaction protocol for a commercial dye primer kit was used, the amount of these fragments was doubled. Augmenting the base-calling expert system with rules specific for low peak resolution also had a significant effect, contributing slightly less than half of the total increase in read length. With full optimization, this read length reached up to 1300 bases (average 1250) with 98.5% accuracy in 2 h for a single-stranded M13 template.

Polymeric matrices for DNA sequencing by capillary electrophoresis.

We review the wide range of polymeric materials that have been employed for DNA sequencing separations by capillary electrophoresis. Intensive research in the area has converged in showing that highly entangled solutions of hydrophilic, high molar mass polymers are required to achieve high DNA separation efficiency and long read length, system attributes that are particularly important for genomic sequencing. The extent of DNA-polymer interactions, as well as the robustness of the entangled polymer network, greatly influence the performance of a given polymer matrix for DNA separation. Further fundamental research in the field of polymer physics and chemistry is needed to elucidate the specific mechanisms by which DNA is separated in dynamic, uncross-linked polymer networks.

Bioinspired polymeric materials: in-between proteins and plastics.

Chemical and biological researchers are making rapid progress in the design and synthesis of non-natural oligomers and polymers that emulate the properties of natural proteins. Whereas molecular biologists are exploring biosynthetic routes to non-natural proteins with controlled material properties, synthetic polymer chemists are developing bioinspired materials with well-defined chemical and physical properties that function or self-organize according to defined molecular architectures. Bioorganic chemists, on the other hand, are developing several new classes of non-natural oligomers that are bridging the gap between molecular biology and polymer chemistry. These synthetic oligomers have both sidechain and length specificity, and, in some cases, demonstrate capability for folding, self-assembly, and specific biorecognition. Continued active exploration of diverse backbone and sidechain chemistries and connectivities in bioinspired oligomers will offer the potential for self-organized materials with greater chemical diversity and biostability than natural peptides. Taken together, advances in molecular bioengineering, polymer chemistry, and bioorganic chemistry are converging towards the creation of useful bioinspired materials with defined molecular properties.

NMR determination of the major solution conformation of a peptoid pentamer with chiral side chains.

Polymers of N-substituted glycines ("peptoids") containing chiral centers at the alpha position of their side chains can form stable structures in solution. We studied a prototypical peptoid, consisting of five para-substituted (S)-N-(1-phenylethyl)glycine residues, by NMR spectroscopy. Multiple configurational isomers were observed, but because of extensive signal overlap, only the major isomer containing all cis-amide bonds was examined in detail. The NMR data for this molecule, in conjunction with previous CD spectroscopic results, indicate that the major species in methanol is a right-handed helix with cis-amide bonds. The periodicity of the helix is three residues per turn, with a pitch of approximately 6 A. This conformation is similar to that anticipated by computational studies of a chiral peptoid octamer. The helical repeat orients the amide bond chromophores in a manner consistent with the intensity of the CD signal exhibited by this molecule. Many other chiral polypeptoids have similar CD spectra, suggesting that a whole family of peptoids containing chiral side chains is capable of adopting this secondary structure motif. Taken together, our experimental and theoretical studies of the structural properties of chiral peptoids lay the groundwork for the rational design of more complex polypeptoid molecules, with a variety of applications, ranging from nanostructures to nonviral gene delivery systems.

Sequence-specific polypeptoids: a diverse family of heteropolymers with stable secondary structure.

We have synthesized and characterized a family of structured oligo-N-substituted-glycines (peptoids) up to 36 residues in length by using an efficient solid-phase protocol to incorporate chemically diverse side chains in a sequence-specific fashion. We investigated polypeptoids containing side chains with a chiral center adjacent to the main chain nitrogen. Some of these sequences have stable secondary structure, despite the achirality of the polymer backbone and its lack of hydrogen bond donors. In both aqueous and organic solvents, peptoid oligomers as short as five residues give rise to CD spectra that strongly resemble those of peptide alpha-helices. Differential scanning calorimetry and CD measurements show that polypeptoid secondary structure is highly stable and that unfolding is reversible and cooperative. Thermodynamic parameters obtained for unfolding are similar to those obtained for the alpha-helix to coil transitions of peptides. This class of biomimetic polymers may enable the design of self-assembling macromolecules with novel structures and functions.

Capillary electrophoresis of DNA in uncrosslinked polymer solutions: evidence for a new mechanism of DNA separation.

The electrophoretic separation of DNA molecules is usually performed in thin slabs of agarose or polyacrylamide gel. However, DNA separations can be achieved more rapidly and efficiently within a microbore fused silica capillary filled with an uncrosslinked polymer solution. An early assumption was that the mechanism of DNA separation in polymer solution-capillary electrophoresis (PS-CE) is the same as that postulated to occur in slab gel electrophoresis, i.e., that entangled polymer chains form a network of "pores" through which the DNA migrates. However, we have demonstrated that large DNA restriction fragments (2.0-23.1 kbp) can be separated by CE in extremely dilute polymer solutions, which contain as little as 6 parts per million [0.0006% (w/w)] of uncrosslinked hydroxyethyl cellulose (HEC) polymers. In such extremely dilute HEC solutions, far below the measured polymer entanglement threshold concentration, pore-based models of DNA electrophoresis do not apply. We propose a transient entanglement coupling mechanism for the electrophoretic separation of DNA in uncrosslinked polymer solutions, which is based on physical polymer/DNA interactions.

The effects of polymer properties on DNA separations by capillary electrophoresis in uncross-linked polymer solutions.

Low-viscosity, aqueous solutions of hydrophilic linear polymers have been shown to be useful for the separation of DNA restriction fragments by capillary electrophoresis (CE). However, the choice of polymer type, size, and concentration remains largely empirical, because the mechanism of high-field electrophoretic DNA separations in polymer solutions is not well understood. To assist in elucidating the mechanism of DNA separation, we experimentally investigated the effects of polymer properties such as stiffness (persistence length), average molecular mass, polydispersity, and hydrophilicity on the separation of DNA ranging from 72 bp to 23 kbp. This was accomplished by comparing the results of DNA separations obtained by counter-migration CE in dilute and semidilute solutions of linear polyacrylamide (PAA), hydroxyethyl-cellulose (HEC), and hydroxypropylcellulose (HPC) polymers of several different average molecular masses.

The use of coated and uncoated capillaries for the electrophoretic separation of DNA in dilute polymer solutions.

We show that both uncoated and polyacrylamide-coated capillaries provide separation of large DNA restriction fragments (2.0-23.1 kbp) by capillary electrophoresis in dilute cellulosic polymer solutions. Uncoated capillaries, however, provide significantly better resolution of DNA fragments, particularly when ultra-dilute polymer solutions are used. This is because electroosmotic flow in uncoated capillaries increases the residence time of DNA in the capillary, without significantly contributing to band-broadening. At a given field strength and polymer concentration in the buffer, the electrophoretic mobilities of DNA restriction fragments in coated capillaries are virtually identical to those previously measured in uncoated capillaries. It is concluded that the fused silica surface of the capillary does not play a significant role in the mechanism of DNA separation by capillary electrophoresis in uncrosslinked polymer solutions. Thus, the separation of large DNA which has been observed to occur in ultra-dilute polymer solutions arises primarily from entanglement interactions between the cellulosic polymers and DNA restriction fragments which occur within the bulk of the polymer solution.

A transient entanglement coupling mechanism for DNA separation by capillary electrophoresis in ultradilute polymer solutions.

Using capillary electrophoresis, large DNA molecules (2.0-23.1 kbp) may be rapidly separated in ultradilute polymer solutions (< 0.002% w/w) under a high-voltage, steady field (265 V/cm). At this polymer concentration, the separation mechanism appears to be significantly different from that postulated to occur in crosslinked gels. Based on experimental results obtained with DNA restriction fragments and with negatively charged latex microspheres, we conclude that the Ogston and reptation models typically used to describe gel electrophoresis are not appropriate for DNA separations in such dilute polymer solutions. Electrophoresis experiments employing solutions of both small and large hydroxyethyl cellulose polymers highlight the importance of polymer length and concentration for the optimum resolution of DNA fragments varying in size from 72 bp to 23.1 kbp. A transient entanglement coupling mechanism for DNA separation in dilute polymer solutions is developed, which suggests that there is no a priori upper size limit to DNA that can be separated by capillary electrophoresis in a constant field.

Capillary electrophoresis of DNA in uncross-linked polymer solutions.

We have used dilute and semi-dilute uncross-linked hydroxyethyl cellulose (HEC) solutions as separation matrices for capillary electrophoresis of DNA restriction fragments. In these experiments, we investigated the effects of HEC molecular weight and concentration on resolution, attempting to relate these parameters to the polymer entanglement threshold concentration. The entanglement thresholds of seven molecular weight fractions of hydroxyethyl cellulose were determined from viscosity-concentration data; the entanglement threshold was found to scale as N-1.2, where N = number of HEC monomers. This finding is not in agreement with classical scaling arguments. We present a relationship to predict the observed entanglement threshold of HEC in solution as a function of number average molecular weight. It was found that excellent separation of phi X174/HaeIII DNA restriction fragments (72-1353 base pairs) by capillary electrophoresis in HEC solutions can be achieved significantly below the entanglement threshold, depending on DNA size and HEC molecular weight. The mechanism of separation in these uncross-linked polymer solutions must therefore be reexamined. Our experiments show that the entanglement threshold is a useful parameter in predicting a range of HEC concentrations which will separate certain DNA fragments for a given HEC molecular weight. However, the presence of a fully entangled network is not a prerequisite for separation.