Discovery of DNA-enzymes dependent on small-molecule cofactors; design, synthesis and evaluation of TLR-7 agonists and their immunoprotein conjugates.
Access changed 3/18/13.
Several catalytic families of DNA enzymes (deoxyribozymes) have previously been isolated through the use of an in vitro selection technique in our laboratory. The activities of these deoxyribozymes appear to be dependent upon the presence of a small organic cofactors. To further characterize these catalytic oligonucleotides, the organic cofactors were re-synthesized and background cleavage experiments were performed in the absence of the enzymatic sequence. Several studies were performed to improve the background cleavage experiment by reacting several variants of the substrate under different conditions. Additionally, enzymatic sequences from the literature were also examined to test the accuracy of our methodology. Results from the background cleavage experiments showed that in the absence of a DNA enzyme, cleavage of the oligonucleotide substrate is almost negligible. In an attempt to isolate new families of deoxyribozymes, an in vitro selection was performed using two concentrations of a new synthetic cofactor. Unfortunately, after eleven rounds of selection, the catalytic activity of the oligonucleotide pool did not improved substantially. In a second project, several adenine derivatives were synthesized as toll-like receptor 7 agonists. These molecules were functionalized for their conjugation to a protein through the use of a heterobifunctional protein linker. The synthesis of these toll-like receptor agonists required a multi-step synthetic route. In vitro studies evaluation of some of these compounds showed excellent potency at inducing cytokine production. An antibody conjugate of one compound, targeted to an internalizing dendritic cell receptor, demonstrated exceptional activity that exceeded the free agonist or the agonist targeted to a non-relevant receptor.