Bioinspired Fabrication of DNA-Inorganic Hybrid Composites Using Synthetic DNA

Abstract

Nucleic acid nanostructures have attracted significant interest as potential therapeutic and diagnostic platforms due to their intrinsic biocompatibility and biodegradability, structural and functional diversity, and compatibility with various chemistries for modification and stabilization. Among the fabrication approaches for such structures, the rolling circle techniques have emerged as particularly promising, producing morphologically round, flower-shaped nucleic acid particles: typically hybrid composites of long nucleic acid strands and inorganic magnesium pyrophosphate (Mg₂PPi). These constructs are known to form via anisotropic nucleic acid-driven crystallization in a sequence-independent manner, rendering monodisperse and densely packed RNA or DNA-inorganic composites. However, it still remains to fully explore how flexible polymer-like RNA or DNA strands (acting as biomolecular additives) mediate the crystallization process of Mg₂PPi and affect the structure and properties of the product crystals. To address this, we closely examined nanoscale details to mesoscopic features of Mg₂PPi/DNA hybrid composites fabricated by two approaches, namely rolling circle amplification (RCA)-based in situ synthesis and long synthetic DNA-mediated crystallization. Similar to the DNA constructs fabricated by RCA, the rapid crystallization of Mg₂PPi was retarded on a short-range order when we precipitated the crystals in the presence of presynthesized long DNA, which resulted in effective incorporation of biomolecular additives such as DNA and enzymes. These findings further provide a more feasible way to encapsulate bioactive enzymes within DNA constructs compared to in situ RCA-mediated synthesis, i.e., by not only protecting them from possible denaturation under the reaction conditions but also preventing nonselective association of proteins arising from the RCA reaction mixtures.

Keywords: DNA inclusion; DNA-inorganic hybrid composites; coprecipitation; crystallization; rolling circle techniques.

Scheme 1. Overview of the Fabrication of DNA-Inorganic Hybrid Composites

Key: (1) DNA flowers (DNF) constructed by a one-pot enzymatic process using rolling circle amplification (RCA) and (2) Mg₂PPi/AmpDNA composites formed by synthetic DNA-driven crystallization, in which abundant Mg and PPi ions precipitated in the presence of amplified DNA (AmpDNA). The AmpDNA was obtained from the RCA reaction with the addition of pyrophosphatase (PPase) and subsequent ethanol precipitation. Deoxynucleoside triphosphate (dNTP), deoxynucleoside monophosphate (dNMP), phi29 DNA polymerase (φ29 DNAP), pyrophosphate (PPi, P₂O₇), phosphate (Pi), magnesium pyrophosphate (Mg₂PPi), and magnesium hydrogen phosphate (MgHPi).

Figure 1

Inclusion of DNA as an organic additive in the formation of Mg₂PPi crystals. Representative SE-SEM images of the grown Mg₂PPi crystals after 20 h crystallization in the absence (left) and presence of AmpDNA (middle) and λ DNA (right) at a fixed concentration of DNA (26.7 μg mL^–1) and PPi ion concentrations of (A) 0.5, (B) 1, and (C) 2 mM, where morphological changes are observed with increasing PPi ion concentrations. Scale bar, 2 μm. (D) SE-SEM and HAADF-STEM images of a nanoflake with ellipsoidal shape observed in the presence of 0.5 mM of Mg₂PPi. The EDS elemental maps confirmed the presence of oxygen (O), magnesium (Mg), and phosphorus (P) in the nanoflake. Scale bar, 200 nm. (E) Schematic illustration of the proposed role of DNA molecules in the regulation of the anisotropic growth of the Mg₂PPi nanoflakes.

Figure 2

Structure and elemental composition of DNA-included Mg₂PPi composites. (A–D) Representative SE-SEM images of the Mg₂PPi crystals precipitated in (A) the absence and presence of (B) 26.7 μg mL^–1 AmpDNA or (C) 26.7 μg mL^–1 λ DNA with 2 mM Mg₂PPi. (D) In comparison, DNF was synthesized through one-step RCA reaction. High-magnification images show the presence of thin lines connecting the nanoflakes (indicated by red arrows) for Mg₂PPi/DNA and DNF, in contrast to the smooth nanoflake surfaces of Mg₂PPi. Scale bars, 500 nm (A–D, left) and 200 nm (A–D, right). (E–H) Representative HAADF-STEM images of each composite. The particle regions from which the higher magnification images (right) originated are indicated by boxes on the lower magnification HAADF-STEM images (left). Scale bars, 500 nm (E–H, left) and 200 nm (E–H, right). (I) EDS spectra recorded from entire individual particles. (J) Relative atomic ratios of each element, C, N, O, Mg, and P to Mg for each particle type. Data represent mean ± standard deviation (s.d.) of the EDS measurements determined over five particles. N.S. (not significant, p > 0.05) and *p < 0.001 based on one-way ANOVA and Tukey test’s multiple comparison.

Figure 3

Structure and elemental composition of enzyme-included Mg₂PPi composites. (A–C) Representative HAADF-STEM images of the Mg₂PPi crystals precipitated in the presence of (A) RNase A and (B) RNase A and AmpDNA at 26.7 μg mL^–1 DNA, 200 μg mL^–1 RNase A and 2 mM Mg₂PPi. (C) In comparison, DNF-R was prepared through RCA reaction with the addition of RNase A. The particle regions from which the higher magnification images (right) originated are indicated by boxes on the lower magnification HAADF-STEM images (left). Scale bars, 500 nm (left) and 100 nm (right). (D) EDS spectra recorded from the whole area of the individual particle. (E) Relative atomic ratios of each element to Mg for each particle type. Data represent mean ± s.d. of the EDS measurements determined over five particles.

Figure 4

Spatially resolved STEM–EELS elemental analysis of (A–E) Mg₂PPi/AmpDNA-R and (F–J) DNF-R. (A, F) Representative HAADF-STEM images of the lamellar Mg₂PPi/AmpDNA-R and DNF-R specimens showing the highly porous interior. Scale bar, 500 nm. (B, G) HAADF images and EELS elemental maps (with an 18–20 nm pixel size) extracted from the area marked as “spectrum image” in (A) and (F), displaying the distribution of carbon (C K-edge), nitrogen (N K-edge), and oxygen (O K-edge). Scale bar, 200 nm. (C, H) Evolution of C K-, N K-, and O K-EELS spectra recorded over an area of 5 × 5 pixels or 4 × 4 pixels from the regions numbered 1–6, as marked in (A) and (F). (D, I) Average C K-edge EELS spectra fitted with Gaussian peaks. Peaks A (284–285 eV), B (286–287 eV), and C (289–290 eV) were assigned to aromatic carbon (aromatic C), organic carbon (organic C), and carbonate bonding, respectively. (E, J) The ratio maps of peak B, i.e., (organic C)/total intensity, and peak C, i.e., (carbonate)/total intensity, show the relative contribution of organic carbon and carbonate bonding within the particle. Ratio maps were generated by dividing intensity maps of peak B (organic C) and C (carbonate), resulting from Gaussian fitting of C K-edges in (D) and (I), by the respective total C K-edge intensities (integrated over 15 eV windows from the edge onsets).

Figure 5

Effects of the inclusion of biomolecules on the growth of Mg₂PPi crystals. (A) Powder XRD patterns and (B, C) Raman spectra of the Mg₂PPi composites grown in the absence/presence of DNA and/or RNase A (Mg₂PPi, Mg₂PPi-R, Mg₂PPi/AmpDNA, Mg₂PPi/AmpDNA-R, and Mg₂PPi/λ DNA) and DNF in the absence/presence of RNase A (DNF and DNF-R). A diffraction pattern of the simulated Mg₂PPi (Mg₂P₂O₇·3.5H₂O) is also shown. The Raman spectra were obtained in (B) dehydrated and (C) hydrated conditions using a 532 nm laser. In (B), the spectra were obtained with a laser power of 13 mW and acquisition time of 5 s and show the average spectra ± s.d. of five different points in the sample area. In (C), the spectra were collected with a laser power of 30 mW and acquisition time of 20 s and show the spectra of one point in the sample area. All Raman spectra are normalized to the area under the curve. (D) UV absorption and (E) CD spectra of AmpDNA, free RNase A (free R), Mg₂PPi/AmpDNA, Mg₂PPi/AmpDNA-R, DNF, and DNF-R. (F) Fluorescence intensity (ΔF = F – F₀, where F and F₀ are the emitted fluorescence of a substrate with and without treatment of RNase-containing samples at λ_ex = 490 nm and λ_em = 520 nm) as a function of time for four different catalytic systems. The concentration of RNA substrate was 4 μM. (G) Reaction rate against various concentrations of the substrate (0.1–8 μM) for four catalytic systems. The concentration of RNase A in each sample was 0.5 ng mL^–1. Results represent mean ± s.d. for four independent experiments.