Nanophotometer p330

For the preparation of DNA origami triangles [20 (link)] and six-helix bundles (6HBs) [28 (link)], the 7249 nt M13mp18 scaffold and about 200 staple strands (Metabion international AG, Planegg/Steinkirchen, Germany) were annealed at a molar ratio of 1:10 in 1 × TAE buffer (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) supplemented with 10 mM MgCl₂ (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) by gradually decreasing the temperature from 80 °C to RT over 1.5 h in a Primus 25 advanced thermocycler (PEQLAB, Erlangen, Germany). The samples were purified using Amicon Ultra-0.5 mL spin filters with 100 kDa molecular weight cut-off (Merck KGaA, Darmstadt, Germany). The concentrations of the obtained DNA origami solutions were determined using an Implen Nanophotometer P330 (Implen GmbH, München, Germany) and adjusted to the desired values.
Genomic dsDNA from salmon testes (Thermo Fisher GmbH, Kandel, Germany) was used as a control with similar GC content as the assembled DNA origami nanostructures [29 (link),30 (link)] but no defined length or secondary structure, in order to distinguish between duplex- and superstructure-specific effects. It was dissolved in 1 × TAE buffer with 10 mM MgCl₂ and the concentration (in bp) adjusted using an Implen Nanophotometer P330 (Implen, München, German).

DNA from leaf material was extracted using 3 plants for each treatment by acetyltrimethyl-ammonium bromide (CTAB) according to [59 ]. The DNA was resuspended in distilled water and quantified by Implen P330 nanophotometer (Implen GmbH, München, Germany). Seven 10-mer primers were used for PCR amplification (Operon Technologies, USA) as presented in Table 8. PCR reactions were carried out in 20 μL volume containing: 2.0 μL of DNA (15 ng μL⁻¹), 7.0 μL of dd.H2O, 10.0 μL of 10 × PCR master mix buffer, and 1.0 μL of single primer (10 pmol). The reaction mixture was subjected to the following conditions: initial denaturation at 94°C for 5 min then 35 cycles of amplification under the following parameters: template denaturation at 94 °C for 1min, primer annealing at 36 °C for 30 s, and extension at 72 °C for 3 min. By the end of the 35th cycle, final extension at 72 °C for 10 min was given, followed by storage at 4 °C. The amplification products were resolved by electrophoresis in 1.5% agarose gels in 0.5 Tris-borate-EDTA (TBE) buffer and documented on Gel Documentation system (Uvitec Cambridge Company, Cambridge, UK). The amplifications were repeated twice and only the reproducible bands were considered. Reproducible fragments were scored as ‘1’ or ‘0’ for presence or absence of the band on the gels, respectively.