Designing DNA nanostar hydrogels with programmable degradation and antibody release

TL;DR

The paper addresses the lack of understanding of how DNAns hydrogel design controls degradation and cargo release in vivo. It designs four three-armed DNAns (A–D) with varied arm length, junction flexibility, and linker connectivity, and probes their degradation under site-specific restriction enzymes versus non-specific DNaseI using gel electrophoresis, time-resolved microrheology, and cargo-release assays. Key findings show that linker-free gels resist site-specific cleavage, while relocating recognition sites to linkers enables programmable degradation; initial viscoelasticity is encoded by architecture, and cargo release (including antibodies) can be selectively triggered by enzymes such as EcoRV, enabling targeted delivery while preserving nuclease resistance to unintended nucleases. Together these results establish concrete design rules for DNAns-based hydrogels with tunable degradation and cargo release, advancing DNA-based scaffolds for tissue regeneration, 3D cell culture, and controlled antibody/drug delivery.

Abstract

DNA nanostar (DNAns) hydrogels are promising materials for in vivo applications, including tissue regeneration and drug and antibody delivery. However, a systematic and quantitative understanding of the design principles controlling their degradation is lacking. Here, we investigate hydrogels made of three-armed DNAns with varying flexible joints, arm lengths, and mesh sizes and use restriction enzymes to cut the DNAns structures while monitoring the gel's degradation. We discover that (i) removing flexible joints, (ii) increasing arm length, or (iii) relocating the RE site along a DNA linker markedly accelerates hydrogel degradation. In contrast, non-specific endonucleases, e.g. DNaseI, quicly degrade DNAns hydrogels regardless of design. Importantly, the release of antibodies from DNAns hydrogels can be modulated by the action of different enzymes, confirming that programmable degradation can be leveraged for responsive drug-delivery systems. These findings provide a better understanding of the design principles for DNAns-based scaffolds with tunable degradation, cargo release, and responsive rheology.

Figures (6)

• Figure 1: DNA nanostar designs. (a)-(d) Schematic representations of the four DNAns designs used in this study. Each design self-assembles into a three-armed DNA nanostar containing recognition sequences for the restriction enzymes EcoRI-HF (two arms, labeled in green), BtgI, and MspA1I (remaining arm, labeled in red and cyan, respectively). For designs B-D an EcoRV-HF site (labelled in magenta) is located on the linker. (e) Summary table of structural parameters for each design. Columns two to four list arm, linker, and sticky-end lengths (in base-pairs (bp) and nucleotides (nt)). Columns five and six indicate the number of unpaired adenines (A) forming flexible joints at the core (FJ1) and before the sticky ends (FJ2). The final three columns report the melting temperatures determined with NUPACK nupack at [NaCl]=150mM, [Mg$^{+2}$]=10mM and [DNA]=250$\mu$M. (f) Schematic of the annealing protocol used for DNAns and hydrogel assembly (shown for design A, without linker).
• Figure 2: DNA hydrogels formed by design A nanostars displays RE resistance.(a) Gel electrophoresis of DNAns hydrogels (250$\mu$M, corresponding to $\sim120\mu$g/$\mu$L) treated with DNaseI for different incubation times (1, 2, 4 and 6 hours) at 35$^\circ$C. "NC" is negative control (no addition of RE). Ladder is NEB low molecular weight. (b) Time-resolved microrheology shows that the gel progressively loses viscoelasticity. (c) Normalized viscosity decreases monotonically with time. (d) Gel electrophoresis of DNAns hydrogels (at 250$\mu$M) treated with the sequence-specific restriction enzymes MspA1I (8h incubation) and EcoRI (16h incubation) shows no detectable degradation. (e) Microrheology confirms no significant change in material properties with respect to control. (f) Normalized viscosity as a function of time display no significant loss of integrity. In panels (c) and (f) the viscosity at digestion time $t$ is normalized by the initial value ($\eta_0$), measured at the earliest available time point.
• Figure 3: Hydrogel pore size does not affect EcoRI-mediated degradation. (a) Left: Schematic of design A DNAns gels below and above the sticky-end melting temperature ($T_{m3}=41^\circ$C). Right: Gel electrophoresis of design A hydrogels incubated with EcoRI for 16h at 37–58$^{\circ}$C. (b) Left: MSD from microrheology for water (gray), design A hydrogel (blue), and hydrogel with ssDNA blockers (red). Right: Gel electrophoresis of design A hydrogels with (+B) and without (-B) blocking strands. All the samples were incubated for 16h with EcoRI at 37$^{\circ}$C. (c) Left: Schematic of a design B nanostar hydrogel displaying larger pores. Right: Gel electrophoresis of design B hydrogels incubated for 16h with EcoRI at 37$^{\circ}$C and showing no degradation with respect to negative control (NC).
• Figure 4: Effect of site location on DNAns hydrogel cleavage by EcoRI and EcoRV.(a) Gel electrophoresis after 21h digestion at 37$^\circ$C and 0.68U/$\mu$g. Left: EcoRV digestion of NS(I)+L(V) (site on linker, purple). Middle: EcoRV digestion of NS(V)+L(I) (site on two arms, magenta). Right: EcoRI digestion of NS(V)+L(I) (site on linker, green). NC: samples without enzyme. (b) Left: MSD from microrheology experiments for NC (blue) and digested samples (EcoRV: purple/magenta, EcoRI: green), showing increased mobility after digestion. Right: Normalized viscosity ($\eta/\eta_\mathrm{NC}$) for each case: EcoRV at linker, EcoRV on DNAns arms, EcoRI on linker.
• Figure 5: EcoRV digestion of DNAns designs B and D. (a) Gel electrophoresis of design B hydrogels digested by EcoRV (100U, at 0.9U/$\mu$g) at 37$^\circ$C. Reactions were stopped at different time points (1h, 2h, 4h, 6h, and 22h). (b) MSD from MR experiments of 500 nm beads over the first 6h of digestion at 37$^\circ$C. (c)-(d) show analogous results as (a)-(b) for design D hydrogels. (e) MSD curves comparing NC with samples digested for 22 h for both designs. (f) Viscosity as function of digestion time.