Deborah Kuchnir Fygenson

Gramicidin Channel Kinetics under Tension

We have measured the effect of tension on dimerization kinetics of the channel-forming peptide gramicidin A. By aspirating large unilamellar vesicles into a micropipette electrode, we are able to simultaneously monitor membrane tension and electrical activity. We find that the dimer formation rate increases by a factor of 5 as tension ranges from 0 to 4 dyn/cm. The dimer lifetime also increases with tension. This behavior is well described by a phenomenological model of membrane elasticity in which tension modulates the mismatch in thickness between the gramicidin dimer and membrane.

Fidelity of Escherichia coli DNA Polymerase III Holoenzyme THE EFFECTS OF β, γ COMPLEX PROCESSIVITY PROTEINS AND ε PROOFREADING EXONUCLEASE ON NUCLEOTIDE MISINCORPORATION EFFICIENCIES

The fidelity of Escherichia coli DNA polymerase III (pol III) is measured and the effects of β, γ processivity and ε proofreading subunits are evaluated using a gel kinetic assay. Pol III holoenzyme synthesizes DNA with extremely high fidelity, misincorporating dTMP, dAMP, and dGMP opposite a template G target with efficiencies f inc = 5.6 × 10−6, 4.2 × 10−7, and 7 × 10−7, respectively. Elevated dGMP·G and dTMP·G misincorporation efficiencies of 3.2 × 10−5 and 5.8 × 10−4, attributed to a “dNTP-stabilized” DNA misalignment mechanism, occur when C and A, respectively, are located one base downstream from the template target G. At least 92% of misinserted nucleotides are excised by pol III holoenzyme in the absence of a next correct “rescue” nucleotide. As rescue dNTP concentrations are increased, pol III holoenzyme suffers a maximum 8-fold reduction in fidelity as proofreading of mispaired primer termini are reduced in competition with incorporation of a next correct nucleotide. Compared with pol III holoenzyme, the α holoenzyme, which cannot proofread, has 47-, 32-, and 13-fold higher misincorporation rates for dGMP·G, dTMP·G, and dAMP·G mispairs. Both the β, γ complex and the downstream nucleotide have little effect on the fidelity of catalytic α subunit. An analysis of the gel kinetic fidelity assay when multiple polymerase-DNA encounters occur is presented in the “Appendix” (see Fygenson, D. K., and Goodman, M. F. (1997) J. Biol. Chem. 272, 27931–27935 (accompanying paper)).

Evidence that Collagen Fibrils in Tendons Are Inhomogeneously Structured in a Tubelike Manner

The standard model for the structure of collagen in tendon is an ascending hierarchy of bundling. Collagen triple helices bundle into microfibrils, microfibrils bundle into subfibrils, and subfibrils bundle into fibrils, the basic structural unit of tendon. This model, developed primarily on the basis of x-ray diffraction results, is necessarily vague about the cross-sectional organization of fibrils and has led to the widespread assumption of laterally homogeneous closepacking. This assumption is inconsistent with data presented here. Using atomic force microscopy and micromanipulation, we observe how collagen fibrils from tendons behave mechanically as tubes. We conclude that the collagen fibril is an inhomogeneous structure composed of a relatively hard shell and a softer, less dense core.

Nanoscale structure and microscale stiffness of DNA nanotubes.

We measure the stiffness of tiled DNA nanotubes (HX-tubes) as a function of their (defined) circumference by analyzing their micrometer-scale thermal deformations using fluorescence microscopy. We derive a model that relates nanoscale features of HX-tube architecture to the measured persistence lengths. Given the known stiffness of double-stranded DNA, we use this model to constrain the average spacing between and effective stiffness of individual DNA duplexes in the tube. A key structural feature of tiled nanotubes that can affect stiffness is their potential to form with discrete amounts of twist of the DNA duplexes about the tube axis (supertwist). We visualize the supertwist of HX-tubes using electron microscopy of gold nanoparticles, attached to specific sites along the nanotube. This method reveals that HX-tubes tend not to form with supertwist unless forced by sequence design, and, even when forced, supertwist is reduced by elastic deformations of the underlying DNA lattice. We compare the hybridization energy gained upon closing a duplex sheet into a tube with the elastic energy paid for deforming the sheet to allow closure. In estimating the elastic energy we account for bending and twisting of the individual duplexes as well as shearing between them. We find the minimum supertwist state has minimum free energy, and global untwisting of forced supertwist is energetically favorable, consistent with our experimental data. Finally, we show that attachment of Cy3 dyes or changing counterions can cause nanotubes to adopt a permanent writhe with micrometer-scale pitch and amplitude. We propose that the coupling of local twist and global counter-twist may be useful in characterizing perturbations of DNA structure.

Few-Atom Fluorescent Silver Clusters Assemble at Programmed Sites on DNA Nanotubes

We show that DNA hairpins template the site-specific assembly of fluorescent few-atom Ag clusters on DNA nanotubes. Fluorescent clusters form only at hairpin sites and not on the double-stranded DNA scaffold, allowing for spatially programmed self-assembly. Ag clusters synthesized on hairpins protruding from DNA nanotubes can have nearly identical fluorescence spectra to those synthesized on free hairpins of identical sequence. Analysis of the stepwise photobleaching of individual clusters suggests a chemical yield of ~45%. Given the well-established sequence-specific optical properties of DNA stabilized Ag clusters, these results point the way toward high yield assembly of metal cluster fluorophores with control over spectra as well as spatial arrangement.

Sialylneolacto-N-tetraose c (LSTc)-bearing Liposomal Decoys Capture Influenza A Virus

Background: Better treatments are needed for combating influenza. Results: LSTc-sialoside-bearing decoy liposomes competitively bind to influenza A virus, as assessed by hemagglutination inhibition, flow cytometry, and growth inhibition studies. Decoy liposomes co-localize with influenza virus, as assessed by confocal imaging. Conclusion: LSTc-sialoside-bearing decoy liposomes are highly effective in capturing influenza virus. Significance: Decoy liposomes may serve as an effective platform for presenting anti-pathogen receptors. Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains.

Design and characterization of 1D nanotubes and 2D periodic arrays self-assembled from DNA multi-helix bundles.

Among the key goals of structural DNA nanotechnology are to build highly ordered structures self-assembled from individual DNA motifs in 1D, 2D, and finally 3D. All three of these goals have been achieved with a variety of motifs. Here, we report the design and characterization of 1D nanotubes and 2D arrays assembled from three novel DNA motifs, the 6-helix bundle (6HB), the 6-helix bundle flanked by two helices in the same plane (6HB+2), and the 6-helix bundle flanked by three helices in a trigonal arrangement (6HB+3). Long DNA nanotubes have been assembled from all three motifs. Such nanotubes are likely to have applications in structural DNA nanotechnology, so it is important to characterize their physical properties. Prominent among these are their rigidities, described by their persistence lengths, which we report here. We find large persistence lengths in all species, around 1-5 μm. The magnitudes of the persistence lengths are clearly related to the designs of the linkages between the unit motifs. Both the 6HB+2 and the 6HB+3 motifs have been successfully used to produce well-ordered 2D periodic arrays via sticky-ended cohesion.

Improving fluorescence detection in lab on chip devices.

Fluorescence is the workhorse for analysis in many of today’s lab-on-chip (LOC) devices. However, LOC fluorescence detection is limited in both spatial resolution and sensitivity, due primarily to sub-optimal light collection. More sensitive, higher resolution LOC devices would enable novel structural and functional biomolecule studies, as well as improve chip-based bioanalytical assays by orders of magnitude. In this focus article, we consider the possible avenues for performing high-sensitivity and high-resolution fluorescence detection within LOC devices.

Active, motor-driven mechanics in a DNA gel

Cells are capable of a variety of dramatic stimuli-responsive mechanical behaviors. These capabilities are enabled by the pervading cytoskeletal network, an active gel composed of structural filaments (e.g., actin) that are acted upon by motor proteins (e.g., myosin). Here, we describe the synthesis and characterization of an active gel using noncytoskeletal components. We use methods of base-pair-templated DNA self assembly to create a hybrid DNA gel containing stiff tubes and flexible linkers. We then activate the gel by adding the motor FtsK50C, a construct derived from the bacterial protein FtsK that, in vitro, has a strong and processive DNA contraction activity. The motors stiffen the gel and create stochastic contractile events that affect the positions of attached beads. We quantify the fluctuations of the beads and show that they are comparable both to measurements of cytoskeletal systems and to theoretical predictions for active gels. Thus, we present a DNA-based active gel whose behavior highlights the universal aspects of nonequilibrium, motor-driven networks.

Tau-isoform dependent enhancement of taxol mobility through microtubules

Tau, a family of microtubule-associated proteins (MAPs), stabilizes microtubules (MTs) and regulates their dynamics. Tau isoforms regulate MT dynamic instability differently: 3-repeat tau is less effective than 4-repeat tau at suppressing the disassembly of MTs. Here, we report another tau-isoform-dependent phenomenon, revealed by fluorescence recovery after photobleaching measurements on a BODIPY-conjugated taxol bound to MTs. Saturating levels of recombinant full-length 3-repeat and 4-repeat tau both cause taxol mobility to be remarkably sensitive to taxol concentration. However, 3-repeat tau induces 2.5-fold faster recovery ( approximately 450s) at low taxol concentrations ( approximately 100 nM) than 4-repeat tau ( approximately 1000 s), indicating that 3-repeat tau decreases the probability of taxol rebinding to its site in the MT lumen. Finding no tau-induced change in the MT-binding affinity of taxol, we conclude that 3-repeat tau either competes for the taxol binding site with an affinity of approximately 1 microM or alters the MT structure so as to facilitate the passage of taxol through pores in the MT wall.