Molecular Logic with a Saccharide Probe on the Few-Molecules Level

In this Communication we describe a two-component saccharide probe with logic capability. The combination of a boronic acid-appended viologen and perylene diimide was able to perform a complementary implication/not implication logic function. Fluorescence quenching and recovery with fructose was analyzed with fluorescence correlation spectroscopy on the level of a few molecules of the reporting dye

Solubilisation of polymerised spidroins.

<p>Dragline silk from <i>N. clavipes</i> was incubated with the indicated solvents and the soluble fraction was analysed by gel-electrophoresis followed by Coomassie staining (A) and western blotting employing antibodies S1Rx and enhanced luminescence (ECL) for detection. The molecular weights of marker proteins are indicated in kilo Daltons (kDa).</p

Treatment of fibres with chaotropic agents and acids.

<p>Panel A. Light Microscopy (LM) phase images of fibres treated with mixtures of different ratios between HAc and HCl. Bar corresponds to 4000 nm. Minor (mi, reinforcement) and major (ma, dragline) ampullate fibres are indicated. Panel B. Light Microscopy (LM) phase contrast images of dragline treated with LiSCN at the stated concentrations for 1 minute. Bar corresponds to 8000 nm. Panel C. Scanning Electron Microscope (SEM) images of dragline incubated for the indicated times with 99% Hfip. Bar corresponds to 1000 nm. Panel D. Video contrast images of draglines (left unstressed, right fractured by applying stress) soaked in 8 M urea and counterstained with Coomassie Brilliant Blue. Bar equals 4000 nm.</p

Compositions and functions of silk layers from the dragline of <i>N. clavipes</i>

<p>MiSp: minor ampullate spidroin; MaSp: major ampullate spidroin</p

Biochemical composition of outer layers.

<p>Panel A. Oil red staining of fibres. Silk fibres were treated with water (upper filament) or ether extracted (lower filament) followed by oil red staining. Bar corresponds to 2000 nm. Panel B. Concavalin A (Con A) staining of fibres. Fibres gently washed in phosphate buffered saline or vigorously washed in 0.1% Triton X-100 were reacted with biotinylated Con A. The presence of α-methyl mannoside (α-MM), an inhibitor of Con A, is indicated by “I”. Bound Con A was visualised by gold conjugated streptavidin and SEM. The bar equals 500 nm.</p

Protein composition of silk layers.

<p>Filter strips obtained by western blotting and loaded with material extracted from the indicated fibre layers were stained with Ponceau S (Pon) or reacted with Concavalin A (ConA), pre-immune serum (PIS), S1Rx, S2Rx and S-pbs. The running position of a 200 kDa marker is indicated by the lines.</p

Localization of components.

<p>Panel A. Fluorescence labelling of silk fibres. Reactivities of Concavalin A (ConA), pre-immune serum (PIS), S-pbs, S1Rx and S2Rx to lesions of vigorously washed silk filaments. Phase contrast (Ph), immunfluorescence (IF) and merged images are shown. The bar indicates 4000 nm. Panel B. Immunogold staining of silk fibre cross-sections. Shown are transmission electron microscopy (TEM) images of dragline cross sections reacted with Concavalin A (ConA) or the indicated antibodies. The bar equals 200 nm.</p

Model of the multilayer organisation of dragline silk.

<p>A dragline can be divided into a shell and a core with five major layers of different material composition (from exterior to interior): a lipid coat, a glyco coat, a skin, an outer core and inner core. The approximate relative extensions of each layer are indicated.</p