DNA hairpins promote temperature controlled cargo encapsulation in a truncated octahedral nanocage structure family

Abstract

In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous demonstration of temperature controlled reversible encapsulation of the cargo enzyme, horseradish peroxidase, in the cage with four hairpin forming strands. However, in this previous study the mechanism of cargo uptake was not directly addressed (Juul, et al., Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724-9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening-closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation and showed that the two hairpin forming strands facilitated extension of at least one of the face surfaces of the cage scaffold, allowing entrance of the cargo protein into the cavity of the structure. Hence, the presented data demonstrate that cargo uptake does not involve a full opening of the structure. Rather, the uptake mechanism represents a feature of increased flexibility integrated in this nanocage structure upon the addition of at least two hairpin-forming strands.

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

“…The H4 DNA nanocage (H4-NC) was designed by starting from a truncated octahedral DNA cage structure [24], composed by eight different oligonucleotides, extensively characterized by our group [11,12,[18][19][20]22]. The DNA cage structure is covalently closed and composed of 12 double-stranded B-DNA helices ( Figure 1A), forming the edges of the structure, connected by short single-stranded thymidine linkers constituting square truncated faces ( Figure 1B).…”

“…Different shape-changing structural modules can be integrated in the DNS, allowing input-induced conformational changes. For example, octahedral DNA cages have been functionalized with temperature-dependent hairpins, to allow the reversible encapsulation and release of a protein [11,12], or with pH-dependent triple helices that allow the Int. J. Mol.…”

“…DNA nanostructures have also been functionalized to selectively interact with intracellular miRNA, mainly to detect their concentration, using electrochemical current or fluorescence signals [15][16][17]. Here, taking advantage of our experience matured in the last years in the characterization of different types of fully covalently octahedral DNA nanocages [11,12,[18][19][20][21], including their receptor-mediated cell targeting and their efficacy in selective drug delivery [5,22,23], we propose a new nanostructure for a possible therapeutic use as an efficient captor of the oncogenic miR21. For this purpose, we have initially engineered in one face of a truncated DNA cage four DNA hairpins complementary to a specific oligonucleotide (Fuel), to form a nanocage (H4-NC) with selective oligonucleotide sequestering activity.…”

Selective targeting and degradation of doxorubicin-loaded folate-functionalized DNA nanocages

“…2,3,4 During the last years our group has in vitro and in silico characterized various types of fully covalently bound truncated octahedral DNA nanocages and studied their behaviour in cells. [5][6][7][8][9][10] DNA nanocages, including octahedral cages, possess many attractive properties for cancer nanotherapy: they are intrinsically nontoxic, with excellent biocompatibility and biodegradability, [11][12][13] are readily internalized by living cells via endocytosis, 10,14 show high resistance to degradation 15,16 and can efficiently intercalate anticancer drugs such as doxorubicin. [17][18][19][20] Doxorubicin (Dox) is one of the most commonly used anticancer agents for the treatment of a wide variety of solid tumours including breast, ovarian, prostate, brain, cervix and lung cancers and haematological malignancies, such as multiple myeloma, several types of leukaemia and lymphomas.…”

The competing effects of core rigidity and linker flexibility in the nanoassembly of trivalent small molecule-DNA hybrids (SMDH3s)–a synergistic experimental-modeling study

“…Because such high concentrations and the long timescale for assembly render full-atomistic simulations infeasible, we adopted a coarse-grained (CG) approach, which reduces the complexity of the system by grouping atoms into beads. 31,32 Previously, we have successfully employed such a CGMD model in MD simulations to unravel the effect of DNA arm length and SMDH concentration on the outcomes of the nanoassemblies of fSMDH 3 s under various conditions. 11 That early model, however, cannot readily be extended to account for the roles that core-centric factors–such as the rigidity and geometry of the organic cores and the connection between the core and the DNA arms–may play in SMDH-based nanoassembly.…”