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 polynucleotide kinase. In-vitro hybridization and restriction enzyme digests were optimized in various concentrations of Tris buffer and water, and confirmed using polyacrylamide gel electrophoresis. Magnesium was determined to be critical for hybridization of the construct. About 10% of the complementary oligonucleotide self-annealed. The functional monomers used for hydrogel synthesis had no effect on hybridization efficiency. Digestion of the DNA helices was carried out by incubating the DNA helices with restriction enzyme BamHI and BamHI buffer. Incubation of the DNA double helix (without BamHI) on ice and at 37°C had no effect on the duplex. BamHI buffer did not disrupt the annealed duplex. Poly(acrylamide–co-N,N' methylene bisacrylamide-co-acrylated DNA) hydrogels were synthesized via redox polymerization and the unincorporated DNA was eluted by electrophoresis. The labeled complementary oligonucleotide, when polymerized into the hydrogel without the anchoring acrylated oligonucleotide, eluted readily from the hydrogel during electrophoresis whereas acrylated DNA duplex was readily incorporated into the network. Quantification of the polymerization process via electrophoresis and phosphoimaging indicated 70% capture of the acrylated DNA, 25 % unincorporated acrylated DNA, and 5% 32P-labeled oligonucleotide. A labeled oligonucleotide bearing a non-complementary sequence to the acrylated oligonucleotide did not get incorporated as annealing did not occur between the two strands. Release studies of 32P-labeled loaded DNA were conducted by incubating the DNA-loaded gels under physiological conditions in the presence of BamHI. 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. Temperature was used to release the 32P-labeled oligonucleotide as an alternative physical trigger. Temperature responsive release characteristics corresponded to the theoretical melting temperature of the helix (58°C). Poly(2-hydroxyethyl methacrylate-co-polyethylene glycol 600 dimethacrylate-co-acrylated DNA) hydrogels of varying crosslinking densities were triggered to release DNA by the non-specific endonuclease DNase I. Release rate of DNA from biocompatible hydrogels varied inversely with the crosslinking densities, and consequently mesh size.
The physiological significance of this platform was demonstrated by delivering a deoxyribozyme, which bore a catalytic 10-23 motif and was specific to a HIV Tat/Rev mRNA. The HIV-1 Tat/Rev RNA was synthesized by in vitro transcription and labeled using [5'-32P]pCp and T4 RNA ligase. Incubation of the deoxyribozyme-loaded hydrogel and the HIV-1 Tat/Rev RNA resulted in down regulation of the gene. BamHI action on a hydrogel lacking the deoxyribozyme construct had no effect on the HIV-1 Tat/Rev RNA.
Recent work is focused on modulation of nucleic acid aptamer structures with programable therapeutic affinity isolated by affinity chromatography, their conjugation to gold nanoparticles, optimization of therapeutic payload and surface coverage, and trigger efficiency. Such novel schemes have strong potential for the intracellular delivery of multiple therapeutics from the same platform, as injectable systems targeted to specific cells using functionalized ligands. These studies were performed using a model therapeutic, fluorescent neomycin. Neomycin molecules were conjugated to gold nanoparticles via an unstructured thiolated oligonucleotide hybridized to the neomycin aptamer. An affinity spectrum of neomycin aptamers was rationally created by introducing point mutations which modulated tertiary structure and hence affected neomycin binding. These mutant neomycin binding aptamers are being conjugated to gold nanoparticles in order to obtain a graded release profile.