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

Abstract

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...

Characterization of Highly Efficient RNA-Cleaving DNAzymes that Function at Acidic pH with No Divalent Metal-Ion Cofactors

“…Further extensive studies have led to the construction of trans ‐acting DNAzymes active at various pH values in the range of 3.0 to 6.0. Note that the selected DNAzymes exhibited broad metal‐ion specificities . When considering the possible mechanisms of catalysis performed by ribozymes or DNAzymes at low pH values, it should be taken into account that under such conditions metal ions might be totally redundant.…”

The Optimization and Characterization of an RNA-Cleaving Fluorogenic DNAzyme Probe for MDA-MB-231 Cell Detection

“…When the target was introduced, the catalytic ability of the probe was activated, followed by the cleaving of the substrate, increasing the distance between DABCYL-dT and fluorescein-dT and increasing the fluorescence signal (Scheme 1). It was found that the cleavage of the substrates could generate a nearly six-fold increase in fluorescence in the PAGE-based experiment [27,29,34,38,39,40,41]. We calculated the percentage of cleavage using the formula [34] Clv% = (F Int[clv] /6)/(F Int[clv] /6 + F Int[unclv] ) × 100% where F Int[clv] is the intensity of the cleaved DNA band and F Int[unclv] represents the intensity of the uncleaved DNA band.…”

“…Many detection systems with high sensitivity and selectivity based on a RFD probe have been reported in recent years, including detection of Escherichia coli ( E. coli ) [26,31], Clostridium difficile , [32], MDA-MB-231 cells [29], T47D cells [33], etc. Although these special RFDs offer an excellent opportunity for designing biosensor-based DNAzymes, only a few studies optimized the RFD probes after in vitro selection [27,34,35,36,37]. Some shortcomings presented by the original probes obtained from the selection include high cost and less sensitivity, which extremely limit the application of the probes.…”

Highly Specific Recognition of Breast Tumors by an RNA-Cleaving Fluorogenic DNAzyme Probe

“…This system is based on both aptamer and DNAzyme technologies. 44,[48][49][50] The RFD probe is a DNA chain with one single RNA linkage inserted. The RNA unit is flanked by nucleotides labelled with a fluorophore and a quencher.…”