Solid-phase oligonucleotide synthesis (SPOS) based on phosphoramidite chemistry is currently the most widespread technique for DNA and RNA synthesis, but suffers from scalability limitations and high reagent consumption. Liquid-phase oligonucleotide synthesis (LPOS) uses soluble polymer supports and has the potential of being scalable. However, at present, LPOS requires 3 separate reaction steps and 4-5 precipitation steps per nucleotide addition. Moreover, long acid exposure times during the deprotection step degrade sequences with high A-content (adenine) due to depurination and chain cleavage. In this work, we present the first one-pot liquid-phase DNA synthesis technique, which allows the addition of one nucleotide in a one-pot reaction of sequential coupling, oxidation and deprotection, followed by a single precipitation step. Furthermore, we demonstrate how to suppress depurination during the addition of adenine nucleotides. We showcase the potential of this technique to prepare high-purity 4-arm PEG-T 20 (T = thymine) and 4-arm PEG-A 20 building blocks in multi-gram scale. Such complementary 4-arm PEG-DNA building blocks reversibly self-assemble into supramolecular model network hydrogels, and facilitate the elucidation of bond lifetimes. These model network hydrogels exhibit new levels of mechanical properties, high stability at room temperature (melting at 44 °C), and thus open up pathways to next-generation, scalable DNA-materials programmable through sequence recognition and available for macroscale applications. File list (2) download file view on ChemRxiv MS final.pdf (7.23 MiB) download file view on ChemRxiv ESI final.pdf (12.53 MiB)
The use of DNA as a building block in synthetic polymer hydrogels promises high levels of programmability regarding sol/gel temperatures, tunable bond lifetimes, biocompatibility, and interaction with biological components (e.g., enzymes, cells, and growth factors). However, scalability and quantitative structure−property relationships for large-scale materials are still challenging to achieve. Building on our recently introduced and scalable one-pot liquid-phase oligonucleotide synthesis of DNA onto star-shaped poly(ethylene glycol) (PEG), we here report hydrogels based on starPEG-DNA conjugates together with divalent DNA linkers of tunable duplex hybridization length. By systematically varying parameters such as the duplex melting temperature, salinity, and building block concentrations, we establish the mechanical phase space of such hydrogels. We elucidate tunable mechanical properties ranging from a few Pa to the kPa regime and discuss time scales of self-healing and bond exchange, as well as tunable sol/gel transition temperatures. These comprehensive investigations shed some light on the future design principles for DNA hydrogel materials based on scalable building blocks, that allow for the formation of quasi-ideal networks due to their starshaped and flexible building block topologies. Such materials can be useful in the field of biomedicine and cell culture.
Solid-phase oligonucleotide synthesis (SPOS) based on phosphoramidite chemistry is currently the most widespread technique for DNA and RNA synthesis, but suffers from scalability limitations and high reagent consumption. Liquid-phase oligonucleotide synthesis (LPOS) uses soluble polymer supports and has the potential of being scalable. However, at present, LPOS requires 3 separate reaction steps and 4-5 precipitation steps per nucleotide addition. Moreover, long acid exposure times during the deprotection step degrade sequences with high A-content (adenine) due to depurination and chain cleavage. In this work, we present the first one-pot liquid-phase DNA synthesis technique, which allows the addition of one nucleotide in a one-pot reaction of sequential coupling, oxidation and deprotection, followed by a single precipitation step. Furthermore, we demonstrate how to suppress depurination during the addition of adenine nucleotides. We showcase the potential of this technique to prepare high-purity 4-arm PEG‑T<sub>20</sub> (T = thymine) and 4-arm PEG-A<sub>20</sub>building blocks in multi-gram scale. Such complementary 4-arm PEG-DNA building blocks reversibly self-assemble into supramolecular model network hydrogels, and facilitate the elucidation of bond lifetimes. These model network hydrogels exhibit new levels of mechanical properties, high stability at room temperature (melting at 44 °C), and thus open up pathways to next-generation, scalable DNA-materials programmable through sequence recognition and available for macroscale applications<i>.</i>
Liquid-liquid phase separation provides a versatile approach to fabricating cell-mimicking coacervates. Recently, it was discovered that phase separation of single-stranded DNA (ssDNA) allows for forming protocells and microgels in multicomponent systems. However, the mechanism of the ssDNA phase separation is not comprehensively understood. Here, we present mechanistic insights into the metal-dependent phase separation of ssDNA and leverage this understanding for a straightforward formation of all-DNA droplets. Two phase separation temperatures are found that correspond to the formation of primary nuclei and a growth process. Ca 2 + allows for irreversible, whereas Mg 2 + leads to reversible phase separation. Capitalizing on these differences makes it possible to control the information transfer of one-component DNA droplets and two-component core-shell protocells. This study introduces new kinetic traps of phase separating ssDNA that lead to new phenomena in cell-mimicking systems.
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