Design

Fig. 1: Supramolecular assembly of DNA

We have designed a biological supramolecular assembly material that uses tetrahedral DNA nanostructures (TDNs) as structural units and G-quadruplexes as linkers. This material can self-assemble into 2D DNA membranes without a matrix and can also spontaneously curl into 3D vesicles under the regulation of ion concentration and other factors, as shown in Fig. 1. Moreover, the inherent properties of TDNs and G-quadruplexes endow this material with a series of advantages, such as excellent biocompatibility, extensive modifiability, and sensitive ion responsiveness.

Fig. 2: The structure of G-quadruplexes [1]. (a) The arrangement of guanine bases in the G-quartet, shown together with a centrally placed metal ion. Hydrogen bonds are shown as dotted lines, and the positions of the grooves are indicated. (b) The poly(dG) four-fold, right-handed helix. (c) Surface view representation of a quadruplex structure comprising eight G-quartets, with the central channel exposed to show an array of metal ions (coloured yellow).

The sequence of a G-quadruplex is typically represented as GₘXₙGₘXₒGₘXₚGₘ, where m denotes the number of G residues in each short G-tract (these G residues are usually directly involved in G-tetrad interactions). Xₙ, Xₒ, and Xₚ can be any combination of residues (including G), forming the loops of the G-quadruplex(Fig. 3). G-quadruplexes can be formed not only by a single DNA strand but also by two DNA sequences each containing the GₘXₙGₘ motif [1].

Fig. 3: G-quadruplex linkage mode (a) Some possible topologies for simple tetramolecular (on the left-hand side) and bimolecular quadruplexes. Strand polarities are shown by arrows.[1] (b) Some possible topologies for simple unimolecular quadruplexes.[2]

In our design, two TDN monomers are linked together via G-quadruplex formation under the action of lithium ions and potassium ions, using the connecting sequences attached to the monomers(Fig. 4).

Fig. 4: The link mode of two TDN monomers through G-quadruplex under the action of metal cations.（yellow: G-quadruplex , blue: TDN , purple: Monovalent metal cation）

The linking method using G-quadruplexes has the following characteristics:

1. Assembly at room temperature: At room temperature, we can mix TDN monomers with the ion solution used for assembly to form the assembled structure. This avoids the damage to the DNA structure and the drugs or enzymes loaded on the assembly material that would be caused by the high temperatures required for other assembly reactions.

2. Controllable opening and closing: G-quadruplexes form and TDN monomers connect to each other under the action of metal cations such as potassium ions. When we add crown ether compounds, monovalent metal cations bind to crown ethers, leading to the disassembly of G-quadruplexes [2–4] (Fig. 5). This property allows us to adjust the functions of the assembled nanostructures more conveniently and flexibly.

Fig. 5: Controlled assembly of TDN monomers. TDN monomers form G-quadruplex and are interconnected under the action of metal cations such as potassium ions. When the crown ether compound is added, the monovalent metal cation combines with the crown ether, resulting in the decomposition of G-quadruplex.

G-quadruplexes formed by the coordination of different metal cations with G-rich sequences vary[5]. Due to its appropriate radius and low dehydration enthalpy, potassium ions can be embedded between two G-quartet layers in the G-quadruplex. They coordinate maximally with oxygen atoms on a total of eight G bases (four from each of the upper and lower G-quartets), neutralizing the negative charges in the G-quadruplex more effectively. This results in the formation of the most compact and strongest G-quadruplex with high binding energy. In contrast, lithium ions have a smaller radius and exhibit weaker stabilizing effects on G-quadruplexes. The G-quadruplex segments involved in assembly are relatively loose, with lower strength and smaller binding energy.

In our design, we can control the formation of two types of G-quadruplexes with different binding energies and rigidities by adjusting the types and concentrations of ions during assembly. This allows us to regulate the critical radius at which the assembly bends during formation, thereby controllably generating 2D membranes and spherical cage structures. During the 2D assembly process, the assembly first forms 2D patch structures. As monomers connect to each other, the radius of the patches continues to increase. Once the critical radius is reached, the reduction in surface energy from further 2D assembly becomes smaller than the increase in edge energy caused by incompletely assembled monomers at the edges, leading to thermodynamically spontaneous bending of the assembly(Fig. 6A). The critical radius is related to the rigidity and energy of the bonds.

Fig. 6: Schematic of the design mechanism[6] (A) Schematic diagram of structural transformation in spherical assembly.(B) Energy surface analysis of G-quadruplex complexes formed by potassium ions.(C) Energy surface analysis of g-quadruplex complexes formed by lithium ions.

Our system uses potassium and lithium ions for monomer assembly, where the binding energies stabilized by the coordination of different ions in the system have significant effects. G-quadruplexes formed with potassium ions have higher binding energy, resulting in greater edge energy at the edges during assembly. This leads to a smaller critical radius and a tendency to bend into tubular structures during assembly(Fig. 6B). In contrast, lithium ions have lower binding energy, allowing for a larger critical radius during assembly and thus maintaining the growth of 2D membrane structures(Fig. 6C). By adjusting the ratio of potassium ions to lithium ions added during assembly, we regulate the edge energy and surface energy of the assembly, enabling it to bend to a certain extent and form spherical cage structures effectively.

The building blocks of our 2D material are modified TDNs. A TDN is a framework DNA structure synthesized from four single-stranded DNAs (ssDNAs) via one-step annealing [7] (Fig. 7). Each ssDNA undergoes complementary base pairing with the other three ssDNAs, forming a tetrahedral DNA structure with high biocompatibility [7], high structural stability [7,8], high cellular uptake efficiency [9], and capacity for functional modification [10–12].

Fig. 7: four single-stranded DNAs form modified TDN.

We extended the GGGGAATGGGG sequence at the 5'-end of three of the four ssDNAs(Fig. 8). After assembling into TDNs, these sequences can connect to each other under the action of monovalent metal cations(Fig. 9).

Fig. 8: Among the 4 ssDNAs, there are 3 extended G-rich sequences at the 5 'end of 3 ssDNAs.

Fig. 9,10: The assembly steps of TDN monomers. Mix TDNs modified with different functions in the required proportion, add the corresponding metal cations, and assemble them to form multifunctional integrated two-dimensional nanomembranes or vesicles.

In addition, we extended a DNA sequence at the 5'-end of the fourth ssDNA of the TDN, which is used to load functional groups(Fig. 11). Through the following steps, we might be able to control the structure of our 2D supramolecular material and modularly integrate the required functions onto it:

1. Synthesize TDNs via one-step annealing.

2. Perform functional modification of TDNs by allowing the extended strand of the TDN to undergo complementary base pairing with the functional group-loaded DNA strand at room temperature.

Fig. 11: Functional modification of TDN. It is achieved by allowing the extended strand of TDN to perform complementary base pairing with the DNA strand loaded with functional groups.

3. Select the desired structures and functions, mix TDNs with different functional modifications in the required ratio, add the corresponding metal cations, and assemble to form a multifunctional integrated 2D nanomembrane or vesicle(Fig. 9,10).

Reference

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