Multi-domain automated patterning of DNA-functionalized hydrogels.

DNA-functionalized hydrogels are capable of sensing oligonucleotides, proteins, and small molecules, and specific DNA sequences sensed in the hydrogels' environment can induce changes in these hydrogels' shape and fluorescence. Fabricating DNA-functionalized hydrogel architectures with multiple domains could make it possible to sense multiple molecules and undergo more complicated macroscopic changes, such as changing fluorescence or changing the shapes of regions of the hydrogel architecture. However, automatically fabricating multi-domain DNA-functionalized hydrogel architectures, capable of enabling the construction of hydrogel architectures with tens to hundreds of different domains, presents a significant challenge. We describe a platform for fabricating multi-domain DNA-functionalized hydrogels automatically at the micron scale, where reaction and diffusion processes can be coupled to program material behavior. Using this platform, the hydrogels' material properties, such as shape and fluorescence, can be programmed, and the fabricated hydrogels can sense their environment. DNA-functionalized hydrogel architectures with domain sizes as small as 10 microns and with up to 4 different types of domains can be automatically fabricated using ink volumes as low as 50 µL. We also demonstrate that hydrogels fabricated using this platform exhibit responses similar to those of DNA-functionalized hydrogels fabricated using other methods by demonstrating that DNA sequences can hybridize within them and that they can undergo DNA sequence-induced shape change.

Programming the Sequential Release of DNA.

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.

Digital Maskless Photolithographic Patterning of DNA-Functionalized Poly(ethylene glycol) Diacrylate Hydrogels with Visible Light Enabling Photodirected Release of Oligonucleotides.

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 (Mn = 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.