Nucleobase-modified deoxyribozymes for amide and peptide hydrolysis

Nature has evolved proteins and RNA as enzymes. These biopolymers can fold into complex structures to enable catalysis. DNA is primarily double-stranded and is non-catalytic in nature. However, considering the structural similarity to RNA, single-stranded DNA should also be able to form complex structures and perform catalysis. In fact, artificial DNA enzymes (deoxyribozymes) have been identified in laboratories by in vitro selection. The identification of new enzymes favors the use of nucleic acids over proteins for several reasons. First, nucleic acids can be amplified by natural enzymes, whereas proteins cannot be amplified in any way. Second, the total number of possible sequences is smaller for nucleic acids than for proteins. Therefore, with a practical amount of material, selection experiments for nucleic acid enzymes can cover a larger fraction of total sequence space. Furthermore, nucleic acid sequences can easily fold into secondary and tertiary structures, whereas most random sequences of proteins will misfold and aggregate. Between the two types of nucleic acid, DNA has several practical advantages over RNA. DNA can be directly amplified by polymerases whereas RNA requires an extra reverse transcription step. DNA is also easier to synthesize and more stable compared to RNA. Proteases, which catalyze cleavage of proteins, are essential enzymes in nature. Natural and engineered proteases are tremendously useful for various academic, therapeutic, and industrial applications. Engineering of natural proteases for novel cleavage sites is an exciting prospect, but this process usually leads to a relaxed, rather than truly altered, substrate specificity. Because deoxyribozymes are identified from pools of random DNA sequences, no inherent peptide sequence biases must be overcome during the selection process, and thus the prospect of truly selective artificial proteases is reasonable. Previous efforts seeking DNA-catalyzed peptide cleavage resulted in the identification of deoxyribozymes that cleave a DNA phosphodiester bond. Subsequent selection experiments for DNA-catalyzed ester and amide bond hydrolysis employed an additional capture step to avoid identifying deoxyribozymes for phosphodiester cleavage, leading to the identification of deoxyribozymes for ester hydrolysis and aromatic amide hydrolysis. However, no deoxyribozymes for aliphatic amide hydrolysis were identified. Chapter 2 describes the identification of nucleobase-modified deoxyribozymes for amide bond hydrolysis. By introducing protein-like functional groups in the nucleobases of deoxyuridines in DNA, amino, hydroxyl, and carboxyl modified deoxyribozymes were identified to catalyze the hydrolysis of a simple amide bond embedded between two DNA anchors. Several efforts to identify modified deoxyribozymes for peptide hydrolysis are described in Chapter 3. In the initial effort, no deoxyribozymes were identified using the same strategy as described in Chapter 2. Subsequent selection experiments were performed to include two types of functional group, among amino, imidazolyl, hydroxyl, carboxyl, benzyl, and thiol, with the anticipation that some combination would function in synergy to enable the catalysis, similar to what natural proteases do in the active sites. Unfortunately, no peptide hydrolysis activity was observed in these selections. One hypothesis is that DNA-catalyzed peptide hydrolysis might require strong substrate binding by the DNA enzyme. Therefore, additional selection experiments were designed, focusing on exploring hydrophobic modifications along with protein-like functional groups. This strategy is based on considerable evidence in DNA aptamer studies that incorporating hydrophobic modifications into DNA can increase the interaction between DNA and protein. Such strong interactions between DNA and the peptide substrate may enable the DNA-catalyzed peptide hydrolysis. These selections are still in progress. In the selections for identifying deoxyribozymes that catalyze amide and peptide hydrolysis, DNA-catalyzed radical-based oxidative DNA cleavage was identified. Surprisingly, no redox active metal ions were required for catalysis. The discovery of the DNA-catalyzed oxidative DNA cleavage indicated the general ability of DNA to catalyze redox reactions