Covalent Tagging of Phosphorylated Peptides by Phosphate‐Specific Deoxyribozymes

Sachdeva, Amit; Chandra, Madhavaiah; Chandrasekar, Jagadeeswaran; Silverman, Scott

doi:10.1002/cbic.201200048

Cited by 13 publications

(13 citation statements)

References 35 publications

(20 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…After each key selection step that used incubation conditions of 70 mM Hepes (pH 7.5), 1 mM ZnCl 2 , 20 mM MnCl 2 , 40 mM MgCl 2 , and 150 mM NaCl at 37°C for 14 h, active DNA catalyst sequences were "captured" with the aid of the 15MZ36 deoxyribozyme that selectively uses dephosphorylated tyrosine (Y OH ) as the nucleophile to attack 5′-triphosphorylated RNA, resulting in a PAGE shift for the corresponding deoxyribozyme sequence ( Fig. S2) (17). The active DNA sequences were amplified by PCR and joined by T4 DNA ligase to the DNA-anchored hexapeptide substrate for entering the next selection round.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Catalytic DNA with phosphatase activity

Chandrasekar

Silverman

2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Catalytic DNA sequences (deoxyribozymes, DNA enzymes, or DNAzymes) have been identified by in vitro selection for various catalytic activities. Expanding the limits of DNA catalysis is an important fundamental objective and may facilitate practical utility of catalysts that can be obtained from entirely unbiased (random) sequence populations. In this study, we show that DNA can catalyze Zn 2+ -dependent phosphomonoester hydrolysis of tyrosine and serine side chains (i.e., exhibit phosphatase activity). The best deoxyribozyme decreases the half-life for phosphoserine hydrolysis from as high as >10 10 y to <1 h. The phosphatase activity also occurs with nonpeptidic substrates but with reduced efficiency, indicating a preference for phosphopeptides. The newly identified deoxyribozymes can function with multiple turnover using free peptide substrates, have activity in the presence of human cell lysate or BSA, and catalyze dephosphorylation of a larger protein substrate, suggesting broader application of DNA catalysts as artificial phosphatases. D evelopment of catalysts is a major impetus for much of modern chemical research. Nature's biomolecular protein and RNA catalysts are responsible for a wide range of chemical reactions, and protein enzymes in particular can achieve large rate enhancements (1, 2). Although DNA catalysts are unknown in nature, in vitro selection [first pioneered for RNA (3)] is readily applied to identify catalytically active artificial DNA sequences (4-6). Importantly, DNA (and RNA) catalysts can be identified by starting with entirely random sequence pools, whereas directed evolution of proteins typically requires a known, catalytically active starting point (7,8). A growing range of chemical reactions has been shown to be catalyzed by DNA (4-6). For DNA phosphodiester hydrolysis, the uncatalyzed (spontaneous) half-life for P-O bond cleavage of ∼30 million y is reduced to as little as 0.5 min by a DNA catalyst (9, 10). However, in this reaction the DNA catalyst interacts with its DNA substrate by extensive Watson-Crick base pairing, and such an approach cannot be generalized to nonoligonucleotide substrates such as peptides and proteins. With the exception of the ribosome, the natural ribozymes catalyze RNA cleavage and ligation, and they generally have more modest rate enhancements (11-13), limited by the relatively high uncatalyzed half-lives of these reactions.DNA catalysts that covalently modify peptide and protein substrates are fundamentally interesting and likely have practical value, especially for biologically relevant chemical modifications.We have initiated studies into DNA-catalyzed modifications of amino acid side chains of peptide substrates, such as nucleopeptide linkage formation involving tyrosine and serine (14-16). A major challenge in such studies is to achieve catalysis even though the DNA catalyst cannot engage in any preprogrammable WatsonCrick binding interactions with the substrate. In this report, we show that DNA can catalyze Zn 2+ -dependent hydrolysis of tyrosine...

show abstract

Section: Resultsmentioning

confidence: 99%

“…The selection procedure, cloning, and initial analysis of individual clones were performed essentially as described previously (36,38), but with a different ligation step as recently reported (17). An overview of the key selection and capture steps of each round is shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

Catalytic DNA with phosphatase activity

Chandrasekar

Silverman

2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…45 (A) Selection strategy, using a capture deoxyribozyme that is highly selective for Tyr over pTyr. (B) Multiple-turnover peptide dephosphorylation by the Zn 2+ -dependent 14WM9 deoxyribozyme (500 μM peptide, 100 μM DNA).…”

Section: Figurementioning

confidence: 99%

Pursuing DNA Catalysts for Protein Modification

Silverman

2015

Acc. Chem. Res.

Self Cite

View full text Add to dashboard Cite

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...

show abstract

“…30,31 Second, we examined DNA-catalyzed nucleopeptide linkage formation, which we have identified as part of our larger efforts that seek DNA-catalyzed covalent modification of amino acid side chains. 32–37 For both types of catalytic activity, our previous efforts used only N 40 random regions, where 4 40 ≈ 10 24 and therefore 10 14 /10 24 = 10 −10 of sequence space was explored. Here, for both kinds of catalysis we examined N 20 through N 60 pools in new, independent selection experiments.…”

Section: Introductionmentioning

confidence: 99%

Systematic Evaluation of the Dependence of Deoxyribozyme Catalysis on Random Region Length

et al. 2012

Self Cite

View full text Add to dashboard Cite

Functional nucleic acids are DNA and RNA aptamers that bind targets, or they are deoxyribozymes and ribozymes that have catalytic activity. These functional DNA and RNA sequences can be identified from random-sequence pools by in vitro selection, which requires choosing the length of the random region. Shorter random regions allow more complete coverage of sequence space but may not permit the structural complexity necessary for binding or catalysis. In contrast, longer random regions are sampled incompletely but may allow adoption of more complicated structures that enable function. In this study, we systematically examined random region length (N20 through N60) for two particular deoxyribozyme catalytic activities, DNA cleavage and tyrosine-RNA nucleopeptide linkage formation. For both activities, we previously identified deoxyribozymes using only N40 regions. In the case of DNA cleavage, here we found that shorter N20 and N30 regions allowed robust catalytic function, either by DNA hydrolysis or by DNA deglycosylation and strand scission via β-elimination, whereas longer N50 and N60 regions did not lead to catalytically active DNA sequences. Follow-up selections with N20, N30, and N40 regions revealed an interesting interplay of metal ion cofactors and random region length. Separately, for Tyr-RNA linkage formation, N30 and N60 regions provided catalytically active sequences, whereas N20 was unsuccessful, and the N40 deoxyribozymes were functionally superior (in terms of rate and yield) to N30 and N60. Collectively, the results indicate that with future in vitro selection experiments for DNA and RNA catalysts, and by extension for aptamers, random region length should be an important experimental variable.

show abstract

Covalent Tagging of Phosphorylated Peptides by Phosphate‐Specific Deoxyribozymes

Cited by 13 publications

References 35 publications

Catalytic DNA with phosphatase activity

Catalytic DNA with phosphatase activity

Pursuing DNA Catalysts for Protein Modification

Systematic Evaluation of the Dependence of Deoxyribozyme Catalysis on Random Region Length

Contact Info

Product

Resources

About