Cellular Uptake of Tile-Assembled DNA Nanotubes

Abstract

DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the GFP-expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the GFP gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing in vitro and in vivo experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.

Self-assembly of hierarchically ordered structures in DNA nanotube systems

“…Clearly this is a physical factor that must be considered in the assembly of well-defined cellular structures from mechanically dissimilar, filamentous, biopolymer components such as actin, microtubules, vimentin and keratin. Additionally, the broad interest in applying DNA-based macromolecules, often those of an extended rod-like geometry and in the size regime subject to crowding effects [16,[45][46][47][48], as intracellular therapeutics, suggests that this basic assembly mechanism should be considered. Figure A3.…”

DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices

“…The structures can also be utilized for transfecting small interfering RNA molecules (siRNA) for silencing genes in a controllable way. Kocabey et al [36] tried to silence a target gene by equipping a small PEGylated folate -modified DNA nanotube ( Figure 1C) with siRNA molecules, but did not succeed with that particular setup. However, Lee et al [37] showed that folate -or peptide -modified tetrahedral DNA nanoparticles loaded with siRNAs ( Figure 1C) can silence the target genes in tumors.…”

“…(B) DNA origami nanostructures for delivering doxorubicin (DOX) into cancer cells: an under-twisted rod-like shape [33], a straight rod [31] and a triangle [31,32]. (C) DNA structures for small interfering RNA (siRNA) delivery: a cage with cell-targeting ligands (folate or peptide) at the end of the siRNA motifs [37], and a PEGylated folatemodified nanotube [36]. (D) DNA structures for CpG-triggered immunostimulation: a DNA origami tube [39] and a polypod-like structure [41].…”

A Molecular Hero Suit for In Vitro and In Vivo DNA Nanostructures

“…Folate modification promotes endocytosis into cancerous cells where folate receptors are overexpressed. DNA nanotubes produced by origami and tile‐based assembly methods that contained folate and fluorescently modified component strands recognized folate receptors on the cell surface, followed by internalization upon binding. This resulted in a tenfold uptake enhancement compared to unmodified origami‐based nanotubes.…”