Characterization of the DNA dodecahedron

The analysis of the annealing mixture via native agarose gel electrophoresis showed one discrete band. Treatment of the self-assembly product with mung bean nuclease, which selectively digests single stranded DNA, showed no effect on the electrophoretic mobility (Figure 33a, lane 7). When subsets of the DNA dodecahedron were treated with mung bean nuclease a shift in electrophoretic mobility was observable (Figure 33a, lane 1-6). In case of the fully assembled DNA dodecahedron all strands of the twenty trisoligonucleotides are hybridized to complementary strands of neighboring trisoligonucleotides and no single-stranded "digestible" DNA remains. Subsets of the DNA dodecahedron necessarily contain single-stranded arms which can be degraded by the nuclease, resulting in an increase of electrophoretic mobility.


Figure 33   (a) Native agarose gel (2%) of the dodecahedron and assembly subsets (a) before and (b) after treatment with mung bean nuclease (digestion of single-stranded DNA). Lane M: 100bp DNA ladder. Lane 1a, b: v14 - v17. Lane 2a, b: v13 - v17. Lane 3a, b: v1, v2, v12 - v17. Lane 4a, b: v1, v2, v11 - v18. Lane 5a, b: v1, v2, v5, v6, v11 - v18. Lane 6a, b: v1, v6, v12 - v18. Lane 7a, b: v1 - v20. (b) Dodecahedra with increasing number of 32P-labeled trisoligonucleotides. Autoradiograph (top) and densitometric analysis (bottom).


The trisoligonucleotides were labeled with 32P using [γ-32P] ATP and T4-polynucleotide kinase. Annealing mixtures of the dodecahedral DNA-object were combined with increasing number of 32P-labeled trisoligonucleotides, underwent the annealing-temperature program and were analyzed in native agarose gels. The recorded autoradiographs showed a linear increase of the band intensity with increasing number of added 32P-labeled trisoligonucleotides. This is consistent with an object which is assembled from the twenty trisoligonucleotides in equal amounts. Atomic force microscopy of the DNA dodecahedra in liquid phase showed uniform particles of the expected lateral size (approximately 20 nm) but limited height, suggesting a certain deformability of the DNA object (Figure 34a). Cryo-EM measurements of the DNA dodecahedra are in agreement with the AFM results, showing discrete particles of approximately 18 nm diameter (Figure 34b).