RESUMEN
We demonstrate a versatile process for assembling micron-scale filament architectures by controlling where DNA tile nanotubes nucleate on DNA origami assemblies. "Nunchucks," potential mechanical magnifiers of nanoscale dynamics consisting of two nanotubes connected by a dsDNA linker, form at yields sufficient for application and consistent with models.
Asunto(s)
ADN/química , Nanotubos/química , Conformación de Ácido NucleicoRESUMEN
AIM: To assess and compare the changes in shape of encapsulated biconvex structures undergoing equatorial traction with those changes reported in the human lens during accommodation. METHODS: Equatorial traction was applied to several different biconvex structures: air, water, and gel filled mylar and rubber balloons and spherical vesicles. In the vesicles, traction was applied externally, using optical tweezers, or from within, by the assembly of encapsulated microtubules. The shape changes were recorded photographically and the change in central radius of curvature of water filled mylar balloons was quantified. RESULTS: Whenever an outward equatorial force was applied to the long axis of long oval biconvex objects, where the minor to major axis ratio was =0.6, the central surfaces steepened and the peripheral surfaces flattened. Similar changes in the shape of the lens have been reported during human in vivo accommodation. CONCLUSIONS: All biconvex structures that have been studied demonstrate similar shape changes in response to equatorial traction. This effect is independent of capsular thickness. The consistent observation of this physical change in the configuration of biconvex structures in response to outward equatorial force suggests that this may be a universal response of biconvex structures, also applicable to the human lens undergoing accommodation.
Asunto(s)
Acomodación Ocular/fisiología , Cristalino/fisiología , Modelos Biológicos , Humanos , Cápsula del Cristalino/anatomía & histología , Estrés MecánicoRESUMEN
DNA nanotubes are crystalline self-assemblies of DNA tiles approximately 10 nm in diameter that readily grow tens of micrometers in length. Easy assembly, programmability, and stiffness make them interesting for many applications, but DNA nanotubes begin to melt at temperatures below 40 degrees C, break open when deposited on mica or scanned by AFM, and disintegrate in deionized water. These weaknesses can be traced to the presence of discontinuities in the phosphate backbone, called nicks. The nanotubes studied here have five nicks, one in the core of a tile and one at each corner. We report the successful ligation of all four corner nicks by T4 DNA ligase. Although ligation does not change the nanotubes' stiffness, ligated nanotubes withstand temperatures over 70 degrees C, resist breaking during AFM, and are stable in pure water for over a month. Ligated DNA nanotubes are thus physically and chemically sturdy enough to withstand the manipulations necessary for many technological applications.
Asunto(s)
ADN Ligasas/química , ADN/química , Nanotubos/química , Silicatos de Aluminio/química , Secuencia de Bases , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Especificidad por Sustrato , Propiedades de SuperficieRESUMEN
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.
Asunto(s)
Gramicidina/química , Canales Iónicos/química , Membrana Dobles de Lípidos , Modelos Biológicos , Dimerización , Electroquímica , Gramicidina/análogos & derivados , Canales Iónicos/fisiología , Modelos Moleculares , Técnicas de Placa-Clamp/instrumentación , Fosfatidilcolinas/química , Probabilidad , Estrés Mecánico , TermodinámicaRESUMEN
The fidelity of Escherichia coli DNA polymerase III (pol III) is measured and the effects of beta, gamma processivity and epsilon 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 finc = 5.6 x 10(-6), 4.2 x 10(-7), and 7 x 10(-7), respectively. Elevated dGMP.G and dTMP.G misincorporation efficiencies of 3.2 x 10(-5) and 5.8 x 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 alpha holoenzyme, which cannot proofread, has 47-, 32-, and 13-fold higher misincorporation rates for dGMP.G, dTMP.G, and dAMP.G mispairs. Both the beta, gamma complex and the downstream nucleotide have little effect on the fidelity of catalytic alpha 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)).