Enhancing immunomodulation on innate immunity by shape transition among RNA triangle, square and pentagon nanovehicles.
Bottom Line: Changing one RNA strand in polygons automatically induced the stretching of the interior angle from 60° to 90° or 108°, resulting in self-assembly of elegant RNA triangles, squares and pentagons.The degree of immunostimulation greatly depended on the size, shape and number of the payload per nanoparticles.Stronger immune response was observed when the number of adjuvants per polygon was increased, demonstrating the advantage of shape transition from triangle to pentagon.
Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Markey Cancer Center, Nanobiotechnology Center, University of Kentucky, Lexington, KY 40536, USA.Show MeSH
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Mentions: The stabilities of polygons were studied using a perpendicular TGGE (Biometra GmbH). This convenient technique has garnered widespread use for measuring melting temperatures of RNA nanoparticles with multiple strands (5,7,14,26,28,55). The preassembled polygons were subjected to 7% native TGGE with a gradient temperature of 30–70°C perpendicular to electrical current. The following apparent TM values were obtained for the polygons at 100 nM total concentration (Ct) in presence of 0.2 mM MgCl2: triangle TM = 56°C, square TM = 53°C and pentagon TM = 50°C (Figure 4A). The triangular nanoscaffolds were more stable than squares and pentagons, although the number of RNA bp was much higher in the pentagon construct, as compared to the square and triangle. Usually, the stability of nucleic acids with the same base-pair content is directly dependent on metal ion and total nucleic acid concentrations. Since these two criteria were the same, it was assumed that the higher the number of bp in a given RNA structure the higher the stability. Therefore, the most stable shape produced should be the pentagon. However, based on TGGE data the opposite was found. This was likely due to the tension caused by the stretching of the native pRNA 3WJ 60° ∠AOB. The triangular construct angle was preserved (60°), the square and pentagon angles were stretched to the wider conformations of 90° and 108°, respectively. Previously it has been shown that any nucleotide mutations or deletions within the native core structure of the pRNA 3WJ motif would also result in the loss of its thermodynamic stability (10). Interestingly, the measured triangle and square TM values differed by +3°C, as did the square and pentagon. Boiling resistance assay in the presence and absence of 8 M urea further confirmed that the triangle was the most stable nanoparticle (Figure 4B). The quantification of nanoparticle bands after heating to 100°C resulted in 75 ± 4% recovery of the triangular assembly, suggesting a TM >100°C. By definition, TM is the measured temperature when half the RNA concentration has melted, i.e. 50% recovery. The percentage of recovery for square was 28 ± 2% and for pentagon was 16 ± 5%, much lower than the value estimated for triangle recovery. The experiment with the presence of 8 M urea in boiling solution showed that the overall trend of stability remained the same, but the percentage of recovery was 55 ± 4% for triangle, 8 ± 3% for square and no pentagon fraction was detected. Overall, the nanoparticle with fewer 3WJ motifs (triangle) resulted in a higher thermostability and resistance in chemical degradation and the change in stability was in large part due to the stretching of the ∠AOB.
Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Markey Cancer Center, Nanobiotechnology Center, University of Kentucky, Lexington, KY 40536, USA.