This study presents a mechanism for releasing a series of different short DNA sequences from sequestered complexes, one after another, using coupled biochemical reactions. The process uses stages of coupled DNA strand-displacement reactions that first release an output molecule and then trigger the initiation of the next release stage. We demonstrate the sequential release of 25 nM of four different sequences of DNA over a day, both with and without a centralized "clock" mechanism to regulate release timing. We then demonstrate how the presence of a target input molecule can determine which of several different release pathways are activated, analogous to branching conditional statements in computer programming. This sequential release circuit offers a means to schedule downstream chemical events, such as steps in the assembly of a nanostructure, or stages in a material's response to a stimulus.

Soft biomaterials possessing structural hierarchy have growing applications in lab-on-chip devices, artificial tissues, and micromechanical and chemomechanical systems. The ability to integrate sets of biomolecules, specifically DNA, within hydrogel substrates at precise locations could offer the potential to form and modulate complex biochemical processes with DNA-based molecular switches in such materials and provide a means of creating dynamic spatial patterns, thus enabling spatiotemporal control of a wide array of reaction-diffusion phenomena prevalent in biological systems. Here we develop a means of photopatterning two-dimensional DNA-functionalized poly­(ethylene glycol) diacrylate (PEGDA) hydrogel architectures with an aim toward these applications. While PEGDA photopatterning methods are well-established for the fabrication of hydrogels, including those containing oligonucleotides, the photoinitiators typically used have significant crosstalk with many UV-photoswitchable chemistries including nitrobenzyl derivatives. We demonstrate the digital photopatterning of PEGDA-co-DNA hydrogels using a blue light-absorbing (470 nm peak) photoinitiator system and macromer comprised of camphorquinone, triethanolamine, and poly­(ethylene glycol) diacrylate (M n = 575) that minimizes absorption in the UV-A wavelength range commonly used to trigger photoswitchable chemistries. We demonstrate this method using digital maskless photolithography within microfluidic devices that allows for the reliable construction of multidomain structures. The method achieves feature resolutions as small as 25 μm, and the resulting materials allow for lateral isotropic bulk diffusion of short single-stranded (ss) DNA oligonucleotides. Finally, we show how the use of these photoinitiators allows for orthogonal control of photopolymerization and UV-photoscission of acrylate-modified DNA containing a 1-(2-nitrophenyl) ethyl spacer to selectively cleave DNA from regions of a PEGDA substrate.

The programmable spatiotemporal activation of molecules could be used to create stimuli-responsive biomaterials capable of executing multistage sensing and computational processes. Here we develop a method for the activation of different DNA molecules in precisely specified locations and times within hydrogels. The activation locations are determined by patterning molecules to be released in their inactive form within hydrogels at resolutions of tens of microns, whereas the time of activation is controlled by a DNA strand displacement reaction cascade that releases the target DNA oligonucleotides at precisely specified time intervals. This programmed activation of DNA from hydrogel substrates thus enables the scalable development of DNA-based reaction-diffusion systems that regulate DNA strand availability in space and time. This system could be used as a platform to autonomously activate predefined chemical signals embedded within a soft material.

The programmable spatiotemporal activation of molecules could be used to create stimuli-responsive biomaterials capable of executing multistage sensing and computational processes. Here we develop a method for the activation of different DNA molecules in precisely specified locations and times within hydrogels. The activation locations are determined by patterning molecules to be released in their inactive form within hydrogels at resolutions of tens of microns, whereas the time of activation is controlled by a DNA strand displacement reaction cascade that releases the target DNA oligonucleotides at precisely specified time intervals. This programmed activation of DNA from hydrogel substrates thus enables the scalable development of DNA-based reaction-diffusion systems that regulate DNA strand availability in space and time. This system could be used as a platform to autonomously activate predefined chemical signals embedded within a soft material.

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