Pursuing DNA Catalysts for Protein Modification

Abstract

Conspectus Catalysis is a fundamental chemical concept, and many kinds of catalysts have considerable practical value. Developing entirely new catalysts is an exciting challenge. Rational design and screening has provided many new small-molecule catalysts, and directed evolution has been used to optimize or redefine the function of many protein enzymes. However, these approaches have inherent limitations that prompt the pursuit of different kinds of catalysts using other experimental methods. Nature evolved RNA enzymes, or ribozymes, for key catalytic roles that in modern biology are limited to phosphodiester cleavage/ligation and amide bond formation. Artificial DNA enzymes, or deoxyribozymes, have great promise for a broad range of catalytic activities. They can be identified from unbiased (random) sequence populations, as long as the appropriate in vitro selection strategies can be implemented for their identification. Notably, in vitro selection is different in key conceptual and practical ways from rational design, screening, and directed evolution. This Account describes the development by in vitro selection of DNA catalysts for many different kinds of covalent modification reactions of peptide and protein substrates, inspired in part by our earlier work with DNA-catalyzed RNA ligation reactions. In one set of studies, we have sought DNA-catalyzed peptide backbone cleavage, with the long-term goal of artificial DNA-based proteases. We originally anticipated that amide hydrolysis should be readily achieved, but in vitro selection instead led surprisingly to deoxyribozymes for DNA phosphodiester hydrolysis; this was unexpected because uncatalyzed amide bond hydrolysis is 105-fold faster. After developing a suitable selection approach that actively avoids DNA hydrolysis, deoxyribozymes were identified for hydrolysis of esters and aromatic amides (anilides). Aliphatic amide cleavage remains an ongoing focus, including via inclusion in the catalyst of chemically modified DNA nucleotides, which we have recently found to enable this cleavage reaction. In numerous other efforts, we have investigated DNA-catalyzed peptide side chain modification reactions. Key successes include nucleopeptide formation (attachment of oligonucleotides to peptide side chains) and phosphatase and kinase activities (removal and attachment of phosphoryl groups to side chains). Through all of these efforts, we have learned the importance of careful selection design, including the frequent need to develop specific “capture” reactions that enable the selection process to provide only those DNA sequences that have the desired catalytic functions. We have established strategies for identifying deoxyribozymes that accept discrete peptide and protein substrates, and we have obtained data to inform the key choice of random region length at the outset of selection experiments. Finally, we have demonstrated the viability of modular deoxyribozymes that include a small-molecule-binding aptamer domain, although the value of such modularity is found t...

DNA-catalyzed glycosylation using aryl glycoside donors

“…1 DNA catalysts have been found for many chemical reactions, including RNA cleavage by transesterification, RNA ligation, and DNA and RNA hydrolysis. 2 More recently, deoxyribozymes have been identified for creation or removal of many common post-translational modifications (PTMs) of peptides, 3 including phosphorylation, 4 dephosphorylation, 5 and formation of dehydroalanine. 6 Glycosylation is an important PTM for which de novo catalysts that allow site-specific peptide and protein modification will have broad utility.…”

Assessing histidine tags for recruiting deoxyribozymes to catalyze peptide and protein modification reactions

“…Following the approach of Rosen et al, 6 we assayed the 8XJ105 deoxyribozyme with each of three different proteins as potential substrates. His 6 -lysozyme was from US Biological (cat.…”

“…1–5 Our laboratory seeks deoxyribozymes for covalent protein modification. 6 In these efforts, a key challenge is achieving DNA-catalyzed reactions using relatively low protein concentrations that enable preparative utility. We recently described the use of azide-functionalized peptide substrates during in vitro selection, which enabled identification of deoxyribozymes with peptide K m of ~100 μM.…”

Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes

“…Some of this work was recently reviewed [22]; selected highlights are provided here (Figure 3B). Deoxyribozymes have been found for conjugation of oligonucleotides to tyrosine [68–70], tyrosine phosphorylation [71,72], phosphotyrosine (pTyr) and phosphoserine (pSer) dephosphorylation [73], formation of dehydroalanine (Dha) by elimination of phosphate from pSer [74], and lysine modification [75].…”

“…(B) Examples of DNA-catalyzed peptide side chain and backbone modification [22,68–74]. (C) Capture step during in vitro selection for identification of deoxyribozymes that form dehydroalanine (Dha) from phosphoserine (pSer) [74].…”