Facile construction of a DNA tetrahedron in unconventional ladder-like arrangements at room temperature

A DNA tetrahedron as the most classical and simplest three-dimensional DNA nanostructure has been widely utilized in biomedicine and biosensing. However, the existing assembly approaches usually require harsh thermal annealing conditions, involve the formation of unwanted by-products, and have poor size control. Herein, a facile strategy to fabricate a discrete DNA tetrahedron as a single, thermodynamically stable product in a quantitative yield at room temperature is reported. This system does not require a DNA trigger or thermal annealing treatment to initiate self-assembly. This DNA tetrahedron was made of three chemically ligated triangular-shaped DNAs in unconventional ladder-like arrangements, with measured heights of ∼4.16 ± 0.04 nm, showing extra protections for enzymatic degradation in biological environment. They show substantial cellular uptake in different cell lines via temperature, energy-dependent and clathrin-mediated endocytosis pathways. These characteristics allow our DNA tetrahedron to be used as vehicles for the delivery of very small and temperature-sensitive cargos. This novel assembly strategy developed for DNA tetrahedra could potentially be extended to other highly complex polyhedra; this indicated its generalizability.


DNA sequences
diisopropylphosphoramidite was site-specifically coupled onto the growing oligonucleotide chain with a prolonged coupling time of 10 min and 3 couplings. The coupling efficiency was monitored by the removed trityl concentration level. All sequences were fully deprotected in concentrated ammonium hydroxide solution at 55°C for over 8 h.

(b) Purification:
Crude DNA strands were purified on 15% polyacrylamide/8M urea polyacrylamide gels at a constant current of 30 mA for 2 h (0.5 h at 250V followed by 1.5 h at 500 V), using 1 X TBE buffer. After

The appearance melting temperature (T m ) assay
The appearance melting temperature T m of DNA tetrahedron was determined with thermal denaturation experiment performed on Cary 300 UV-Vis spectrophotometer. The DNA tetrahedron sample was formed with 4.40 μM of each of the component strand T1, T2, T3, L1, L2 and L3 in 1 X PBS buffer was heated at 1 °C/min from 15 °C to 80 °C with a 1 °C interval and hold for 1 min at each degree. This experiment was performed twice. The appearance melting temperature T m was calculated by taking the first derivative of the melting curve.

Quantitation of gold nanoparticle-DNA monoconjugates
To confirm the band excised from the agarose gel containing only one DNA strand per AuNP, we conducted additional experiments to quantify the unlabeled DNA strand bound to gold nanoparticle by measuring the UV absorbance at 260 nm and at 520 nm. The coverage quantification was based on a UVvisible spectroscopy method reported by Hutchison and coworkers .1 and performed on NanoDrop 1000 spectrophotometer. KCN treatment is used to decompose the purified AuNP-L1, conjugates in order to eliminate the UV/vis absorbance at 260 nm from AuNP core which can interfere with the UV absorbance of DNA at 260 nm. Take AuNP-L1 as an example.
Next, to determine the absorbance of AuNP core contributed at 260 nm after KCN treatment, calibration curve of the decomposed AuNPs was obtained. As we have performed identical operations in our previous research work with the same gold nanoparticles. 2  The same procedure was performed to confirm that we have also obtained the DNA-AuNP monoconjugate for both L2-SH and L3-SH. We found that the number of DNA strand per gold nanoparticle for L2-SH and L3-SH was 1.38 and 1.42 respectively.     Figure S7. Flow cytometry analysis of the cellular uptake of py-THDs in HeLa cells.

Theoretical height calculation
To facilitate the analysis of the physical parameters obtained from characterization on the DNA tetrahedron, it is necessary to discuss and clarify some geometrical parameters associated with standard tetrahedron model. This DNA tetrahedron constructed by our strategy could be considered as a standard