Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA

Xiao, Ying; Wehrmann, Rebecca J.; Ibrahim, Nora A.; Silverman, Scott

doi:10.1093/nar/gkr860

Cited by 40 publications

(60 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Between 0.2 and 2 mM Zn 2+ , k obs varied by ~2-fold, with inhibition above 2 mM (Figure 2C), as observed with several other deoxyribozymes and perhaps due to precipitation of Zn(OH) 2 . 8a,15 Assays under multiple-turnover conditions did not provide evidence of turnover by RadDz3 (data not shown).…”

Section: Resultsmentioning

confidence: 94%

“…2 The first report 3 of a deoxyribozyme was for nonhydrolytic RNA cleavage by mediating attack of a 2′-hydroxyl on an adjacent phosphodiester (the ribonuclease mechanism 4 ), and many RNA-cleaving deoxyribozymes were subsequently found. 5 While seeking DNA-catalyzed amide bond hydrolysis, 6 eventually with success, 7 we initially identified deoxyribozymes that hydrolyze specific phosphodiester linkages of single-stranded DNA (ssDNA) substrates 8 and RNA substrates. 9 We and others also found deoxy-ribozymes that deglycosylate DNA, with subsequent elimination reactions that lead to strand scission.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

DNA-Catalyzed DNA Cleavage by a Radical Pathway with Well-Defined Products

et al. 2016

Self Cite

View full text Add to dashboard Cite

We describe an unprecedented DNA-catalyzed DNA cleavage process in which a radical-based reaction pathway cleanly results in excision of most atoms of a specific guanosine nucleoside. Two new deoxyribozymes (DNA enzymes) were identified by in vitro selection from N40 or N100 random pools initially seeking amide bond hydrolysis, although they both cleave simple single-stranded DNA oligonucleotides. Each deoxyribozyme generates both superoxide (O2-• or HOO•) and hydrogen peroxide (H2O2) and leads to the same set of products (3′-phosphoglycolate, 5′-phosphate, and base propenal) as formed by the natural product bleomycin, with product assignments by mass spectrometry and colorimetric assay. We infer the same mechanistic pathway, involving formation of the C4′ radical of the guanosine nucleoside that is subsequently excised. Consistent with a radical pathway, glutathione fully suppresses catalysis. Conversely, adding either superoxide or H2O2 from the outset strongly enhances catalysis. The mechanism of generation and involvement of superoxide and H2O2 by the deoxyribozymes is not yet defined. The deoxyribozymes do not require redox-active metal ions and function with a combination of Zn2+ and Mg2+, although including Mn2+ increases the activity, and Mn2+ alone also supports catalysis. In contrast to all of these observations, unrelated DNA-catalyzed radical DNA cleavage reactions require redox-active metals and lead to mixtures of products. This study reports an intriguing example of a well-defined, DNA-catalyzed, radical reaction process that cleaves single-stranded DNA and requires only redox-inactive metal ions.

show abstract

Section: Resultsmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

DNA-Catalyzed DNA Cleavage by a Radical Pathway with Well-Defined Products

et al. 2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…For all of these applications, the most general substrate sequence scope is desired, either from an individual DNA enzyme or collectively from a set of DNA enzymes. Such generality has been achieved with RNA-cleaving deoxyribozymes 49–51 and RNA ligase deoxyribozymes 52,53 and seems likely for DNA-hydrolyzing deoxyribozymes as well, 45,54 suggesting the viability of general DNA-catalyzed oligonucleotide 3′-phosphorylation.…”

Section: Discussionmentioning

confidence: 92%

DNA Oligonucleotide 3′-Phosphorylation by a DNA Enzyme

et al. 2016

Self Cite

View full text Add to dashboard Cite

T4 polynucleotide kinase is widely used for 5′-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3′-phosphorylation. Here, we report the in vitro selection of deoxyribozymes (DNA enzymes) capable of DNA oligonucleotide 3′-phosphorylation, using a 5′-triphosphorylated RNA transcript (pppRNA) as the phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3′-phosphorylated DNA substrate terminus by 2-methylimidazole activation of the 3′-phosphate (forming 3′-MeImp) and subsequent splint ligation with a 5′-amino DNA oligonucleotide. Competing and precedented DNA-catalyzed reactions were DNA phosphodiester hydrolysis or deglycosylation, each also leading to a 3′-phosphate but at a different nucleotide position within the DNA substrate. One oligonucleotide 3′-kinase deoxyribozyme, obtained from an N40 random pool and named 3′Kin1, can 3′-phosphorylate nearly any DNA oligonucleotide substrate for which the 3′-terminus has the sequence motif 5′-NKR-3′, where N denotes any oligonucleotide sequence, K = T or G, and R = A or G. These results establish the viabilty of in vitro selection for identifying DNA enzymes that 3′-phosphorylate DNA oligonucleotides.

show abstract

“…A growing range of chemical reactions has been shown to be catalyzed by DNA (4)(5)(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.…”

mentioning

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

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

Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA

Cited by 40 publications

References 41 publications

DNA-Catalyzed DNA Cleavage by a Radical Pathway with Well-Defined Products

DNA-Catalyzed DNA Cleavage by a Radical Pathway with Well-Defined Products

DNA Oligonucleotide 3′-Phosphorylation by a DNA Enzyme

Catalytic DNA with phosphatase activity

Contact Info

Product

Resources

About