Academic Appointments


  • Research Engineer, Bioengineering

Professional Education


  • BS, Cornell University, Chemical Engineering
  • PhD, Northwestern University, Chemical Engineering

All Publications


  • Potent Antiviral Activity against HSV-1 and SARS-CoV-2 by Antimicrobial Peptoids Pharmaceuticals Diamond, G., Molchanova, N., Herlan, C., Fortkort, J. A., Lin, J. S., Figgins, E., Bopp, N., Ryan, L. K., Chung, D., Adcock, R. S., Sherman, M., Barron, A. E. 2021; 14 (4): 304

    View details for DOI 10.3390/ph14040304

  • The human cathelicidin LL-37 is a nanomolar inhibitor of amyloid self-assembly of islet amyloid polypeptide (IAPP) Angewandte Chemie International Edition, https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.202000148 Armiento, V., Hille, K., Naltsas, D., Lin, J. S., Barron, A. E., Kapurniotu, A. 2020; In Press: 6

    View details for DOI 10.1002/anie.202000148

  • The human cathelicidin LL-37 is a nanomolar inhibitor of amyloid self-assembly of islet amyloid polypeptide (IAPP). Angewandte Chemie (International ed. in English) Armiento, V. n., Hille, K. n., Naltsas, D. n., Lin, J. S., Barron, A. E., Kapurniotu, A. n. 2020

    Abstract

    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 immunomodu-latory polypeptide human cathelicidin LL-37 binds IAPP with nano-molar affinity and effectively suppresses its amyloid self-assembly and related pancreatic β-cell damage in vitro. In addition, we identify key LL-37 segments mediating 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 combining antimicrobial, immunomodulatory, and T2D-related anti-amyloid functions as promising candidates for multifunc-tional drugs.

    View details for DOI 10.1002/anie.202000148

    View details for PubMedID 31999880

  • Helical side chain chemistry of a peptoid-based SP-C analogue: Balancing structural rigidity and biomimicry BIOPOLYMERS Brown, N. J., Lin, J. S., Barron, A. E. 2019; 110 (6)

    View details for DOI 10.1002/bip.23277

    View details for Web of Science ID 000477676800006

  • Helical side chain chemistry of a peptoid-based SP-C analogue: Balancing structural rigidity and biomimicry. Biopolymers Brown, N. J., Lin, J. S., Barron, A. E. 2019: e23277

    Abstract

    Surfactant protein C (SP-C) is an important constituent of lung surfactant (LS) and, along with SP-B, is included in exogenous surfactant replacement therapies for treating respiratory distress syndrome (RDS). SP-C's biophysical activity depends upon the presence of a rigid C-terminal helix, of which the secondary structure is more crucial to functionality than precise side-chain chemistry. SP-C is highly sequence-conserved, suggesting that the beta-branched, aliphatic side chains of the helix are also important. Nonnatural mimics of SP-C were created using a poly-N-substituted glycine, or "peptoid," backbone. The mimics included varying amounts of alpha-chiral, aliphatic side chains and alpha-chiral, aromatic side chains in the helical region, imparting either biomimicry or structural rigidity. Biophysical studies confirmed that the peptoids mimicked SP-C's secondary structure and replicated many of its surface-active characteristics. Surface activity was optimized by incorporating both structurally rigid and biomimetic side chain chemistries in the helical region indicating that both characteristics are important for activity. By balancing these features in one mimic, a novel analogue was created that emulates SP-C's in vitro surface activity while overcoming many of the challenges related to natural SP-C. Peptoid-based analogues hold great potential for use in a synthetic, biomimetic LS formulation for treating RDS.

    View details for PubMedID 30972750

  • Effective in vivo treatment of acute lung injury with helical, amphipathic peptoid mimics of pulmonary surfactant proteins SCIENTIFIC REPORTS Czyzewski, A. M., McCaig, L. M., Dohm, M. T., Broering, L. A., Yao, L., Brown, N. J., Didwania, M. K., Lin, J. S., Lewis, J. F., Veldhuizen, R., Barron, A. E. 2018; 8: 6795

    Abstract

    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.

    View details for PubMedID 29717157

  • Evidence that the Human Innate Immune Peptide LL-37 May Be a Binding Partner of Abeta and Inhibitor of Fibril Assembly De Lorenzi, E., Chiari, M., Colombo, R., Cretich, M., Sola, L., Vanna, R., Gagni, P., Bisceglia, F., Morasso, C., Lin, J. S., Lee, M., McGeer, P. L., Barron, A. E. CELL PRESS. 2018: 393A
  • Intracellular biomass flocculation as a key mechanism of rapid bacterial killing by cationic, amphipathic antimicrobial peptides and peptoids. Scientific reports Chongsiriwatana, N. P., Lin, J. S., Kapoor, R. n., Wetzler, M. n., Rea, J. A., Didwania, M. K., Contag, C. H., Barron, A. E. 2017; 7 (1): 16718

    Abstract

    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.

    View details for PubMedID 29196622

    View details for PubMedCentralID PMC5711933

  • Evidence that the Human Innate Immune Peptide LL-37 may be a Binding Partner of Amyloid-β and Inhibitor of Fibril Assembly. Journal of Alzheimer's disease : JAD De Lorenzi, E. n., Chiari, M. n., Colombo, R. n., Cretich, M. n., Sola, L. n., Vanna, R. n., Gagni, P. n., Bisceglia, F. n., Morasso, C. n., Lin, J. S., Lee, M. n., McGeer, P. L., Barron, A. E. 2017; 59 (4): 1213–26

    Abstract

    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.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.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).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.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.

    View details for PubMedID 28731438

  • Evidence that the Human Innate Immune Peptide LL-37 may be a Binding Partner of Amyloid-β and Inhibitor of Fibril Assembly Journal of Alzheimer's Disease De Lorenzi, E., Chiari, M., Colombo, R., Cretich, M., Sola, L., Vanna, R., Gagni, P., Bisceglia, F., Morasso, C., Lin, J. S., Lee, M., McGeer, P. L., Barron, A. E. 2017; 59 (4): 1213-1226

    Abstract

    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.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.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).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.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.

    View details for DOI 10.3233/JAD-170223

    View details for PubMedCentralID PMC5611894

  • Simultaneous detection of 19 K-ras mutations by free-solution conjugate electrophoresis of ligase detection reaction products on glass microchips ELECTROPHORESIS Albrecht, J. C., Kotani, A., Lin, J. S., Soper, S. A., Barron, A. E. 2013; 34 (4): 590-597

    Abstract

    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 to 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 min (E = 310 V/cm) and by microchip electrophoresis in 140 s (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 less than 70 s 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.

    View details for DOI 10.1002/elps.201200462

    View details for Web of Science ID 000315169800016

    View details for PubMedID 23192597

  • Divergent dispersion behavior of ssDNA fragments during microchip electrophoresis in pDMA and LPA entangled polymer networks ELECTROPHORESIS Fredlake, C. P., Hert, D. G., Niedringhaus, T. P., Lin, J. S., Barron, A. E. 2012; 33 (9-10): 1411-1420

    Abstract

    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 dependence 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.

    View details for DOI 10.1002/elps.201100686

    View details for Web of Science ID 000304600500010

    View details for PubMedID 22648809

  • Peptoid transporters: effects of cationic, amphipathic structure on their cellular uptake MOLECULAR BIOSYSTEMS Huang, W., Seo, J., Lin, J. S., Barron, A. E. 2012; 8 (10): 2626-2628

    Abstract

    Two cationic, amphipathic peptoids (poly-N-substituted glycines) were developed as new molecular transporters, which have extensive cellullar uptake and utilize different internalization mechanisms from purely cationic polyguanidine comparators.

    View details for DOI 10.1039/c2mb25197c

    View details for Web of Science ID 000308098600019

    View details for PubMedID 22828784

    View details for PubMedCentralID PMC3431920

  • Monodisperse, "Highly" Positively Charged Protein Polymer Drag-Tags Generated in an Intein-Mediated Purification System Used in Free-Solution Electrophoretic Separations of DNA BIOMACROMOLECULES Wang, X., Albrecht, J. C., Lin, J. S., Barron, A. E. 2012; 13 (1): 117-123

    Abstract

    Free-solution conjugate electrophoresis (FSCE) is a method of DNA sequencing that eliminates the need for viscous polymer solutions by tethering a carefully designed, mobility modifying "drag-tag" to each DNA molecule to achieve size-based separations of DNA. The most successful drag-tags to date are genetically engineered, highly repetitive polypeptides ("protein polymers") that are designed to be large, water-soluble, and completely monodisperse. Positively charged arginines were deliberately introduced at regular intervals into the amino acid sequence to increase the hydrodynamic drag without increasing drag-tag length. Additionally, a one-step purification method that combines affinity chromatography and on-column tag cleavage was devised to achieve the required drag-tag monodispersity. Sequencing with a read length of approximately 180 bases was successfully achieved with a known sequence in free-solution electrophoresis using one of these positively charged drag-tags. This preliminary result is expected to lead to further progress in FSCE sequencing with ~400 bases read length possible when more "highly" positively charged protein polymers of larger size are generated with the intein system.

    View details for DOI 10.1021/bm2013313

    View details for Web of Science ID 000298897300013

    View details for PubMedID 22168388

    View details for PubMedCentralID PMC3270947

  • Ultrafast, efficient separations of large-sized dsDNA in a blended polymer matrix by microfluidic chip electrophoresis: A design of experiments approach ELECTROPHORESIS Sun, M., Lin, J. S., Barron, A. E. 2011; 32 (22): 3233-3240

    Abstract

    Double-stranded (ds) DNA fragments over a wide size range were successfully separated in blended polymer matrices by microfluidic chip electrophoresis. Novel blended polymer matrices composed of two types of polymers with three different molar masses were developed to provide improved separations of large dsDNA without negatively impacting the separation of small dsDNA. Hydroxyethyl celluloses with average molar masses of ∼27  kDa and ∼1  MDa were blended with a second class of polymer, high-molar mass (∼7  MDa) linear polyacrylamide. Fast and highly efficient separations of commercially available DNA ladders were achieved on a borosilicate glass microchip. A distinct separation of a 1-kb DNA extension ladder (200-40,000  bp) was completed in 2  min. An orthogonal design of experiments was used to optimize experimental parameters for DNA separations over a wide size range. We find that the two dominant factors are the applied electric field strength and the inclusion of a high concentration of low-molar mass polymer in the matrix solution. These two factors exerted different effects on the separations of small dsDNA fragments below 1  kbp, medium dsDNA fragments between 1 and 10  kbp, and large dsDNA fragments above 10  kbp.

    View details for DOI 10.1002/elps.201100260

    View details for Web of Science ID 000298098700020

    View details for PubMedID 22009451

  • Blinded study determination of high sensitivity and specificity microchip electrophoresis-SSCP/HA to detect mutations in the p53 gene ELECTROPHORESIS Hestekin, C. N., Lin, J. S., Senderowicz, L., Jakupciak, J. P., O'Connell, C., Rademaker, A., Barron, A. E. 2011; 32 (21): 2921-2929

    Abstract

    Knowledge of the genetic changes that lead to disease has grown and continues to grow at a rapid pace. However, there is a need for clinical devices that can be used routinely to translate this knowledge into the treatment of patients. Use in a clinical setting requires high sensitivity and specificity (>97%) in order to prevent misdiagnoses. Single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two DNA-based, complementary methods for mutation detection that are inexpensive and relatively easy to implement. However, both methods are most commonly detected by slab gel electrophoresis, which can be labor-intensive, time-consuming, and often the methods are unable to produce high sensitivity and specificity without the use of multiple analysis conditions. Here, we demonstrate the first blinded study using microchip electrophoresis (ME)-SSCP/HA. We demonstrate the ability of ME-SSCP/HA to detect with 98% sensitivity and specificity >100 samples from the p53 gene exons 5-9 in a blinded study in an analysis time of <10 min.

    View details for DOI 10.1002/elps.201100396

    View details for Web of Science ID 000298101000001

    View details for PubMedID 22002021

    View details for PubMedCentralID PMC3416029

  • Completely Monodisperse, Highly Repetitive Proteins for Bioconjugate Capillary Electrophoresis: Development and Characterization BIOMACROMOLECULES Lin, J. S., Albrecht, J. C., Meagher, R. J., Wang, X., Barron, A. E. 2011; 12 (6): 2275-2284

    Abstract

    Protein-based polymers are increasingly being used in biomaterial applications because of their ease of customization and potential monodispersity. These advantages make protein polymers excellent candidates for bioanalytical applications. Here we describe improved methods for producing drag-tags for free-solution conjugate electrophoresis (FSCE). FSCE utilizes a pure, monodisperse recombinant protein, tethered end-on to a ssDNA molecule, to enable DNA size separation in aqueous buffer. FSCE also provides a highly sensitive method to evaluate the polydispersity of a protein drag-tag and thus its suitability for bioanalytical uses. This method is able to detect slight differences in drag-tag charge or mass. We have devised an improved cloning, expression, and purification strategy that enables us to generate, for the first time, a truly monodisperse 20 kDa protein polymer and a nearly monodisperse 38 kDa protein. These newly produced proteins can be used as drag-tags to enable longer read DNA sequencing by free-solution microchannel electrophoresis.

    View details for DOI 10.1021/bm200358r

    View details for Web of Science ID 000291499900038

    View details for PubMedID 21553840

    View details for PubMedCentralID PMC3129339

  • Free-solution electrophoretic separations of DNA-drag-tag conjugates on glass microchips with no polymer network and no loss of resolution at increased electric field strength ELECTROPHORESIS Albrecht, J. C., Kerby, M. B., Niedringhaus, T. P., Lin, J. S., Wang, X., Barron, A. E. 2011; 32 (10): 1201-1208

    Abstract

    Here, we demonstrate the potential for high-resolution electrophoretic separations of ssDNA-protein conjugates in borosilicate glass microfluidic chips, with no sieving media and excellent repeatability. Using polynucleotides of two different lengths conjugated to moderately cationic protein polymer drag-tags, we measured separation efficiency as a function of applied electric field. In excellent agreement with prior theoretical predictions of Slater et al., resolution is found to remain constant as applied field is increased up to 700 V/cm, the highest field we were able to apply. This remarkable result illustrates the fundamentally different physical limitations of free-solution conjugate electrophoresis (FSCE)-based DNA separations relative to matrix-based DNA electrophoresis. ssDNA separations in "gels" have always shown rapidly declining resolution as the field strength is increased; this is especially true for ssDNA > 400 bases in length. FSCE's ability to decouple DNA peak resolution from applied electric field suggests the future possibility of ultra-rapid FSCE sequencing on chips. We investigated sources of peak broadening for FSCE separations on borosilicate glass microchips, using six different protein polymer drag-tags. For drag-tags with four or more positive charges, electrostatic and adsorptive interactions with poly(N-hydroxyethylacrylamide)-coated microchannel walls led to appreciable band-broadening, while much sharper peaks were seen for bioconjugates with nearly charge-neutral protein drag-tags.

    View details for DOI 10.1002/elps.201000574

    View details for Web of Science ID 000290586200014

    View details for PubMedID 21500207

    View details for PubMedCentralID PMC3416026

  • A 265-Base DNA Sequencing Read by Capillary Electrophoresis with No Separation Matrix ANALYTICAL CHEMISTRY Albrecht, J. C., Lin, J. S., Barron, A. E. 2011; 83 (2): 509-515

    Abstract

    Electrophoretic DNA sequencing without a polymer matrix is currently possible only with the use of some kind of "drag-tag" as a mobility modifier. In free-solution conjugate electrophoresis (FSCE), a drag-tag attached to each DNA fragment breaks linear charge-to-friction scaling, enabling size-based separation in aqueous buffer alone. Here we report a 265-base read for free-solution DNA sequencing by capillary electrophoresis using a random-coil protein drag-tag of unprecedented length and purity. We identified certain methods of protein expression and purification that allow the production of highly monodisperse drag-tags as long as 516 amino acids, which are almost charge neutral (+1 to +6) and yet highly water-soluble. Using a four-color LIF detector, 265 bases could be read in 30 min with a 267-amino acid drag-tag, on par with the average read of current next-gen sequencing systems. New types of multichannel systems that allow much higher throughput electrophoretic sequencing should be much more accessible in the absence of a requirement for viscous separation matrix.

    View details for DOI 10.1021/ac102188p

    View details for Web of Science ID 000286129800007

    View details for PubMedID 21182303

    View details for PubMedCentralID PMC3271724

  • Sequencing of DNA by free-solution capillary electrophoresis using a genetically engineered protein polymer drag-tag ANALYTICAL CHEMISTRY Meagher, R. J., Won, J., Coyne, J. A., Lin, J., Barron, A. E. 2008; 80 (8): 2842-2848

    Abstract

    We demonstrate the first use of a non-natural, genetically engineered protein polymer drag-tag to sequence DNA fragments by end-labeled free-solution electrophoresis (ELFSE). Fluorescently labeled DNA fragments resulting from the Sanger cycle sequencing reaction were separated by free-solution capillary electrophoresis, with much higher resolution and cleaner results than previously reported for this technique. With ELFSE, size-based separation of DNA in the absence of a sieving matrix is enabled by the end-on attachment of a polymeric "drag-tag" that modifies the charge-to-friction ratio of DNA in a size-dependent fashion. Progress in ELFSE separations has previously been limited by the lack of suitable large, monodisperse drag-tags. To address this problem, we designed, constructed, cloned, expressed, and purified a non-natural, genetically engineered 127mer protein polymer for use as an ELFSE drag-tag. The Sanger cycle sequencing reaction is performed with the drag-tag covalently attached to the sequencing primer, a major advance over previous strategies for ELFSE sequencing. The electrophoretic separation is diffusion-limited, without significant adsorption of the drag-tag to capillary walls. Although the read length (at about 180 bases) is still short, our results provide evidence that larger protein polymer drag-tags, currently under development, could extend the read length of ELFSE to more competitive levels. ELFSE offers the possibility of very rapid DNA sequencing separations without any of the difficulties associated with viscous polymeric sieving networks and hence will be amenable to implementation in microchannel and chip-based electrophoresis systems.

    View details for DOI 10.1021/ac702591t

    View details for Web of Science ID 000254969100026

    View details for PubMedID 18318549