Anti digoxigenin fab fragments

Constant-trap-position experiments have been performed on a dual optical tweezer (C-Trap^TM from Lumicks, The Netherland). Opposite helical domains of the DNA origami structure were linked to two double-stranded 3 kbp-long DNA handles, functionalized at the ends either with digoxygenin or biotin. Digoxygenin- and biotin-functionalized handles were obtained by PCR amplification of the pET-28a vector (5369 bp). DNA handles, bridging the DNA origami to the beads, contained a central PEG9 spacer flanked by two ca. 30 nt-long stretches, one partially complementary to the M13 scaffold and the other partially complementary to the tether. For the tweezers experiment, DNA origami-handles constructs were tethered between 1.76 µm streptavidin polystyrene beads (Spherotech, Inc) and 1 µm polystyrene beads (Spherotech, Inc), previously covalently functionalized with anti-digoxigenin Fab fragments (Roche). Measurements were carried out at room temperature in 1× TEMg buffer with the addition of an oxygen scavenger system (26 U/ml glucose oxidase, 17,000 U/ml catalase).

DNA constructs were incubated for 2 h with 2 μm silica beads (Bangs Laboratories, Inc), which were previously covalently functionalized with anti-digoxigenin F_ab fragments (Roche). The mixture was diluted in optical tweezers buffer (50 mM sodium phosphate buffer, 250 mM NaCl, 1% (w/v) d-glucose, pH 7.1) and mixed with streptavidin-coated 1 μm silica beads (Bangs Laboratories, Inc.). Measurements were carried out at room temperature in optical tweezers buffer after addition of an oxygen scavenger system (26 U/ml glucose oxidase, 17 000 U/ml catalase). DNA conjugates concentration was adjusted to only sparsely cover the beads leading mainly to single-tether formation. The beads were trapped in the foci of a dual beam optical tweezers platform (JPK NanoTracker) (9 (link)). Both trapped beads were brought into close proximity for tether formation. The beads were then separated with a constant velocity yielding force vs. extension traces. Trap stiffness was determined using a previously described calibration protocol (10 ). The effective trap stiffness was determined with an error of ∼10% and varied from 0.056 to 0.062 pN/nm. Data were acquired at a sampling rate of 50 kHz. The signals were corrected for crosstalk.

A standard protocol was followed for in situ hybridization of embryos after fixation and dechorionation at the indicated stages. Probes were prepared by in vitro transcription. Whole-mount in situ hybridization was performed using Digoxigenin labelled antisense RNA probe and visualized using anti-Digoxigenin Fab fragments conjugated with alkaline phosphatase (Roche Molecular Biochemicals) as described [17 (link), 18 (link)]. Embryos were processed and hybridized as described [19 (link)], except that 10 mg/ml of proteinase K in PBS/0.1% Tween-20 was used for 10 to 30 minutes depending on the age of the collected embryos.
For histology, zebrafish embryos were fixed in 4% paraformaldehyde and dehydrated prior embedding in JB-4 (Polysciences). 5-μm sections were cut using Leica RM2255 microtome (Leica, Wetzlar, Germany), dried, and stained with Hematoxylin and Eosin as described [20 ]. Still images were taken with an Olympus SZX 16 and Zeiss stereomicroscope (AxioSkop 2 plus).

Flow channel was first made from two #1 glass coverslips and the bottom one was functionalized by 3-Aminopropyl triethoxy silane (APTES). The channel was then flushed by Silane-PEG-NHS (PG2-NSSL-5k, Nanocs) dissolved in DMSO and incubated for 1 h. It was followed by flushing anti-digoxigenin Fab fragments (Roche) in the channel to create covalent bonds with NHS group on PEG and incubated for another 1 h. The channel was then incubated with 1% BSA solution in 1× phosphate buffered saline (PBS) buffer (pH 7.4) at 4°C before experiments to avoid non-specific binding of DNA to the surface.

The carboxylated polystyrene microspheres (0.58 μm diameter; Bangs Laboratory) were labeled with anti-digoxigenin Fab fragments (Roche) based on a two-step functionalization method described in^{70 (link)} with the following modifications. First, we used a 3:1 molar ratio of 2 kDa α-methoxy-ω-amino PEG: 3 kDa α-amino-ω-carboxy PEG (Rapp Polymere) in the first functionalization step. Second, we used 0.16 mg/mL anti-digoxigenin Fab in phosphate buffer saline (PBS) pH 7.4 for the antibody conjugation step. The Fab-conjugated microspheres were sonicated briefly (~15 s) and incubated with 1 mg/mL BSA on ice for at least 10 min prior to the optical tweezer experiments to enhance the bead surface passivation.