Chemists at the University of Illinois, Urbana-Champaign, have identified a deoxyribozyme that catalyzes amide hydrolysis, the first step toward making a DNA-based enzyme that can cleave proteins in specific places. Such a DNA enzyme could help researchers process proteins for proteomic studies.
Scott K. Silverman and his team have been trying unsuccessfully to find an amide-hydrolyzing deoxyribozyme for years. Their first attempt yielded a DNA that performed phosphodiester cleavage instead. “There’s something about an amide bond that is difficult to cleave—at least by a nucleic acid catalyst,” Silverman says.
Now, Silverman’s team has managed to produce DNA sequences that cleave amide bonds by incorporating modified nucleotides with amine, hydroxyl, or carboxyl groups (J. Am. Chem. Soc. 2016, DOI: 10.1021/jacs.5b12647). The modifications mimic the side chains on amino acids that are involved in peptide bond cleavage by natural proteases.
“There’s a mechanistic basis for expecting some benefit from including these side chains on the nucleobases,” Silverman says. But because these modified bases are still DNA, the researchers could use tools to amplify the nucleic acids, as well as methods developed for selecting and evolving DNAs for specific functions.
Through these selection methods, Silverman’s team screened a library of catalysts consisting of 40-nucleotide-long random sequences flanked by regions with fixed sequences that could bind a model substrate. The substrate comprised an amide bond inserted between DNA sequences. They found multiple catalysts that enhanced the rate of amide hydrolysis by about 1,000-fold.
“It’s not a blazing catalyst,” says Gerald F. Joyce, whose group studies functional nucleic acids at Scripps Research Institute, California. “But it has a nice acceleration compared to the uncatalyzed reaction.”
In addition, one of the catalysts with hydroxyl-modified bases retained some activity even when resynthesized with unmodified DNA. “So the story comes full circle, and it seems that unmodified DNA can do this reaction after all,” Joyce says.