The incorporation of nucleic acid constructs into hydrogels can result in novel biomaterials with programmable on-demand switches or modulatory mechanisms, with unprecedented control of therapeutic loading and release. Custom oligonucleotides, which were partially complementary to each other and had a programmed recognition site for the restriction endonuclease BamHI, were modified with a polymerizable acrylate functionality and 32P-labeled using T4 RNA ligase. In-vitro hybridization and restriction enzyme digests were performed and confirmed using polyacrylamide gel electrophoresis. Approximately 10-11% of the 32P-labeled oligonucleotide self-annealed. Capture layers in a polyacrylamide gel enabled the highly efficient separation of unincorporated double stranded DNA and unhybridized single strands from the double stranded DNA incorporated into the networks. Quantification via phosphoimaging indicated-70% capture, 25 % unincorporated acrylated DNA, and 5% 32P-labeled oligonucleotide. The release of DNA due to the penetration of restriction enzyme was shown to be highly specific, with no release in the absence of BamHI, and the presence of another endonuclease, EcoRI. As an alternative trigger, temperature was used to release the 32P-labeled oligonucleotide. Temperature responsive release characteristics corresponded to the theoretical melting temperature of the helix (58°C). The physiological significance of these biomaterials was demonstrated by delivering a deoxyribozyme using BamHI in order to cleave a HIV Tat/Rev mRNA. Scission digests due to restriction enzyme or deoxyribozyme treatment were analyzed using electrophoretic mobility shift assays on denaturing polyacrylamide gels. These nucleic acid constructs were incorporated into nanoparticles, were passivated using polyethylene glycol, and are being targeted to specific cells by transferring/galactosamine functionalization on the surface of the nanoparticles.

We have also produced modulatory devices by programming the swelling states of the hydrogels, based on the binding and release of ligands and aptamers selected for them. Ligand-binding RNA pseudoknots were identified using SELEX and affinity chromatography. They were synthesized in vitro using the T7 RNA polymerase and synthetic DNA templates. Transcription of these pseudoknots was controlled by promoters that are known for producing RNA with very high efficiency and yield. Templates and transcripts were examined by agarose and polyacrylamide gel electrophoresis, respectively. Modified nucleotides were co-transcriptionally incorporated into RNA pseudoknots to render them resistant against ribonucleases. A pseudoknot-hairpin-pseudoknot construct was shown to bind and release the ligand by metal ion-chelating agent switching. This new strategy falls under the paradigm of biomimesis, wherein one attempts mimicry of the biological processes, where the molecular recognitive principles are understood, so that they can be exploited and regulated in siRNA-treatment regimes.