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Amyloid self‐assembly of islet amyloid polypeptide (IAPP) is linked to pancreatic inflammation, β‐cell degeneration, and the pathogenesis of type 2 diabetes (T2D). The multifunctional host‐defence peptides (HDPs) cathelicidins play crucial roles in inflammation. Here, we show that the antimicrobial and immunomodulatory polypeptide human cathelicidin LL‐37 binds IAPP with nanomolar affinity and effectively suppresses its amyloid self‐assembly and related pancreatic β‐cell damage in vitro. In addition, we identify key LL‐37 segments that mediate its interaction with IAPP. Our results suggest a possible protective role for LL‐37 in T2D pathogenesis and offer a molecular basis for the design of LL‐37‐derived peptides that combine antimicrobial, immunomodulatory, and T2D‐related anti‐amyloid functions as promising candidates for multifunctional drugs., One for all: A high‐affinity amyloid‐suppressing interaction between the human antimicrobial and immunomodulatory polypeptide cathelicidin LL‐37 and the key type 2 diabetes (T2D) amyloid polypeptide IAPP was identified. The results suggest a protective role for LL‐37 in T2D pathogenesis and offer a molecular basis for the design of novel molecules combining antimicrobial, immunomodulatory, and anti‐amyloid functions as multifunctional drug candidates.

Viral infections, such as those caused by Herpes Simplex Virus-1 (HSV-1) and SARS-CoV-2, affect millions of people each year. However, there are few antiviral drugs that can effectively treat these infections. The standard approach in the development of antiviral drugs involves the identification of a unique viral target, followed by the design of an agent that addresses that target. Antimicrobial peptides (AMPs) represent a novel source of potential antiviral drugs. AMPs have been shown to inactivate numerous different enveloped viruses through the disruption of their viral envelopes. However, the clinical development of AMPs as antimicrobial therapeutics has been hampered by a number of factors, especially their enzymatically labile structure as peptides. We have examined the antiviral potential of peptoid mimics of AMPs (sequence-specific N-substituted glycine oligomers). These peptoids have the distinct advantage of being insensitive to proteases, and also exhibit increased bioavailability and stability. Our results demonstrate that several peptoids exhibit potent in vitro antiviral activity against both HSV-1 and SARS-CoV-2 when incubated prior to infection. In other words, they have a direct effect on the viral structure, which appears to render the viral particles non-infective. Visualization by cryo-EM shows viral envelope disruption similar to what has been observed with AMP activity against other viruses. Furthermore, we observed no cytotoxicity against primary cultures of oral epithelial cells. These results suggest a common or biomimetic mechanism, possibly due to the differences between the phospholipid head group makeup of viral envelopes and host cell membranes, thus underscoring the potential of this class of molecules as safe and effective broad-spectrum antiviral agents. We discuss how and why differing molecular features between 10 peptoid candidates may affect both antiviral activity and selectivity.

Antimicrobial peptides have attracted considerable interest as potential new class of antibiotics against multi-drug resistant bacteria. However, their therapeutic potential is limited, in part due to susceptibility towards enzymatic degradation and low bioavailability. Peptoids (oligomers of N-substituted glycines) demonstrate proteolytic stability and better bioavailability than corresponding peptides while in many cases retaining antibacterial activity. In this study, we synthesized a library of 36 peptoids containing fluorine, chlorine, bromine and iodine atoms, which vary by length and level of halogen substitution in position 4 of the phenyl rings. As we observed a clear correlation between halogenation of an inactive model peptoid and its increased antimicrobial activity, we designed chlorinated and brominated analogues of a known peptoid and its shorter counterpart. Short brominated analogues displayed up to 32-fold increase of the activity against S. aureus and 16- to 64-fold against E. coli and P. aeruginosa alongside reduced cytotoxicity. The biological effect of halogens seems to be linked to the relative hydrophobicity and self-assembly properties of the compounds. By small angle X-ray scattering (SAXS) we have demontrated how the self-assembled structures are dependent on the size of the halogen, degree of substitution and length of the peptoid, and correlated these features to their activity.

Acute lung injury (ALI) leads to progressive loss of breathing capacity and hypoxemia, as well as pulmonary surfactant dysfunction. ALI’s pathogenesis and management are complex, and it is a significant cause of morbidity and mortality worldwide. Exogenous surfactant therapy, even for research purposes, is impractical for adults because of the high cost of current surfactant preparations. Prior in vitro work has shown that poly-N-substituted glycines (peptoids), in a biomimetic lipid mixture, emulate key biophysical activities of lung surfactant proteins B and C at the air-water interface. Here we report good in vivo efficacy of a peptoid-based surfactant, compared with extracted animal surfactant and a synthetic lipid formulation, in a rat model of lavage-induced ALI. Adult rats were subjected to whole-lung lavage followed by administration of surfactant formulations and monitoring of outcomes. Treatment with a surfactant protein C mimic formulation improved blood oxygenation, blood pH, shunt fraction, and peak inspiratory pressure to a greater degree than surfactant protein B mimic or combined formulations. All peptoid-enhanced treatment groups showed improved outcomes compared to synthetic lipids alone, and some formulations improved outcomes to a similar extent as animal-derived surfactant. Robust biophysical mimics of natural surfactant proteins may enable new medical research in ALI treatment.

Peptoids are peptidomimetic polymers that are resistant to proteolysis and less prone to immune responses; thus, they can provide a practical alternative to peptides. Among the various therapeutic applications that have been explored, cationic amphipathic peptoids have demonstrated broad-spectrum antibacterial activity, including activity towards drug-resistant bacterial strains. While their potency and activity spectrum can be manipulated by sequence variations, bacterial selectivity and systemic toxicity need to be improved for further clinical development. To this aim, we incorporated various hydrophobic or cationic residues to improve the selectivity of the previously developed antibacterial peptoid 1. The analogs with hydrophobic residues demonstrated non-specific cytotoxicity, while those with an additional cationic residue showed improved selectivity and comparable antibacterial activity. Specifically, compared to 1, peptoid 7 showed much lower hemolysis and cytotoxicity, while maintaining the antibacterial activity. Therefore, we believe that peptoid 7 has the potential to serve as a promising alternative to current antimicrobial therapies.

Background Identifying physiologically relevant binding partners of amyloid-β (Aβ) that modulate in vivo fibril formation may yield new insights into Alzheimer's disease (AD) etiology. Human cathelicidin peptide, LL-37, is an innate immune effector and modulator, ubiquitous in human tissues and expressed in myriad cell types. Objective We present in vitro experimental evidence and discuss findings supporting a novel hypothesis that LL-37 binds to Aβ42 and can modulate Aβ fibril formation. Methods Specific interactions between LL-37 and Aβ (with Aβ in different aggregation states, assessed by capillary electrophoresis) were demonstrated by surface plasmon resonance imaging (SPRi). Morphological and structural changes were investigated by transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy. Neuroinflammatory and cytotoxic effects of LL-37 alone, Aβ42 alone, and LL-37/Aβ complexes were evaluated in human microglia and neuroblastoma cell lines (SH-SY5Y). Results SPRi shows binding specificity between LL-37 and Aβ, while TEM shows that LL-37 inhibits Aβ42 fibril formation, particularly Aβ's ability to form long, straight fibrils characteristic of AD. CD reveals that LL-37 prevents Aβ42 from adopting its typical β-type secondary structure. Microglia-mediated toxicities of LL-37 and Aβ42 to neurons are greatly attenuated when the two peptides are co-incubated prior to addition. We discuss the complementary biophysical characteristics and AD-related biological activities of these two peptides. Conclusion Based on this body of evidence, we propose that LL-37 and Aβ42 may be natural binding partners, which implies that balanced (or unbalanced) spatiotemporal expression of the two peptides could impact AD initiation and progression.

Many organisms rely on antimicrobial peptides (AMPs) as a first line of defense against pathogens. In general, most AMPs are thought to kill bacteria by binding to and disrupting cell membranes. However, certain AMPs instead appear to inhibit biomacromolecule synthesis, while causing less membrane damage. Despite an unclear understanding of mechanism(s), there is considerable interest in mimicking AMPs with stable, synthetic molecules. Antimicrobial N-substituted glycine (peptoid) oligomers (“ampetoids”) are structural, functional and mechanistic analogs of helical, cationic AMPs, which offer broad-spectrum antibacterial activity and better therapeutic potential than peptides. Here, we show through quantitative studies of membrane permeabilization, electron microscopy, and soft X-ray tomography that both AMPs and ampetoids trigger extensive and rapid non-specific aggregation of intracellular biomacromolecules that correlates with microbial death. We present data demonstrating that ampetoids are “fast killers”, which rapidly aggregate bacterial ribosomes in vitro and in vivo. We suggest intracellular biomass flocculation is a key mechanism of killing for cationic, amphipathic AMPs, which may explain why most AMPs require micromolar concentrations for activity, show significant selectivity for killing bacteria over mammalian cells, and finally, why development of resistance to AMPs is less prevalent than developed resistance to conventional antibiotics.

We demonstrate here the power and flexibility of free-solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild-type DNA. Here, four large drag-tags are used to achieve free-solution electrophoretic separation of 19 LDR products ranging in size from 42–66 nt that correspond to mutations in the K-ras oncogene. LDR-FSCE enabled electrophoretic resolution of these 19 LDR-FSCE products by CE in 13.5 minutes (E = 310 V/cm) and by microchip electrophoresis in 140 seconds (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free-solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR-FSCE products were separated in < 70 seconds with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K-ras mutations on integrated “sample-in/answer-out” devices with amplification, LDR, and detection all on one platform.

Biomolecule encapsulation in droplets is important for miniaturizing biological assays to reduce reagent consumption, cost and time of analysis, and can be most effectively achieved by using microfabricated devices. Microfabricated fluidic devices can generate emulsified drops of uniform size with controlled dimensions and contents. Biological and chemical components such as cells, microgels, beads, hydrogel precursors, polymer initiators, and other droplets can be encapsulated within these drops. Encapsulated emulsions are appealing for a variety of applications since drops can be used as tiny reaction vessels to perform high-throughput reactions at fast rates, consuming minimal sample and solvent amounts due to the small size (micron diameters) of the emulsion drops. Facile mixing and droplet coalescence allow for a diversity of assays to be performed on-chip with tunable parameters. The simplicity of operation and speed of analysis with microencapsulated drops lends itself well to an array of quantitative biomolecular studies such as directed evolution, single-molecule DNA amplification, single-cell encapsulation, high-throughput sequencing, enzyme kinetics, and microfluidic cell culture. This review highlights recent advances in the field of microfabricated encapsulating devices, emphasizing the development of emulsifying encapsulations, device design, and current assays that are performed using encapsulating droplets.

Protein polymer-based hydrogels have shown potential for tissue engineering applications, but require biocompatibility testing for in vivo use. Enzymatically crosslinked protein polymer-based hydrogels were tested in vitro and in vivo to evaluate their biocompatibility. Endotoxins present in the hydrogel were removed by Trition X-114 phase separation. The reduction of endotoxins decreased TNF-α production by a macrophage cell line in vitro; however, significant inflammatory response was still present compared to collagen control gels. A branched PEG molecule and dexamethasone were added to the hydrogel to reduce the response. In vitro testing showed a decrease in the TNF-α levels with the addition of dexamethasone. In vivo implantations into the epididymal fat pad of C57/BL6 mice, however, indicated a decreased inflammatory mediated immune response with a hydrogel treated with both PEGylation and endotoxin reduction. This study demonstrates the importance of endotoxin testing and removal in determining the biocompatibility of biomaterials.

Networks of discrete, genipin-crosslinked gelatin microfibers enveloping pancreatic islets were incorporated within barium alginate microcapsules. This novel technique enabled encapsulation of cellular aggregates in a spherical fibrous matrix

Resolution of DNA fragments separated by electrophoresis in polymer solutions (“matrices”) is determined by both the spacing between peaks and the width of the peaks. Prior research on the development of high-performance separation matrices has been focused primarily on optimizing DNA mobility and matrix selectivity, and gave less attention to peak broadening. Quantitative data are rare for peak broadening in systems in which high electric field strengths are used (> 150 V/cm), which is surprising since capillary and microchip-based systems commonly run at these field strengths. Here, we report results for a study of band broadening behavior for ssDNA fragments on a glass microfluidic chip, for electric field strengths up to 320 V/cm. We compare dispersion coefficients obtained in a poly(N,N-dimethylacrylamide) (pDMA) separation matrix that was developed for chip-based DNA sequencing with a commercially available linear polyacrylamide (LPA) matrix commonly used in capillaries. Much larger DNA dispersion coefficients were measured in the LPA matrix as compared to the pDMA matrix, and the dependences of dispersion coefficient on DNA size and electric field strength were found to differ quite starkly in the two matrices. These observations lead us to propose that DNA migration mechanisms differ substantially in our custom pDMA matrix compared to the commercially available LPA matrix. We discuss the implications of these results in terms of developing optimal matrices for specific separation (microchip or capillary) platforms.

Wound healing is a natural process involving several signaling molecules and cell types over a significant period of time. Although current dressings help to protect the wound from debris or infection, they do little in accelerating the healing process. Insulin has been shown to stimulate the healing of damaged skin. We have developed an alginate sponge dressing (ASD) that forms a hydrogel capable of providing a moist and protective healing environment. By incorporating insulin-loaded poly(d,l-lactide-co-glycolide) (PLGA) microparticles into ASD, we successfully stabilized and released insulin for up to 21 days. Insulin release and water absorption and transfer through the ASD were influenced by altering the levels of poly(ethylene glycol) (PEG) in the dressing matrix. Bioactivity of released insulin can be maintained for at least 10 days, demonstrated using a human keratinocyte migration assay. Results showed that insulin-loaded PLGA microparticles, embedded within PEG-ASD, functioned as an effective long-term delivery platform for bioactive insulin.

Adiponectin has many beneficial effects on cardiovascular and obesity-related disorders. It is part of a class of proteins that contains short collagenous domains, along with surfactant proteins A and D, and complement protein C1q. This class of biomacromolecules requires post-translational modifications to form biologically active assemblies. By introducing a set of post-translational modifying enzymes into Escherichia coli , we have created a prokaryotic expression system that functionally assembles adiponectin, as assessed by the ability of produced adiponectin multimers to suppress human endothelial cell apoptosis. This study represents the first example of the assembly of functional high order multimers of any member of this class of proteins outside of eukaryotic cells. Furthermore, the results give fundamental insight into the process of assembly such as the necessity and sufficiency of various post-translational steps for functional assembly. We expect that fine-tuning of the expression system will allow for efficient production and functional assembly of biomolecules that assemble via short collagenous domains.

DNA barcodes are short, unique ssDNA primers that "mark" individual biomolecules. To gain better understanding of biophysical parameters constraining primer-dimer formation between primers that incorporate barcode sequences, we have developed a capillary electrophoresis method that utilizes drag-tag-DNA conjugates to quantify dimerization risk between primer-barcode pairs. Results obtained with this unique free-solution conjugate electrophoresis approach are useful as quantitatively precise input data to parameterize computation models of dimerization risk. A set of fluorescently labeled, model primer-barcode conjugates were designed with complementary regions of differing lengths to quantify heterodimerization as a function of temperature. Primer-dimer cases comprised two 30-mer primers, one of which was covalently conjugated to a lab-made, chemically synthesized poly-N-methoxyethylglycine drag-tag, which reduced electrophoretic mobility of ssDNA to distinguish it from ds primer-dimers. The drag-tags also provided a shift in mobility for the dsDNA species, which allowed us to quantitate primer-dimer formation. In the experimental studies, pairs of oligonucleotide primer barcodes with fully or partially complementary sequences were annealed, and then separated by free-solution conjugate CE at different temperatures, to assess effects on primer-dimer formation. When less than 30 out of 30 base-pairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive base-pairs formed, yet non-consecutive base-pairs did not create stable dimers even when 20 out of 30 possible base-pairs bonded. The use of free-solution electrophoresis in combination with a peptoid drag-tag and different fluorophores enabled precise separation of short DNA fragments to establish a new mobility shift assay for detection of primer-dimer formation.

Antimicrobial peptides (AMPs) are integral components of innate immunity and are typically found in combinations in which they can synergize for broader-spectrum or more potent activity. Previously, we reported peptoid mimics of AMPs with potent and selective antimicrobial activity. Using checkerboard assays, we demonstrate that peptoids and AMPs can interact synergistically, with fractional inhibitory concentration indices as low as 0.16. These results strongly suggest that antimicrobial peptoids and peptides are functionally and mechanistically analogous.

Peptoids are a developing class of peptide-like oligomers originally invented for drug discovery in the early 1990s. While peptides hold great promise for therapeutic applications, current development of peptide-based pharmaceuticals is hindered by their potential for misfolding and aggregation, and particularly, for rapid in vivo degradation post-administration. Researchers have investigated alternative peptide-like constructs that may be able to circumvent such complications. Peptoids comprise a peptide-based backbone and N-substituted glycines for side chain residues, resulting in complete protease-resistance. Synthesis of peptoid sequences up to 50 units in length allows for controlled sequence composition and incorporation of diverse side chain chemistries. Though the landscape of peptoid structure is not clearly defined, secondary, tertiary, loop, turn, and random structures have been identified. As protease-resistant isomers of peptides, peptoids are being developed as versatile molecular tools in biochemistry and biophysics, and are becoming attractive candidates for therapeutic and diagnostic applications. Peptoids have thus far demonstrated bioactivity as protein mimics and as replacements for small molecule drugs. In this review, we discuss the most recent advances in peptoid research on the therapeutic front in the last few years, including in vitro and in vivo studies in the fields of lung surfactant therapy, antimicrobial agents, diagnostics, and cancer. We particularly focus on the biophysical activity of lipid-associated peptoids and their potential therapeutic applications.

Tuberculosis is a leading cause of death worldwide. Resistance of Mycobacterium to antibiotics can make treatments less effective in some cases. We tested selected oligopeptoids—previously reported as mimics of natural host defense peptides—for activity against Mycobacterium tuberculosis and assessed their cytotoxicity. A tetrameric, alkylated, cationic peptoid (1-C13 4mer ) was most potent against M. tuberculosis and least cytotoxic, whereas an unalkylated analogue, peptoid 1 4mer , was inactive. Peptoid 1-C13 4mer thus merits further study as a potential antituberculosis drug.

Prolyl 4-hydroxylases are ascorbate-dependent oxygenases that play key roles in a variety of eukaryotic biological processes including oxygen sensing, siRNA regulation, and collagen folding. They perform their functions by catalyzing the post-translational hydroxylation of specific proline residues on target proteins to form (2S,4R)-4-hydroxyproline. Thus far, our ability to study these post-translational modifications has been limited by the lack of a prokaryotic recombinant expression system for producing hydroxylated proteins. By introducing a biosynthetic shunt to produce ascorbate-like molecules in Eschericia coli cells that heterologously express human prolyl 4-hydroxylase (P4H), we have created a strain of Escherichia coli that produces collagenous proteins with high levels of (2S,4R)-4-hydroxyproline. Using this new system, we have observed hydroxylation patterns indicative of a processive catalytic mode for P4H that is active even in the absence of ascorbate. Our results provide insights into P4H enzymology, and create a foundation for better understanding how post-translational hydroxylation affects proteins.

Significant progress has been made in recent years toward creating interesting, unique, and in some cases, predictable oligopeptoid/polypeptoid secondary, tertiary, and in one case, quaternary structures. This article describes this progress, identifies a few of the many remaining challenges, and discusses potentially interesting or fruitful strategies for the peptoid biomimetics research community. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 96: 556–560, 2011.

We have evaluated "NMEGylation"--the covalent attachment of an oligo-N-methoxyethylglycine (NMEG) chain--as a new form of peptide/protein modification to enhance the bioavailability of short peptides. OligoNMEGs are hydrophilic polyethylene glycol-like molecules made by solid-phase synthesis, typically up to 40 monomers in length. They have been studied as nonfouling surface coatings and as monodisperse mobility modifiers for free-solution conjugate capillary electrophoresis. However, polyNMEGs have not been demonstrated before this work as modifiers of therapeutic proteins. In prior published work, we identified a short peptide, "C20," as a potential extracellular inhibitor of the fusion of human respiratory syncytial virus with mammalian cells. The present study was aimed at improving the C20 peptide's stability and solubility. To this end, we synthesized and studied a series of NMEGylated C20 peptide-peptoid bioconjugates comprising different numbers of NMEGs at either the N- or C-terminus of C20. NMEGylation was found to greatly improve this peptide's solubility and serum stability; however, longer polyNMEGs (n > 3) deleteriously affected peptide binding to the target protein. By incorporating just one NMEG monomer, along with a glycine monomer as a flexible spacer, at C20's N-terminus (NMEG-Gly-C20), we increased both solubility and serum stability greatly, while recovering a binding affinity comparable to that of unmodified C20 peptide. Our results suggest that NMEGylation with an optimized number of NMEG monomers and a proper linker could be useful, more broadly, as a novel modification to enhance bioavailability and efficacy of therapeutic peptides.

We report the creation of alkylated poly- N -substituted glycine (peptoid) mimics of antimicrobial lipopeptides with alkyl tails ranging from 5 to 13 carbons. In several cases, alkylation significantly improved the selectivity of the peptoids with no loss in antimicrobial potency. Using this technique, we synthesized an antimicrobial peptoid only 5 monomers in length with selective, broad-spectrum antimicrobial activity as potent as previously reported dodecameric peptoids and the antimicrobial peptide pexiganan.

We report a capillary-based DNA sequencing read length of 100 bases in 16 min using end-labeled free-solution conjugate electrophoresis (FSCE) with a monodisperse poly-N-substituted glycine (polypeptoid) as a synthetic drag-tag. FSCE enabled rapid separation of single-stranded (ss) DNA sequencing fragments with single-base resolution without the need for a viscous DNA separation matrix. Protein-based drag-tags previously used for FSCE sequencing, for example, streptavidin, are heterogeneous in molar mass (polydisperse); the resultant band-broadening can make it difficult to obtain the single-base resolution necessary for DNA sequencing. In this study, we synthesized and HPLC-purified a 70mer poly-N-(methoxyethyl)glycine (NMEG) drag-tag with a molar mass of - 11 kDa. The NMEG monomers that comprise this peptoid drag-tag are interesting for bioanalytical applications, because the methoxyethyl side chain's chemical structure is reminiscent of the basic monomer unit of polyethylene glycol, a highly biocompatible commercially available polymer, which, however, is not available in monodisperse preparation at an - 11 kDa molar mass. This is the first report of ssDNA separation and of four-color, base-by-base DNA sequencing by FSCE through the use of a chemically synthesized drag-tag. These results show that high-molar mass, chemically synthesized drag-tags based on the polyNMEG structure, if obtained in monodisperse preparation, would serve as ideal drag-tags and could help FSCE reach the commercially relevant read lengths of 100 bases or more.

Biomaterials that mimic the extracellular matrix in both modularity and crosslinking chemistry have the potential to recapitulate the instructive signals that ultimately control cell fate. Toward this goal, modular protein polymer-based hydrogels were created through genetic engineering and enzymatic crosslinking. Animal derived tissue transglutaminase (tTG) and recombinant human transglutaminase (hTG) enzymes were used for coupling two classes of protein polymers containing either lysine or glutamine, which have the recognition substrates for enzymatic crosslinking, evenly spaced along the protein backbone. Utilizing tTG under physiological conditions, crosslinking occurred within two minutes, as determined by particle tracking microrheology. Hydrogel composition impacted the elastic storage modulus of the gel over 4-fold and also influenced microstructure and degree of swelling, but did not appreciably effect degradation by plasmin. Mouse 3T3 and primary human fibroblasts were cultured in both 2- and 3-dimensions without a decrease in cell viability and displayed spreading in 2D. The properties of these gels, which are controlled through the specific nature of the protein polymer precursors, render these gels valuable for in situ therapies. Furthermore, the modular hydrogel composition allows tailoring of mechanical and physical properties for specific tissue engineering applications.

Hydrophobic lung surfactant proteins B and C (SP-B and SP-C) are critical for normal respiration in vertebrates, and each comprises specific structural attributes that enable the surface-tension-reducing ability of the lipid–protein mixture in lung surfactant. The difficulty in obtaining pure SP-B and SP-C on a large scale has hindered efforts to develop a non-animal-derived surfactant replacement therapy for respiratory distress. Although peptide-based SP-C mimics exhibit similar activity to the natural protein, helical peptide-based mimics of SP-B benefit from dimeric structures. To determine if in vitro surface activity improvements in a mixed lipid film could be garnered without creating a dimerized structural motif, a helical and cationic peptoid-based SP-B mimic was modified by SP-C-like N-terminus alkylation with octadecylamine. “Hybridized” mono- and dialkylated peptoids significantly decreased the maximum surface tension of the lipid film during cycling on the pulsating bubble surfactometer relative to the unalkylated variant. Peptoids were localized in the fluid phase of giant unilamellar vesicle lipid bilayers, as has been described for SP-B and SP-C. Using Langmuir–Wilhelmy surface balance epifluorescence imaging (FM) and atomic force microscopy (AFM), only lipid-alkylated peptoid films revealed micro- and nanostructures closely resembling films containing SP-B. AFM images of lipid-alkylated peptoid films showed gel condensed-phase domains surrounded by a distinct phase containing “nanosilo” structures believed to enhance re-spreading of submonolayer material. N-terminus alkylation may be a simple, effective method for increasing lipid affinity and surface activity of single-helix SP-B mimics.

Non-natural oligomers have recently shown promise as functional analogues of lung surfactant proteins B and C (SP-B and SP-C), two helical and amphiphilic proteins that are critical for normal respiration. The generation of non-natural mimics of SP-B and SP-C has previously been restricted to step-by-step, sequence-specific synthesis, which results in discrete oligomers that are intended to manifest specific structural attributes. Here we present an alternative approach to SP-B mimicry that is based on sequence-random copolymers containing cationic and lipophilic subunits. These materials, members of the nylon-3 family, are prepared by ring-opening polymerization of beta-lactams. The best of the nylon-3 polymers display promising in vitro surfactant activities in a mixed lipid film. Pulsating bubble surfactometry data indicate that films containing the most surface-active polymers attain adsorptive and dynamic-cycling properties that surpass those of discrete peptides intended to mimic SP-B. Attachment of an N-terminal octadecanoyl unit to the nylon-3 copolymers, inspired by the post-translational modifications found in SP-C, affords further improvements by reducing the percent surface area compression to reach low minimum surface tension. Cytotoxic effects of the copolymers are diminished relative to that of an SP-B-derived peptide and a peptoid-based mimic. The current study provides evidence that sequence-random copolymers can mimic the in vitro surface-active behavior of lung surfactant proteins in a mixed lipid film. These findings raise the possibility that random copolymers might be useful for developing a lung surfactant replacement, which is an attractive prospect given that such polymers are easier to prepare than are sequence-specific oligomers.

Magnetic resonance imaging is a noninvasive imaging modality with high spatial and temporal resolution. Contrast agents (CAs) are frequently used to increase the contrast between tissues of interest. To increase the effectiveness of MR agents, small molecule CAs have been attached to macromolecules. We have created a family of biodegradable, macromolecular CAs based on protein polymers, allowing control over the CA properties. The protein polymers are monodisperse, random coil, and contain evenly spaced lysines that serve as reactive sites for Gd(III) chelates. The exact sequence and length of the protein can be specified, enabling controlled variation in lysine spacing and molecular weight. Relaxivity could be modulated by changing protein polymer length and lysine spacing. Relaxivities of up to approximately 14 mM(-1) s(-1) per Gd(III) and approximately 461 mM(-1) s(-1) per conjugate were observed. These CAs are biodegradable by incubation with plasmin, such that they can be easily excreted after use. They do not reduce cell viability, a prerequisite for future in vivo studies. The protein polymer CAs can be customized for different clinical diagnostic applications, including biomaterial tracking, as a balanced agent with high relaxivity and appropriate molar mass.

Peptoids, oligo-N-substituted glycines, can fold into well-defined helical secondary structures. The design and synthesis of new peptoid building blocks that are capable of both (a) inducing a helical secondary structure and (b) decorating the helices with chemical functionalities are reported. Peptoid heptamers containing carboxamide, carboxylic acid or thiol functionalities were synthesized, and the resulting peptoids were shown to form stable helices. A thiol-containing peptoid readily formed the homodisulfide, providing a convenient route to prepare peptoid helix homodimers.

Numerous strategies exist to prevent biological fouling of surfaces in physiological environments; the authors’ strategy focuses on the modification of surfaces with poly-N-substituted glycine oligomers (polypeptoids). The authors previously reported the synthesis and characterization of three novel polypeptoid polymers that can be used to modify titanium oxide surfaces, rendering the surfaces resistant to adsorption of proteins, to adhesion of mammalian and bacterial cells, and to degradation by common protease enzymes. In this study, they investigated the effect of polypeptoid chain length on the antifouling properties of the modified surfaces. For these experiments, they used poly(N-methoxyethyl) glycines with lengths between 10 and 50 repeat units and determined the influence of chain length on coating thickness and density as well as resistance to protein adsorption and cellular adhesion. Short-term protein resistance remained low for all polymers, as measured by optical waveguide light mode spectroscopy, while fibroblast adhesion after several weeks indicated reduced fouling resistance for the polypeptoid-modified surfaces with the shortest chain length polymer. Experimental observations were compared to predictions obtained from a molecular theory of polymer and protein adsorption. Good agreement was found between experiment and theory for the chain length dependence of peptoid grafting density and for protein adsorption as a function of peptoid grafting density. The theoretical predictions provide specific guidelines for the surface coverage for each molecular weight for optimal antifouling. The predictions show the relationship between polymer layer structure and fouling.

In 2009, electrophoretically driven DNA separations in slab gels and capillaries have the sepia tones of an old-fashioned technology in the eyes of many, even while they remain ubiquitously used, fill a unique niche, and arguably have yet to reach their full potential. For comic relief, what is old becomes new again: agarose slab gel separations are used to prepare DNA samples for "next-gen" sequencing platforms (e.g. the Illumina and 454 machines) - dsDNA molecules within a certain size range are "cut out" of a gel and recovered for subsequent "massively parallel" pyrosequencing. In this review, we give a Barron lab perspective on how our comprehension of DNA migration mechanisms in electrophoresis has evolved, since the first reports of DNA separations by CE ( approximately 1989) until now, 20 years later. Fused-silica capillaries and borosilicate glass and plastic microchips quietly offer increasing capacities for fast (and even "ultra-fast"), efficient DNA separations. While the channel-by-channel scaling of both old and new electrophoresis platforms provides key flexibility, it requires each unique DNA sample to be prepared in its own micro or nanovolume. This Achilles' heel of electrophoresis technologies left an opening through which pooled sample, next-gen DNA sequencing technologies rushed. We shall see, over time, whether sharpening understanding of transitions in DNA migration modes in crosslinked gels, nanogel solutions, and uncrosslinked polymer solutions will allow electrophoretic DNA analysis technologies to flower again. Microchannel electrophoresis, after a quiet period of metamorphosis, may emerge sleeker and more powerful, to claim its own important niche applications.

A family of peptoid dimers developed to mimic SP-B is presented, where two amphipathic, cationic helices are linked by an achiral octameric chain. SP-B is a vital therapeutic protein in lung surfactant replacement therapy, but its large-scale isolation or chemical synthesis is impractical. Enhanced biomimicry of SP-B’s disulfide-bonded structure has been previously attempted via disulfide-mediated dimerization of SP-B1-25 and other peptide mimics, which improved surface activity relative to the monomers. Herein, the effects of disulfide- or ‘click’-mediated (1,3-dipolar cycloaddition) dimerization, as well as linker chemistry, on the lipid-associated surfactant activity of a peptoid monomer are described. Results revealed that the ‘clicked’ peptoid dimer enhanced in vitro surface activity in a DPPC:POPG:PA lipid film relative to its disulfide-bonded and monomeric counterparts in both surface balance and pulsating bubble surfactometry studies. On the pulsating bubble surfactometer, the film containing the ‘clicked’ peptoid dimer outperformed all presented peptoid monomers and dimers, and two SP-B derived peptides, attaining an adsorbed surface tension of 22 mN m −1 , and maximum and minimum cycling values of 42 mN m −1 and near-zero, respectively.

In recent years, there has been an increasing demand for a wide range of DNA separations that require the development of materials to meet the needs of high resolution and high throughput. Here, we demonstrate the use of thermoresponsive N-alkoxyalkylacrylamide polymers as a sieving matrix for DNA separations on a microfluidic chip. The viscosities of the N-alkoxyalkylacrylamide polymers are more than an order of magnitude lower than that of a linear polyacrylamide (LPA) of corresponding molecular weight, allowing rapid loading of the microchip. At 25 degrees C, N-alkoxyalkylacrylamide polymers can provide improved DNA separations compared with LPA in terms of reduced separation time and increased separation efficiency, particularly for the larger DNA fragments. The improved separation efficiency in N-alkoxyalkylacrylamide polymers is attributed to the peak widths increasing only slightly with DNA fragment size, while the peak widths increase appreciably above 150 bp using an LPA matrix. Upon elevating the temperature to 50 degrees C, the increase in viscosity of the N-alkoxyalkylacrylamide solutions is dependent upon their overall degree of hydrophobicity. The most hydrophobic polymers exhibit a lower critical solution temperature below 50 degrees C, undergoing a coil-to-globule transition followed by chain aggregation. DNA separation efficiency at 50 degrees C therefore decreases significantly with increasing hydrophobic character of the polymers, and no separations were possible with solutions with a lower critical solution temperature below 50 degrees C. The work reported here demonstrates the potential for this class of polymers to be used for applications such as PCR product and RFLP sizing, and provides insight into the effect of polymer hydrophobicity on DNA separations.

By using a microfluidic electrophoresis platform to perform DNA sequencing, genomic information can be obtained more quickly and affordably than the currently employed capillary array electrophoresis (CAE) instruments. Previous research in our group has shown that physically cross-linked, hydrophobically modified polyacrylamide (HMPAM) matrices separate double-stranded DNA (dsDNA) more effectively than linear polyacrylamide (LPA) solutions. Expanding upon this work, we have synthesized a series of linear polyacrylamide-co-dihexylacrylamide (LPA-co-DHA) block copolymers specifically designed to electrophoretically sequence single-stranded DNA (ssDNA) quickly and efficiently on a microfluidic device. By incorporating very small amounts of N,N-dihexylacrylamide, a hydrophobic monomer, these copolymer solutions achieved up to ~10% increases in average DNA sequencing read length over LPA homopolymer solutions of matched molar mass. Additionally, the inclusion of the small amount of hydrophobe does not significantly increase the polymer solution viscosities, relative to LPA solutions, so that channel loading times between the copolymers and the homopolymers are similar. The resulting polymer solutions are capable of providing enhanced sequencing separations in a short period of time without compromising the ability to rapidly load and unload the matrix from a microfluidic device.

Previous studies have reported that mixed molar mass polymer matrices show enhanced DNA sequencing fragment separation compared to matrices formulated from a single average molar mass. Here, we describe a systematic study to investigate the effects of varying the amounts of two different average molar mass polymers on the DNA sequencing ability of poly(N,N-dimethylacrylamide) (pDMA) sequencing matrices in microfluidic chips. Two polydisperse samples of pDMA, with weight-average molar masses of 3.5 MDa and 770 kDa, were mixed at various fractional concentrations while maintaining the overall polymer concentration at 5% (w/v). We show that although the separation of short DNA fragments depends strongly on the overall solution concentration of the polymer, inclusion of the high-molar mass polymer is essential to achieve read lengths of interest (> 400 bases) for many sequencing applications. Our results also show that one of the blended matrices, comprised of 3% 3.5 MDa pDMA and 2% 770 kDa pDMA, yields similar sequencing read lengths (> 520 bases on average) to the high molar mass matrix alone, while also providing a five-fold reduction in zero-shear viscosity. These results indicate that the long read lengths achieved in a viscous, high molar mass polymer matrix are also possible to achieve in a tuned, blended matrix of high and low molar mass polymers with a much lower overall solution viscosity.

Peptides can potentiate lipid-mediated gene delivery by modifying lipoplex physiochemical properties to overcome rate-limiting steps to gene transfer. The objectives of this study were to determine the regimes over which cationic peptides enhance lipofection and to investigate the mechanism of action, such as increased cellular association resulting from changes in lipoplex physical properties. Short, cationic peptides were incorporated into lipoplexes by mixing peptide, lipid and DNA. Lipoplexes were characterized using gel retardation, dynamic light scattering, and fluorescent microscopy, and the amount of surface-displayed amines was quantified by fluorescamine. Size, zeta potential, and surface amines for lipoplexes were dependent on peptide/DNA ratio. Inclusion of peptides in lipoplexes resulted in up to a 13-fold increase in percentage of cells transfected, and up to a 76-fold increase in protein expression. This transfection enhancement corresponded to a small particle diameter and positive zeta potential of lipoplexes, as well as increased amount of surface-displayed amines. Relative to lipid alone, these properties of the peptide-modified lipoplexes enhanced cellular association, which has been reported as a rate-limiting step for transfection with lipoplexes. The addition of peptides is a simple method of lipofection enhancement, as direct chemical modification of lipids is not necessary for increased transfection.

We have studied the effects of polymer molar mass and concentration on the electrophoretic migration modalities of individual molecules of DNA in LPA, HEC, and PEO solutions via epifluorescent videomicroscopy. While both transient entanglement coupling (TEC) and reptation have been studied in the past, the transition between them has not. Understanding this transition will allow for polymer network properties to be optimized to enhance the speed and resolution of DNA separations in microfluidic devices. Near the overlap threshold concentration, C*, TEC is the dominant observed mode of DNA migration, and the observation frequency of TEC increases with increasing polymer molar mass. As polymer concentration is increased, observed TEC events reduce to zero while DNA reptation events become the only detected mechanism. Individual DNA molecules undergoing both migration mechanisms were counted in solutions of varying polymer molar masses and concentrations and were plotted against a dimensionless polymer concentration, C/C*. The data for LPA reduce to form universal curves with a sharp increase in DNA reptation at approximately 6.5C*. Analogous transition concentrations for PEO and HEC were observed at 5C* and 3.5C*, respectively, reflecting the different physical properties of these polymers. This transition correlates closely with the polymer network entanglement concentration, Ce, as measured by rheological techniques. The electrophoretic mobility of lambda-DNA in LPA polymer solutions was also measured and shows how a balance can be struck between DNA resolution and separation speed by choosing the desired prevalence of DNA reptation.

We report a new, bioconjugate approach to performing highly multiplexed single-base extension (SBE) assays, which we demonstrate by genotyping a large panel of point mutants in exons 5-9 of the p53 gene. A series of monodisperse polyamide "drag-tags" was created using both chemical and biological synthesis and used to achieve the high-resolution separation of genotyping reaction products by microchannel electrophoresis without a polymeric sieving matrix. A highly multiplexed SBE reaction was performed in which 16 unique drag-tagged primers simultaneously probe 16 p53 gene loci, with an abbreviated thermal cycling protocol of only 9 min. The drag-tagged SBE products were rapidly separated by free-solution conjugate electrophoresis (FSCE) in both capillaries and microfluidic chips with genotyping accuracy in excess of 96%. The separation requires less than 70 s in a glass microfluidic chip, or about 20 min in a commercial capillary array sequencing instrument. Compared to gel electrophoresis, FSCE offers greater freedom in the design of SBE primers by essentially decoupling the length of the primer and the electrophoretic mobility of the genotyping products. FSCE also presents new possibilities for the facile implementation of SBE on integrated microfluidic electrophoresis devices for rapid, high-throughput genetic mutation detection or SNP scoring.

Surfactant protein B (SP-B) is one of two helical, amphipathic proteins critical for the biophysical functioning of lung surfactant (LS) and hence is an important therapeutic protein. This small, complex 79mer has three internal disulfide bonds and homodimerizes via another disulfide bridge. A helical, amphipathic 25mer from the amino terminus (SP-B(1-25)) exhibits surface-active properties similar to those of full-length, synthetic SP-B. In previous work, we created helical, non-natural mimics of SP-B(1-25) based on sequence-specific peptoid 17mers and demonstrated their biomimetic surface activity. Like SP-B(1-25), the peptoids were designed to adopt helical structures with cationic and nonpolar faces. Here, we compare the surface activities of six different helical peptoid analogues of SP-B(1-25) to investigate the importance of mimicking its N-terminal insertion domain as well as its two arginine residues, both thought to be important for the peptide's proper function. Although the peptoid analogues of SP-B(1-25) studied here share many similar features and all functionally mimic SP-B(1-25) to some degree, it is notable that small differences in their sequences and side chain chemistries lead to substantial differences in their observed interactions with a lipid film. A peptoid comprising a hydrophobic, helical insertion region with aromatic side chains shows more biomimetic surface activity than simpler peptoids, and even better activity, by comparison to natural LS, than SP-B(1-25). However, the substitution of lysine-like side chains for arginine-like side chains in the peptoid has little effect on biomimetic surface activity, indicating that interactions of the guanidino groups with lipids may not be critical for the function of these SP-B mimics.

We describe a novel class of DNA separation media for microchip electrophoresis, "physically cross-linked" block copolymer networks, which provide rapid (4.5 min) and remarkably enhanced resolution of DNA in a size range critical for genotyping. Linear poly(acrylamide-co-dihexylacrylamide) (LPA-co-DHA) comprising as little as 0.13 mol % dihexylacrylamide yields substantially improved electrophoretic DNA separations compared to matched molar mass linear polyacrylamide. Single-molecule videomicroscopic images of DNA electrophoresis reveal novel chain dynamics in LPA-co-DHA matrixes, resembling inchworm movement, to which we attribute the increased DNA resolution. Substantial improvements in DNA peak separation are obtained, in particular, in LPA-co-DHA solutions at polymer/copolymer concentrations near the interchain entanglement threshold. Higher polymer concentrations yield enhanced separations only for small DNA molecules (120 base pairs). Hydrophobically cross-linked networks offer advantages over conventional linear polymers based on enhanced separation performance (or speed) and over chemically cross-linked gels because hydrophobic cross-links can be reversibly broken, allowing facile microchannel loading.

In end-labeled free-solution electrophoresis (ELFSE), DNA molecules are labeled with a frictional modifier or "drag-tag", allowing their size-based electrophoretic separation in free solution. Among the interesting observations from early work with dsDNA using streptavidin as a drag-tag was that the drag induced by including a streptavidin label at both ends was significantly more than double that from a single streptavidin (Heller, C. et al.., J. Chromatogr. A 1998, 806, 113-121). This finding was assumed to be in error, and subsequent work focused on experiments in which only a single drag-tag is appended to one end of the DNA molecule. Recent theoretical work (McCormick, L. C., Slater, G. W., Electrophoresis 2005, 26, 1659-1667) has examined the contribution of end-effects to the free-solution electrophoretic mobility of charged-uncharged polymer conjugates, reopening the question of enhanced drag from placing a drag-tag at both ends. In this study, this effect is investigated experimentally, using custom-synthesized ssDNA oligonucleotides allowing the attachment of drag-tags to one or both ends, as well as dsDNA PCR products generated with primers appropriate for the attachment of drag-tags at one or both ends. A range of sizes of drag-tags are used, including synthetic polypeptoid drag-tags as well as genetically engineered protein polymer drag-tags. The enhanced drag arising from labeling both ends has been confirmed, with 6-9% additional drag for the ssDNA and 10-23% additional drag for the dsDNA arising from labeling both ends than would be expected from simply doubling the size of the drag-tag at one end. The experimental results for ssDNA labeled at both ends are compared to the predictions of the recent theory of end-effects, with reasonably good quantitative agreement. These experimental findings demonstrate the feasibility of enhancing ELFSE separations by labeling both ends of the DNA molecule, leading to greater resolving power and a wider range of applications for this technique.

We present the rapid single-strand conformation polymorphism (SSCP) analysis by capillary and microchip electrophoresis to detect the mutations in K-ras gene. Parameters that might affect the analysis of mutation in K-ras gene, such as the polymer and the additive in the sieving matrix, have been studied systematically. Under the optimal conditions, the analysis of seven mutants of K-ras gene could be finished within 10 min by capillary electrophoresis (CE). Furthermore, with the wild-type gene as the inner standard, the analysis accuracy of mutations could be improved. In addition, by studying the properties of polymer solutions, the matrix suitable for microchip electrophoresis was found, and the detection of mutations in K-ras gene could be further shortened to 1 min.

We demonstrate the feasibility of end-labeled free-solution electrophoresis (ELFSE) separation of DNA using genetically engineered protein polymers as drag-tags. Protein polymers are promising candidates for ELFSE drag-tags because their sequences and lengths are controllable not only to generate monodisperse polymers with high frictional drag, but also to meet other drag-tag requirements for high-resolution separations by microchannel electrophoresis. A series of repetitive polypeptides was designed, expressed in Escherichia coli, and purified. By performing an end-on conjugation of the protein polymers to a fluorescently labeled DNA oligomer (22 bases) and analyzing the electrophoretic mobilities of the conjugate molecules by free-solution capillary electrophoresis (CE), effects of the size and charge of the protein polymer drag-tags were investigated. In addition, the electrophoretic behavior of bioconjugates comprising relatively long DNA fragments (108 and 208 bases) and attached to uncharged drag-tags was observed, by conjugating fluorescently labeled polymerase chain reaction (PCR) products to charge-neutral protein polymers, and analyzing via CE. We calculated the amount of friction generated by the various drag-tags, and estimated the potential read-lengths that could be obtained if these drag-tags were used for DNA sequencing in our current system. The results of these studies indicate that larger and uncharged drag-tags will have the best DNA-resolving capability for ELFSE separations, and that theoretically, up to 233 DNA bases could be sequenced using one of the protein polymer drag-tags we produced, which is electrostatically neutral with a chain length of 337 amino acids. We also show that denatured (unfolded) polypeptide chains impose much greater frictional drag per unit molecular weight than folded proteins, such as streptavidin, which has been used as a drag-tag before.

DNA is a free-draining polymer. This subtle but "unfortunate" property of highly charged polyelectrolytes makes it impossible to separate nucleic acids by free-flow electrophoresis. This is why one must typically use a sieving matrix, such as a gel or an entangled polymer solution, in order to obtain some electrophoretic size separation. An alternative approach consists of breaking the charge to friction balance of free-draining DNA molecules. This can be achieved by labeling the DNA with a large, uncharged molecule (essentially a hydrodynamic parachute, which we also call a drag-tag) prior to electrophoresis; the resulting methodology is called end-labeled free-solution electrophoresis (ELFSE). In this article, we review the development of ELFSE over the last decade. In particular, we examine the theoretical concepts used to predict the ultimate performance of ELFSE for single-stranded (ssDNA) sequencing, the experimental results showing that ELFSE can indeed overcome the free-draining issue raised above, and the technological advances that are needed to speed the development of competitive ELFSE-based sequencing and separation technologies. Finally, we also review the reverse process, called free-solution conjugate electrophoresis (FSCE), wherein uncharged polymers of different sizes can be analyzed using a short DNA molecule as an electrophoretic engine.

In an earlier study we showed that a blend of thermoresponsive and nonthermoresponsive hydroxyalkylcelluloses could be used to create a thermally tunable polymer network for double-stranded (ds) DNA separation. Here, we show the generality of this approach using a family of polymers suited to a wider range of DNA separations: a blended mixture of N,N-dialkylacrylamide copolymers with different thermoresponsive behaviors. A mixture of 47% w/w N,N-diethylacrylamide (DEA)/53% w/w N,N-dimethylacrylamide (DMA) (DEA47; thermoresponsive, transition temperature = 55 degrees C in water) and 30% w/w DEA/70% w/w DMA (DEA30; nonthermoresponsive, transition temperature > 85 degrees C in water) copolymers in the ratio of 1:5 w/w DEA47:DEA30 was used to separate a dsDNA restriction digest (PhiX174-HaeIII). We investigated the effects of changing mesh size on dsDNA separation, as controlled by temperature. We observed good DNA separation performance with the copolymer blend at temperatures ranging from 25 degrees C to 48 degrees C. The separation selectivity was evaluated quantitatively for certain DNA fragment pairs as a function of temperature. The results were compared with those obtained with a control matrix consisting only of the nonthermoresponsive DEA30. Different DNA fragment pairs of various sizes show distinct temperature-dependent selectivities. Over the same temperature range, no significant temperature dependence of selectivity is observed for these DNA fragment pairs in the nonthermoresponsive control matrix. Overall, the results show similar trends in the temperature dependency of separation selectivity to what was previously observed in hydroxyalkylcellulose blends, for the same DNA fragment pairs. Finally, we showed that a ramped temperature scheme enables improved separation in the blended copolymer matrix for both small and large DNA fragments, simultaneously in a single capillary electrophoresis (CE) run.

We present data showing a substantial narrowing of the polydispersity index (PDI) of high polymers occurring as a consequence of random chain scission events in a transient elongational flow field. In our experiments, semi-dilute aqueous solutions of high-molar mass, polydisperse polymers (PDI>1.4) were injected under pressure through an elongational flow field at the entrance of a capillary tube (i.d. 250 μm). Chain scission events occurring during multiple passes through the capillary entrance cause a marked decrease in PDI, to values as low as 1.12, along with the expected decrease of the average molar mass. The phenomenon appears to be entirely physical and independent of the chemical nature of the polymer, since similar results are obtained with polyacrylamide, polydimethylacrylamide, and poly(ethylene oxide). Statistical modeling of the evolution of the polymer molar mass distribution shows the results to be consistent with the random scission, near the mid-point, of those polymer chains that exceed a certain flow field-dependent critical chain length.

We have developed sparsely cross-linked "nanogels", sub-colloidal polymer structures composed of covalently linked, linear polyacrylamide chains, as novel DNA sequencing matrices for capillary electrophoresis. The presence of covalent cross-links affords nanogel matrices with enhanced network stability relative to standard, linear polyacrylamide (LPA), improving the separation of large DNA fragments. Nanogels were synthesized via inverse emulsion (water-in-oil) copolymerization of acrylamide and N,N-methylenebisacrylamide (Bis). In order to retain the fluidity necessary in a replaceable polymer matrix for capillary array electrophoresis (CAE), a low percentage of the Bis cross-linker (10(-4) mol%) was used. Nanogels were characterized by multiangle laser light scattering and rheometry, and were tested for DNA sequencing by CAE with four-color laser-induced fluorescence (LIF) detection. The properties and performance of nanogel matrices were compared to those of a commercially available LPA network, which was matched for both weight-average molar mass (Mw) and extent of interchain entanglements (c/c*). Nanogels presented in this work have an average radius of gyration of 226 nm and a weight-average molar mass of 8.8 x 10(6) g/mol. At concentrations above the overlap threshold, nanogels form a clear, viscous solution, similar to the LPA matrix (Mw approximately 8.9 x 10(6) g/mol). The two matrices have similar flow and viscosity characteristics. However, because of the physical network stability provided by the internally cross-linked structure of the nanogels, a substantially longer read length ( approximately 63 bases, a 10.4% improvement) is obtained with the nanogel matrix at 98.5% accuracy of base-calling. The nanogel network provides higher-selectivity separation of ssDNA sequencing fragments longer than 375 bases. Moreover, nanogel matrices require 30% less polymer per unit volume than LPA. This is the first report of a sequencing matrix that provides better performance than LPA, in a side-by-side comparison of polymer matrices matched for Mw and extent of interchain entanglements.

Among the families of peptidomimetic foldamers under development as novel biomaterials and therapeutics, poly-N-substituted glycines (peptoids) with α-chiral side chains are of particular interest for their ability to adopt stable, helical secondary structure in organic and aqueous solution. Here, we show that a peptoid 22-mer with a biomimetic sequence of side chains and an amphipathic, helical secondary structure acts as an excellent mimic of surfactant protein C (SP-C), a small protein that plays an important role in surfactant replacement therapy for the treatment of neonatal respiratory distress syndrome. When integrated into a lipid film, the helical peptoid SP mimic captures the essential surface-active behaviors of the natural protein. This work provides an example of how an abiological oligomer that closely mimics both the hydrophobic/polar sequence patterning and the fold of a natural protein can also mimic its biophysical function.