Gel Shift Analysis

Gametophore and protonema tissue from 3- to 4-wk-old moss clones were dispensed into 96-well Microtube Rack plates (National Scientific Supply) containing two steel balls per well. Frozen or lyophilized moss tissue was ground using a TissueLyserII system (Qiagen). The high-throughput DNA extraction fast method was performed as follows: 150 μl of extraction buffer (0.2 M Tris-HCl pH 7.5, 0.25 M NaCl, 0.025 M EDTA, 0.5% SDS) was added to each well. After mixing and 5 min of centrifugation (3220 × g), 100 μl of supernatant was transferred to a 96 v-shape well microplate (Greiner bio-one). DNA was precipitated by adding 100 μl of isopropanol and centrifuging for 15 min at 3220 × g. The supernatant was discarded, and precipitated DNA was cleaned by adding 200 μl of ethanol 75%, and centrifuged 15 min at 3220 × g. The ethanol was discarded, and precipitated DNA was dried for 20 min at 37°. Finally, DNA was dissolved in 80–100 μl of TE buffer. PCR reactions (50 µl) were performed using 1 μl of extracted DNA solution as template. Primers for PCR gel shift analysis were designed to obtain 150–200 bp fragments surrounding the on-target crRNA sequence (Table S3). Primer3plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) was used to design primers (Untergasser et al. 2007 ). PCR fragment shifts were evaluated in 3% agarose TBE gel-electrophoresis. When a shift was detected, the fragment was sequenced. Sequences were aligned by CodonCode Aligner V5.1.5 from LI-COR. Alignments were curated manually to find mutations around the PAM sequence of the corresponding on-target genomic sequence. Clones with a confirmed mutation were also sequenced for all on-target genomic sequences.

As previously reported, the binding affinity of selected peptides to siRNA was investigated using the SYBR green exclusion assay [16 (link)]. The MLPs and scrambled siRNA were mixed in normal saline (in triplicates) with different N/P ratios ranging from 0.05 to 40. The mixture was incubated at room temperature for 30 min to ensure complete complexation of peptides and siRNA. After the incubation time, the complexes were transferred to a black 96-well plate. SYBR green dye solution for this assay was made by diluting 1 part of SYBR green dye with 10,000 parts of purified water. Then, 200 µL of the freshly made SYBR green dye dilution was added to each siRNA complex in the black 96-well plate. The plate was covered with aluminum foil before reading in a micro-plate reader that detected the fluorescent signal (485 nm excitation and 527 nm emission). SYBR green only binds to free siRNA, which significantly enhances the fluorescence signal. By creating a standard curve, the intensity of the fluorescent signal was translated to the concentration of free siRNA, and an indication of the percentage of siRNA bond to the protein. The percentage of the siRNA complexed with the peptide was determined using the following equation: $$\% \mathrm{siRNA} \mathrm{bond} \mathrm{to} \mathrm{peptide}=100-\left(\frac{\mathrm{Fluorescence} \mathrm{signal} \mathrm{for} \mathrm{complexed} \mathrm{siRNA} }{\mathrm{Fluorescence} \mathrm{signal} \mathrm{for} \mathrm{free} \mathrm{siRNA}}\times 100\right)$$
% siRNA bound to the peptide was plotted against a range of nitrogen to phosphate (N/P) ratios used in the experiment, and BR50 (the N/P ratio of the complex required to bind 50% of siRNA) was determined using a sigmoidal model. The negatively charged nucleic acids interact and form complexes with positively charged carriers via interionic interactions. Therefore, the N/P ratio can be a deciding factor, not only in the percentage of siRNA bond to the carrier, but also in how the complexes interact with the cell.
In addition to the SYBR green dye exclusion assay described above, the binding affinity of the peptides to siRNA was also determined using the gel retardation/gel shifting assay. Briefly, the MLPs-siRNA complexes were prepared at different N to P ratios ranging from 0 to 60. The N/P ratio of zero represents free siRNA. The tubes containing complexes were incubated at room temperature for 30 min for complete complex formation. After this incubation time, the complexes were mixed with gel-loading dye. Each of the samples was loaded into the wells of 1% agarose gel with (1 μg/mL) ethidium bromide, and 400 Amperes and 70 Volts were used to run the gel for 20 min. After the run, the gels were visualized under a Bio-Rad imager. The intensity of the bands was quantified by Image Lab software.

To assess the in vitro cold-triggered drug release profile, aliquots of 10 mg of CPT&Cy5-siR CRNPs were dispersed in 3 ml of acetate buffer (pH 5.0), phosphate buffer (pH 6.5), and phosphate buffer (pH 7.4) in a 15-ml centrifuge tube, respectively, and then placed in an Incubating Orbital Shaker (VWR, Radnor, PA, USA) at 100 rpm and 37 °C. For cold treatment at 8 h, the samples were immersed into cold NaCl solution (−4 °C) for 10 min. At various times, 400 µl of the supernatant of the samples were collected for release measurement after centrifuging the samples at room temperature or 0 °C (only for the time point right after the cold treatment) at 13800 g for 20 min. A total of 400 µl of fresh buffer was added into the sample right after each collection of the supernatant. The fluorescence intensity of CPT in the collected supernatant was measured using the Spark Multimode Microplate Reader, with an excitation wavelength at 370 nm and an emission wavelength of 434 nm. The amount of Cy5-siR released at different time points was also quantified by the Spark Multimode Microplate Reader with an excitation wavelength of 651 nm and an emission wavelength of 670 nm. The cold-triggered release of siR from the nanoparticles at −4, 22, and 37 °C was also investigated with the aforementioned gel retardation assay. The unprocessed scans of the gels are provided in the Source Data file.