Random sequence-guided crosslinking for on-demand injectable HA–DNA hydrogels supporting neural progenitor cells

Hyaluronic acid (HA) hydrogels crosslinked through DNA hybridization (HA-DNA hydrogels) offer a programmable and cell-compatible platform for regenerative medicine. However, their practical use has been limited by undesired premature network formation arising from nonspecific hybridization among tethered strands. Here, we introduce an on-demand injectable HA-DNA hydrogel engineered through a dual-tube hybrid-bridge design coupled with sequence-level randomization to regulate gelation kinetics. Randomization was expected to disrupt excessive complementarity among identical crosslinkers, reducing nonspecific self-dimerization. Each HA chain was conjugated with an anchor DNA strand and pre-hybridized with a one crosslinker type (Tube A or Tube B), after which the two solutions were mixed to trigger on-demand gelation. In silico analysis identified that introducing randomized bases into the crosslinker overlap domain, with a total randomized length of N = 8, minimized undesired self-dimerization while preserving productive hybridization. Consistently, rheological analyses confirmed these predictions, showing suppressed pre-gelation in single-tube condition and rapid, homogeneous gelation upon precursor mixing. Furthermore, the optimized N = 8 hydrogel enabled uniform encapsulation of neural progenitor cells, supporting high viability and sustained expression of neural stemness-associated markers (SOX2, NESTIN). Above all, the hydrogel further exhibited injectable capability with shear-thinning, rapid self-healing, and robust post-extrusion structural integrity, enabling minimally invasive delivery without compromising cell proliferation. Collectively, this sequence-optimized dual-tube strategy establishes a versatile and programmable HA-DNA hydrogel platform for controlled gelation and injectable neural tissue engineering applications.