Phosphoester-transfer mechanism of an RNA-cleaving acidic deoxyribozyme revealed by radioactivity tracking and enzymatic digestion

Kandadai, Srinivas A.; Chiuman, William; Li, Yingfu

doi:10.1039/b604682g

Cited by 12 publications

(12 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…From these analyses, we conclude that the cleavage of S3 by ETB.2 produces a 5′-cleavage fragment with a 2′,3′-cyclic phosphate and a 3′-cleavage fragment with a 5′-OH. It is interesting to note that the same products are found for all naturally occurring self-cleaving ribozymes and all man-made RNA-cleaving nucleic acid enzymes (47,52).…”

Section: Characterization Of Cleavage Productsmentioning

confidence: 80%

“…We next examined whether the terminal phosphate of P1 was in the form of a 2′,3′-cyclic phosphate or a 2′-and (or) 3′-monophosphate through treatment of three protein enzymes: T4 polynucleotide kinase (T4 PNK, which has both 2′,3′-cyclic nucleotide 3′-phosphodiesterase activity and 2′-and 3′-phosphatase activity), calf intestine alkaline phosphatase (CIAP, which has 2′-and 3′-phosphatase activity but does not have 2′,3′-cyclic phosphodiesterase activity), and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase, which has the ability to hydrolyze 2′,3′-cyclic phosphate solely to 2′-phosphate) (52). When the ETB.2-S3 reaction mixture (Fig.…”

Section: Characterization Of Cleavage Productsmentioning

confidence: 99%

“…To characterize the cleavage products produced by pH3DZ1, we followed a previously published protocol that utilizes a specially labeled substrate containing 32 P-labeled phosphodiester linkage at the cleavage site that permits the tracing of the transfer of the phosphorous atom from the substrate to the cleavage products (52). The substrate (denoted S3) was produced by ligating a matching 5′-32 Plabeled oligonucleotide to a relevant 3′-ribo-terminated Fig.…”

Section: Characterization Of Cleavage Productsmentioning

confidence: 99%

See 2 more Smart Citations

Characterization of pH3DZ1 — An RNA-cleaving deoxyribozyme with optimal activity at pH 3

Ali¹,

Kandadai²,

Li³

2007

Can. J. Chem.

Self Cite

View full text Add to dashboard Cite

We previously described a cis-acting RNA-cleaving deoxyribozyme known as pH3DZ1 that exhibits optimal catalytic activity at pH 3.0 (. Am. Chem. Soc. 125, 7539 (2003)). This DNA catalyst was made of a 99-nucleotide (nt) catalytic domain covalently linked to a 23-nt DNA-RNA chimeric substrate containing a single ribonucleotide as the cleavage site. In the present work, we conducted an extensive sequence examination of this deoxyribozyme via nucleotide truncation and reselection experiments, with a goal to minimize its size and identify the nucleotides that are crucial to its catalytic function. A trans-acting deoxyribozyme that can process an external substrate was also successfully designed. Stretches of 30 and 17 nucleotides from the 5′ and 3′ ends of the trans catalyst, respectively, were found to be completely dispensable; in contrast, few nucleotides could be deleted internally without producing a detrimental effect. The reselection experiment led to the discovery of 7 and 5 absolutely conserved nucleotides located at the 5′ and 3′ ends of the minimized catalyst, respectively, separated by a 31-nt element in which 14 highly conserved nucleotides were scattered among 17 variable nucleotides. The shortened deoxyribozyme and the original catalyst showed a similar pH profile with the optimal activity at pH 3; however, the minimized deoxyribozyme still exhibited strong catalytic activity at pH 2.5, while the fulllength catalyst was barely active at this pH. Finally, it was found that this deoxyribozyme generated two cleavage fragments, one with 2′,3′-cyclic phosphate and the other with 5′-OH. a décrit un désoxyribozyme connu sous le nom de code pH3DZ1, qui est capable de cliver l'ARN, qui agit d'une façon cis et dont l'activité catalytique est optimale à un pH de 3,0; ce catalyseur d'ADN a été fait en liant le domaine catalytique 99-nucléotide (nt) à un substrat chimérique 23-nt ADN-ARN ne contenant qu'un seul ribonucléotide comme site de clivage. Dans le travail présenté maintenant, on a fait appel à des expériences de tronquage et de resélection pour effectuer un examen extensif de la séquence de ce désoxyribozyme dans le but de minimiser sa taille et d'identifier les nucléotides qui sont cruciaux à son fonctionnement catalytique. On a aussi déve-loppé avec succès un désoxyribozyme agissant de façon trans qui peut traiter un substrat extérieur. On a identifié des blocs respectivement de 30 et de 17 nucléotides des extrémités 5′ et 3′ du catalyseur trans dont on peut se dispenser complètement; par opposition à cette situation, il n'y a que peu de nucléotides internes qui peuvent être éliminés sans produire des effets néfastes. L'expérience de resélection a permis de découvrir des blocs de 7 et 5 nucléotides absolument nécessaires qui sont situés respectivement aux extrémités 5′ et 3′ du catalyseur minimisé et qui sont séparés par un élément de 31-nt dans lequel 14 des nucléotides à conserver sont éparpillés parmi les 17 nucléotides variables. Le désoxyribozyme raccourci et le catalyseur original présentent tous l...

show abstract

Section: Characterization Of Cleavage Productsmentioning

confidence: 80%

Section: Characterization Of Cleavage Productsmentioning

confidence: 99%

Section: Characterization Of Cleavage Productsmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of pH3DZ1 — An RNA-cleaving deoxyribozyme with optimal activity at pH 3

Ali¹,

Kandadai²,

Li³

2007

Can. J. Chem.

Self Cite

View full text Add to dashboard Cite

show abstract

“…28,[41][42][43] We sought to characterize the cleavage product of CT10.3.29M4 by exploiting the enzymatic properties of T4 polynucleotide kinase (PNK), calf intestine alkaline phosphatase (CIAP), and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) using a method previously published by our group. 44 Figure 5 shows the result of combinatory treatments with PNK, CNPase, and CIAP. "Uncleaved" refers to the uncleaved substrate that was internally labeled with 32 P at the cleavage site, and "cleaved" denotes the expected 5′ cleavage fragment.…”

Section: Secondary Structure Confirmation By Mutagenesismentioning

confidence: 99%

A Complex RNA-Cleaving DNAzyme That Can Efficiently Cleave a Pyrimidine–Pyrimidine Junction

Lam

Withers

2010

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

“…T4 polynucleotide kinase (PNK) cleaves the 2',3'-cyclic phosphate. [16] Angewandte Chemie Because of the fluorophore and quencher attached to the two T residues flanking the cleavage site in pH6-ET4 (F and Q in Figure 3 a), the DNAzyme could also report target binding through the generation of a fluorescence signal. The activity of pH6-ET4 was confirmed to be dependent on the presence of ATP, as revealed by PAGE (polyacrylamide gel electrophoresis; Figure 3 a): A much larger quantity of P1 (which is fluorescent and can be detected by fluorimaging) was produced in the mixture containing 1 mm ATP than in the mixture containing no ATP or 1 mm guanosine triphosphate (GTP, which has no affinity for the DNA aptamer).…”

Section: Monsur Ali and Yingfu Li*mentioning

confidence: 99%

Colorimetric Sensing by Using Allosteric‐DNAzyme‐Coupled Rolling Circle Amplification and a Peptide Nucleic Acid–Organic Dye Probe

Ali

2009

Angew Chem Int Ed

Self Cite

133

View full text Add to dashboard Cite

Target detection by the naked eye: The action of an RNA-cleaving allosteric DNAzyme in response to ligand binding was coupled to a rolling circle amplification process to generate long single-stranded DNA molecules for colorimetric sensing (see scheme). Upon hybridization of the resulting DNA with a complementary PNA sequence in the presence of a duplex-binding dye, the color of the dye changed from blue to purple.

show abstract

Phosphoester-transfer mechanism of an RNA-cleaving acidic deoxyribozyme revealed by radioactivity tracking and enzymatic digestion

Cited by 12 publications

References 34 publications

Characterization of pH3DZ1 — An RNA-cleaving deoxyribozyme with optimal activity at pH 3

Characterization of pH3DZ1 — An RNA-cleaving deoxyribozyme with optimal activity at pH 3

A Complex RNA-Cleaving DNAzyme That Can Efficiently Cleave a Pyrimidine–Pyrimidine Junction

Colorimetric Sensing by Using Allosteric‐DNAzyme‐Coupled Rolling Circle Amplification and a Peptide Nucleic Acid–Organic Dye Probe

Contact Info

Product

Resources

About