The 1 kb ladder from Invitrogen was used as the source of all linear dsDNA fragments. Ladder was prepared according to manufacturer instructions and run on a 1.0% agarose gel stained with Gel Red (0.5x) for 1 h in 1xTAE buffer at 90 V. The desired linear dsDNA fragments (0.5, 1, and 3 kb) were visualized using an ultraviolet light and extracted from the gel using a scalpel. The DNA fragments were then purified from the agarose using the Qiagen gel extraction kit, following the manufacturer instructions, eluting all final fragments from columns using Ultrapure Water (Sigma-Aldrich). Isolated DNA stocks were diluted to 20 ng/μl.

The pMS plasmid with a segment of 3,033 bp inserted at the multiclonal site (total length 5,556 bp), was used for study of pDNA size and PDI in complex with Tat₂. The plasmid was cloned using E. coli, strain DH5α. E. coli containing pMS were taken from a glycerol stock and cultured overnight in 200 ml of LB media and purified from cells using a plasmid maxiprep kit according to the manufacturers instructions (Sigma-Aldrich). Plasmid was eluted from the included columns using Ultrapure water (Sigma) and diluted to 20 ng/μl for use in complex formation.

The 21-mer siRNA duplex was purchased from IDT as the sequence, 5′-AUGGAGACGCCGUCGUU-3′ and its complementary strand 5′-CGAGGACGGCGUCUCCAUGUU-3′ producing a duplex (molecular weight of 13,368.1 Da) with double U overhangs. The lyophilized product was dissolved in Ultrapure Water and subsequently diluted to a stock concentration of 2.0 μM (26.7 ng/μl). Final concentrations of all nucleic acids were determined by NanoDrop 8000 UV-Vis spectrophotometer (Thermo Scientific).

Sample preparation and analysis methods were based on a previous work involving siRNA nano-complexes (Jafari et al., 2014). Individual samples were prepared in a PCR well plate. Maltose-mannitol (MM, 90 mg/ml maltose, 9 mg/ml mannitol) solutions with varying amounts of CaCl₂ (0.00, 78.43, 156.86, 235.29, 313.73, and 392.16 mM) were prepared in ddH₂O at pH 7.0 and filtered twice through 0.2 μm cellulose syringe filters (VWR) using a 5 ml syringe. From these stock solutions 17 μL were aliquoted into well plates for each CaCl₂ concentration and 1 μl of stock dsRNA or DNA was added. Finally, 1 μl of the appropriate amount of Tat₂ peptide (varied by N:P ratio) was added and 1 μl of MM solution with no CaCl₂ was added to bring the final volume to 20 μl The N:P ratio for Tat₂ to nucleic acid was defined as the ratio of cationic lysine and arginine residues of Tat₂ (N) to the number of nucleic acid backbone phosphates (P). The N:P ratios used were 1:1, 4:1, and 8:1 for dsRNA experiments and only the 8:1 N:P ratio was used for experiments with DNA. All these preparations resulted in 20 μl samples with final CaCl₂ concentrations of 0.00, 66.66, 133.3, 200.0, 266.6, and 333.3 mM, corresponding to ionic strengths (I_c) of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 M respectively. The reaction mixtures were allowed to incubate for 15 min before measurement.

In the case of formulations containing tetrabutylammonium bromide (TBAB, Sigma) or tetrabutylphosphonium bromide (TBPB, Sigma), instead of MM, 1 μl of 8.4 nmol/μl TBAB, or TBPB dissolved in MM was added after 10 min of incubation time between the nucleic acid and Tat₂ and allowed a further 5 min of incubation time. This was a 100:1 molar charge ratio of N⁺ or P⁺ core atoms of each salt cation to backbone phosphates (P). This ratio was kept stationary for all experiments involving TBPB or TBAB. The final concentrations of both TBAB and TBPB were 420 μM, contributing little to the ionic strength of solution. Experiments with TBPB and TBAB were performed for dsRNA formulations and only experiments involving TBPB were performed with DNA formulations.

Size analysis was carried out immediately after incubation in a wetted (with 200 μl MM solution) low volume quartz sizing cuvette (ZEN 2112). The entire sample was loaded into the cuvette and measured on a Zetasizer Nano ZS (Malvern) with a 633 nm laser at 173° backscatter. Data was analyzed using the CONTIN algorithm in the Zetasizer software v.7.02 (Malvern). Three repeat measurements were made of each sample and means and standard deviations of both the primary particle size distribution peak, and PDI were calculated and plotted.

Hydrodynamic diameters (mean measured sizes, D_H) from formulations which resulted in minimally sized nano-complexes for each size of nucleic acid were used to calculate hydrodynamic volumes (V_H) under the assumption of a spheroid for all nano-complexes. V_H was plotted as a function of the approximate length of the nucleic acid based on the assumption of 0.33 nm/bp. In the case of circular plasmid the length was halved to account for supercoiling and circular shape. Linear regression was used to determine a mathematical relationship between nucleic acid length and theoretical nano-complex volume, and the slope was used to determine the accuracy of fit of each individual data point.

Zeta-potentials (symbol-ζ) were analyzed for circular plasmid, and for 21-mer siRNA in formulations as previously described but with an increase in the concentration of nucleic acid and Tat₂ by five-fold (5 ng/μl, nucleic acid). This was done at a 1:1, 4:1, and 8:1 N:P ratio for each with and without TBPB (100:1 molar charge ratio as previously described), as well as nucleic acid alone. Zeta-potential of samples were read using a capillary zeta-cuvette with gold electrodes in a Zetasizer-Nano (Malvern). The samples were prepared to a volume of 700 μl to fill the cuvette. Zeta-potential measurements were recorded and displayed to a number of significant figures provided by and to the accuracy of the Zetasizer-Nano instrument.

Seeds of Triticosecale sp. Whittmack cv Sunray, were planted in 8 × 4 root trainers and allowed to grow in a growth cabinet at 12–15°C, and a photoperiod of 19 h/day at an intensity of 300 μE/m²/s. At 13 days post planting, at the two leaf stage, plants were inoculated using a 1 ml sterile syringe (VWR) a formulation of one of the following in either ddH₂O or maltose-mannitol solution; solution only (Control), TBPB only, siRNA only, Tat₂ only, TBPB+Tat₂, TBPB:siRNA, siRNA+Tat₂, Tat₂:siRNA:TBPB, scsiRNA (scrambled siRNA), and Tat₂:scsiRNA:TBPB. Together there were a total of 18 treatments performed in a minimum of four replicates, where a single repetition consisted of 18 individual plants. A total of 250 pmol of siRNA in 100 μl was injected in all siRNA treatments, with TBPB, and Tat₂ being added proportional to the siRNA at the 8:1 N:P ratio of Tat₂ to siRNA and TBPB at the 100:1 ratio of positive to negative ratio of phosphonium ions to phosphates. The injections were directed toward the base of the youngest leaf after which the plants were returned to the growth cabinet (12–15°C, photoperiod of 19 h/day at an intensity of 300 μE/m²/s) until sample collection 24 h later.

The 21-mer siRNA duplexwas targeted to the phytoene desaturase (PDS) endogenous gene and was designed using pssRNAit online tool provided by the Zhao Bioinformatics Laboratory based on a chinese spring wheat PDS mRNA sequence (acc. FJ517553.1) with the sequence 5′-CAUGUUGUGAAGACACCCGAG-3′ (sense) and 5′-CGGUGUCUUCACAACAUGGU-3′ (anti-sense).

To generate the scrambled siRNA duplex (scsiRNA) (5′-AGCCGGUACGAAUAGTGAGUC-3′ and 3′-UCGGCCAUGCUUAUCACUCAG-5′), the siRNA sense sequence was scrambled using the Genscript® online sequence scrambler; with rice being used as a reference genome. Both strands of the resultant sequence were tested for potential off-target sequence alignment with the Ensembl Plants database (Triticum aestivum and Hordeum vulgare genomes, genome assemblies TGACv1 and ASM32608v1), as well as the general NCBI database using BLASTn.

The tip of the inoculated leaves (3–4 cm length by 1 cm width) was cut using scissors and placed into screwcap “tough tubes” (VWR) with 3 stainless steel beads (3 mm width) and immediately frozen in liquid nitrogen and stored at −80°C. To extract RNA the Nucleomag RNA 96 well plate (Macherey-Nagel) extraction kit was used, according to the manufacturers instructions with automation of extraction on a Biosprint 96 well plate robot (Qiagen). Purified RNA was quantified using UV-vis spectroscopy on a Nanodrop 8000 (ThermoFisher Scientific). cDNA was synthesized using a Superscript® VILO cDNA synthesis kit (ThermoFisher Scientific) according the manufacturers instructions with a total of 1.2 μg of RNA template per 20 μl reaction. RT-qPCR analysis was performed using SYBR-Green master mix (Life Technologies) with 40 ng of template cDNA per PCR reaction in 96 well plates. Each treatment and biological replicate was evaluated in three technical PCR replicates, with primers (500 nM forward and reverse) designed to amplify the target cDNA phytoene desaturase (PDS) and for three reference genes; ADP-ribosylation factor (ADP-RF), cell division control protein (CDC) and RNase L inhibitor protein (RLI) (see Table 1 for details). Thermocycling conditions were as follows: 95°C for 15 min, 40 cycles of 15 s of 95°C, 30 s of 60°C, and 30 s of 72°C, followed by a melting temperature series to evaluate amplicon content; 15 s at 95°C, 15 s at 60°C, and 15 s 95°C. All RT-qPCR was performed using an Applied Biosystems 7900HT RT-PCR machine and SDS 2.4 software. Reference genes were analyzed for stability by global averages of Ct-values (see Table S1) and the most stable was used for normalization of the target PDS gene expression level, based on the ΔΔCt method as amplification efficiencies were substantially similar (within <2%). Data was displayed as relative expression of PDS using 2^−ΔΔCt for conversion.

Data was analyzed using ANOVA and unpaired t-test to detect significant differences between pairs of means, with calibration of all treatments to an n = 3 group of untreated controls. This was done to maintain a balanced system for ANOVA. Statistical significance was rated at the 95 and 99.99% confidence intervals, p < 0.05 and p < 0.0001 respectively. Two-way ANOVA was applied twice to evaluate factors affecting relative expression of PDS. The initial application evaluated variation of sample treatment (Control, TBPB, siRNA, Tat₂, Tat₂:siRNA, siRNA:TBPB, Tat₂:siRNA:TBPB, scsiRNA, and Tat₂:scsiRNA:TBPB), versus solvent used (ddH₂O or maltose-mannitol). The second application of two way ANOVA gauged possible interaction between sample treatments (Control, siRNA, Tat₂, Tat₂:siRNA, and scsiRNA) with and without TBPB. In cases where no statistical significance between factors could be determined, data sets were combined and eight biological replicates were used per treatment in unpaired Student's t-test (see Table 3).

Hydrodynamic diameters and PDI of Tat₂:dsRNA nano-complexes were measured using DLS. Tat₂ complexation with 21-mer siRNA showed significant reduction in size and PDI upon the use of TBPB as an additional agent. It can be seen from Figure 3 that at all charge ratios of Tat₂:dsRNA, TBPB decreased the size of the nano-complexes but was most effective at the 8:1 Tat₂:dsRNA N:P ratio, producing nano-complexes with a D_H of ~10 nm without addition of CaCl₂ (see Figure 1). Generally it was observed that PDI correlates with size, lower size measurements correlating with lower PDI measurements. A PDI of ~0.1 indicates a strongly homogeneous size of the particles, and was only achieved using TBPB, with TBAB only improving complex formation at an I_c of 0.6 M before it was indistinguishable from the effect of CaCl₂ alone. This trend was observed with CaCl₂ throughout. It can be seen that as I_c increases, the addition of TBPB had a less dramatic effect that was indistinguishable from the effect of CaCl₂ alone at I_c exceeding 0.6 M (see Figure 1).

Tat₂:DNA nano-complex D_H were measured under different ionic strengths of CaCl₂ and in the presence or absence of TBPB. The addition of TBPB to Tat₂:DNA nano-complexes showed a noted lesser effect than that observed with dsRNA (see Figures 1, 2). The behavior of linear DNA did not strongly resemble the patterns observed with dsRNA. In the case of 0.5 kb, it was shown that TBPB had a weak effect on PDI, but reduced the size by ~ 50%, from 189.3 to 96.0 nm. This effect became more pronounced with an increase in ionic strength; however, at ionic strength of 0.8 the size increased to 811 nm, with reduction to a minimum in size at an I_c of 1.0 M of 46.95 nm. The PDI was found again to generally correlate inversely with I_c with a reduction to 0.155 at an I_c of 1.0 M.

In the case of the 1 kb DNA fragment (see Figure 2B), a true disparity in size and PDI can only be seen at the 0.0 M I_c limit, where size was at a minimum (62.6 nm) for the TBPB formulation and PDI was 0.538. A similar minimum was reached for formulation without TBPB at 0.4 M ionic strength with a size of 72.6 nm. The PDI was generally too high at all I_c higher than 0.0 M for appropriate analysis.

At 3 kb both MM and MM+TPBP produced nearly identical results in regards to PDI, while size was generally reduced to 90–120 nm under all I_c except for 0.2 M where MM and MM+TPBP formulations showed the same size particles (~ 250 nm) and 0.6 M, where the MM only formulation was shown to be significantly smaller (135.9 nm) and the MM+TBPB formula was substantially larger (461.5 nm). A complete divergence in size was noted after I_c 0.8 M.

For circular plasmid, the results show a close correlation in size under both MM and MM+TBPB condition, up to 0.2 M ionic strength and at 0.6 M. PDI was lowered substantially by addition of TBPB (a reduction of 0.3), but became smaller and more similar between MM and MM+TBPB formulations after an I_c of 0.4 M.

Generally it was shown that TBPB decreases particle size marginally, while often having a more significant effect on PDI up to <3 kb sized linear DNA and has less substantial effects at 3 kb linear DNA, in combination with Tat₂. Increasing ionic strength generally decreases PDI, but is highly dependent on the size of the nucleic acid. Additionally, large sizes can be observed to occur at anomalous positions, particularly with 0.5 kb linear DNA, where the 0.8 M I_c increment was substantially larger than all other formulations (nearly eight-fold). As can be seen by comparing the TBPB positive samples to the TBPB lacking samples, the size of nano-complexes was more affected at the smaller size ranges, with the effect becoming very slight with circular plasmid, though all minimized sizes were found only in the TBPB containing formulations.

Theoretical number distribution based hydrodynamic volumes (V_H) were calculated from the smallest Tat₂ nano-complex sizes measured for each nucleic acid under their specific conditions (see Table 2). The volumes allow for a robust linear regression when related to the length of the nucleic acid (see Figure 3). It was also interesting that the approximation of the size of the circular plasmid to one half of its actual sequential length to account for secondary structure and circular shape, was well approximated to the regression shown (see Figure 3).

By solving for the slope of the linear regression fit based on the equation V_H = 380.01(L), where the calculated slope of the regression appears to indicate a hydrodynamic volume increase of ~380 nm³/bp. Additionally, all the smallest measured nano-complexes (numerical values in Table 2) required the use of TBPB, though other formulations at high I_c were also found to give similar results (see Figure 2). It must be noted that this simplistic mathematical model does not take into account the entropic or enthalpic complexity of nucleic acid-cation interactions.

To determine the apparent charge character of the dsRNA nano-complexes and to determine if differences in how large dsDNA behaves in the presence of CPP and TBPB versus dsRNA as far as apparent charge character, zeta-potentials of Tat₂:circular plasmid, and Tat₂:dsRNA nano-complexes were measured at different N:P ratios, both with and without TBPB. At low N:P ratios (1:1 and 4:1), zeta-potential was greatly affected by TBPB, where circular plasmid was found to have virtually no change at the 1:1 N:P ratio level in the presence of TBPB while without TBPB a change of 22.2 mV occurred with the addition of Tat₂. With dsRNA the addition of TBPB had no effect on zeta-potential at the 1:1 N:P ratio, with a measure of ~ −24 mV. At the 4:1 N:P ratio, the circular plasmid was overcharged to a zeta-potential of 30.9 without TBPB but significantly less at 22.0 mV with TBPB. The opposite trend was observed in the case of 21-mer siRNA, as the 4:1 ratio without TBPB resulted in a zeta-potential of neutralization at −0.241 mV, while with TBPB the potential was measured at 4.54 mV. Overcharging continued at the 8:1 ratio in the case of circular plasmid and dsRNA without TBPB to 30.9 and 33.6 mV respectively. Increase in zeta-potential was shown in the case of circular plasmid up to 38.4 mV in the presence of TBPB at the 8:1 N:P ratio. The increase in zeta-potential was comparatively marginal in the case of dsRNA at an average of 8.56 mV, an increase of only 4.02 mV compared to circular plasmid's increase of 16.4 mV (see Figure 4).

The expression level of the phytoene desaturase gene was evaluated using RT-qPCR, and converted to relative expression values using the 2^−ΔΔCt method. A reduction in relative transcript abundance by 20–30 % was observed in all samples that were treated by injection. Samples treated with TBPB, showed up to 45% reduction in expression with Tat₂:TBPB, TBPB:siRNA, and Tat₂:scsiRNA:TBPB, showing significances of p < 0.05. An approximately 75% reduction in PDS expression (p < 0.0001 compared to injected control) was only observed from the Tat₂:siRNA:TBPB treatments (see Figure 5). Additionally, only the Tat₂:siRNA:TBPB treatment was found to show a statistical difference from all other samples except for TBPB alone (p = 0.068) in unpaired comparisons using Student's t-test (see Table 3). Furthermore, it must also be noted that an especially high degree of statistical significance was noted between the Tat₂:siRNA:TBPB treatment and the Tat₂:scsiRNA:TBPB treatment at p = 4.21·10⁻⁴. This was noteworthy as these two treatments were the closest in composition, the only difference being the sequence of the RNA duplex (see Table 3). Still, it should be noted that the Tat₂:scsiRNA:TBPB control was significantly different from the injection control and scsiRNA alone, which reflects the apparent effect of TBPB observed in the other treatments (see Table 3 and Figure 5).

ADP-RF expression was found to be the most stable reference for normalization, with CDC and RLI being far less stable. ADP-RF had a standard deviation amongst all tested samples of less than 1 Ct, while all other reference genes displayed standard deviations of greater than 1.3 Ct. Additionally it was found that for CDC and RLI samples that utilized TBPB had significantly lower Ct values than those without, with differences of up to 5 Ct, which represents an approximately 2⁵-fold difference in expression level. In the case of CDC this was a change of 22–24 Ct to 19–22 Ct, and in the case of RLI a change from 24–26 Ct to 20–22 Ct and as low as 18 Ct (see details in Table S1).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.