Pursuing DNA catalysts for protein modification

Abstract

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 have 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 surprisingly led to deoxyribozymes for DNA phosphodiester hydrolysis; this was unexpected because uncatalyzed amide bond hydrolysis is 10(5)-fold faster. After developing a suitable selection approach that actively avoids DNA hydrolysis, we were able to identify deoxyribozymes for hydrolysis of esters and aromatic amides (anilides). Aliphatic amide cleavage remains an ongoing focus, including via inclusion of chemically modified DNA nucleotides in the catalyst, 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 to be minimal, with implications for many selection endeavors. Advances such as those summarized in this Account reveal that DNA has considerable catalytic abilities for biochemically relevant reactions, specifically including covalent protein modifications. Moreover, DNA has substantially different, and in many ways better, characteristics than do small molecules or proteins for a catalyst that is obtained "from scratch" without demanding any existing information on catalyst structure or mechanism. Therefore, prospects are very strong for continued development and eventual practical applications of deoxyribozymes for peptide and protein modification.

Figure 1

RNA cleavage and ligation reactions. (A) RNA cleavage by transesterification. (B) RNA ligation, illustrated with 3′-hydroxy + 5′-triphosphate. The reverse of the RNA cleavage reaction in panel A is also possible.

Figure 2

Uncatalyzed half-lives of individual linkages in RNA, protein, and DNA (at near-neutral pH, near-ambient temperature, in the absence of divalent metal ions).

Figure 3

Initial foray toward DNA-catalyzed peptide bond cleavage, and unexpected identification of DNA catalysts for DNA phosphodiester hydrolysis. (A) Selection design with tripeptide substrate embedded between two DNA binding arms. (B) Selection outcome of DNA-catalyzed DNA phosphodiester hydrolysis. Each deoxyribozyme catalyzes hydrolysis specifically at one of the marked phosphodiester bonds.

Figure 4

DNA can catalyze an otherwise-disfavored reaction, RNA cleavage by hydrolysis rather than transesterification. When uncatalyzed, transesterification is favored by >10^6-fold.

Figure 5

DNA-catalyzed ester and amide cleavage. (A) Capture reaction for the carboxylic acid product of ester and amide hydrolysis. (B) Outcome of selection. With an aromatic amide (anilide) substrate, we observed DNA-catalyzed hydrolysis of the anilide but not the aliphatic amide.

Figure 6

Structures of chemically modified nucleotides for enhancing DNA catalyst function.

Figure 7

DNA-catalyzed nucleopeptide formation. (A) Reaction between tyrosine and RNA to form a nucleopeptide. (B) Two schematic depictions of the 3HJ architecture that juxtaposes nucleophile (peptide side chain) and electrophile (RNA 5′-triphosphate).

Figure 8

Nucleopeptide formation with a discrete peptide substrate. (A) Tethered peptide substrate, and selection arrangement. Various tether compositions and lengths were used; a short tether is illustrated. (B) Selection with discrete amino-peptide and reductive amination capture; outcome of nucleobase reductive amination. (C) Selection with discrete azido-peptide and CuAAC capture; outcome of nucleopeptide formation.

Figure 9

DNA-catalyzed peptide dephosphorylation (phosphatase activity). (A) Selection strategy, using a capture deoxyribozyme that is highly selective for Tyr over pTyr. (B) Multiple-turnover peptide dephosphorylation by the Zn²⁺ -dependent 14WM9 deoxyribozyme (500 μM peptide, 100 μM DNA).

Figure 10

DNA-catalyzed peptide phosphorylation (kinase activity). (A) Selection strategy. 5′-Triphosphorylated RNA is shown as the phosphoryl donor; in separate selections, GTP or ATP can be provided instead. (B) GTP concentration dependence of the 8EA101 (N₃₀) and 16EC103 (N₅₀) deoxyribozymes.

Figure 11

DNA-catalyzed lysine reactivity, using a reactive 5′-phosphorimidazolide (Imp) as the electrophile.

Figure 12

Selection design for identifying modular deoxyribozymes that integrate a distinct small-molecule binding site (aptamer) near an initially random catalytic region.