In Silico and In Cell Analysis of Openable DNA Nanocages for miRNA Silencing

Abstract

A computational and experimental integrated approach was applied in order to study the effect of engineering four DNA hairpins into an octahedral truncated DNA nanocage, to obtain a nanostructure able to recognize and bind specific oligonucleotide sequences. Modeling and classical molecular dynamics simulations show that the new H4-DNA nanocage maintains a stable conformation with the closed hairpins and, when bound to complementary oligonucleotides produces an opened conformation that is even more stable due to the larger hydrogen bond number between the hairpins and the oligonucleotides. The internal volume of the open conformation is much larger than the closed one, switching from 370 to 650 nm3, and the predicted larger conformational change is experimentally detectable by gel electrophoresis. H4-DNA nanocages display high stability in serum, can efficiently enter the cells where they are stable and maintain the ability to bind, and sequester an intracellular-specific oligonucleotide. Moreover, H4-DNA nanocages, modified in order to recognize the oncogenic miR21, are able to seize miRNA molecules inside cells in a selective manner.

Combined and selective miR-21 silencing and doxorubicin delivery in cancer cells using tailored DNA nanostructures

“…2). As reported, the conformational change of Fol-NCs toward the open state leads to a lower degree of stability 31 . Here we use Fol-scr-NCs as a model to initially study internalization and intracellular stability, since Fol-miR21-NCs, once entered in the cells, undergo a conformational change toward the bound open state due to the intracellular presence of miR-21 ( Fig.…”

“…In line, Fig. 2 shows that the highest concentration of intact Fol-NCs inside the cells occurs after 24 h of incubation, unlike what happens to pristine H4-DNA-NCs administered by transfection 31 . In the latter case, most of the nanocages enter in 2-3 h and are then quickly degraded.…”

“…Since each nanocage has four sequestering units, the "miR-21 sequestering units: endogenous miR-21" ratio ranges between 240:1 and 120:1, so there are always more than 100x molar excess of sequestering units. This ratio permits an efficient miR-21 binding, as recently demonstrated in vitro 31 .…”

“…The Fol-miR21-NC was designed starting from a recently described H4-DNA cage 31 , where four miRNA sequestering units were introduced in a corner of a truncated octahedral DNA nanocage 22 . The structure, resulting from the assembly of eight oligonucleotides, is characterized by a scaffold consisting of 12 doublestranded B-DNA helices, which form the main edges, connected by short single-stranded five-thymidine linkers, constituting the square truncated faces ( Supplementary Fig.…”

“…We recently obtained openable nanostructures that are able to recognize and bind specific oligonucleotide sequences in vitro and in cells, suitable for specifically sequestering intracellular oligonucleotides, such as miR-NAs 31 . Here, we propose folate-functionalized NCs harboring DNA sequestering units complementary to miR-21 (Fol-miR21-NCs), to form nanostructures with a specific miR-21 silencing activity.…”