The Mechanism of Cleavage of RNA Phosphodiesters by a Gold Nanoparticle Nanozyme.

The cleavage of uridine 3'-phosphodiesters bearing alcohols with pKa ranging from 7.14 to 14.5 catalyzed by AuNPs functionalized with 1,4,7-triazacyclononane-Zn(II) complexes has been studied to unravel the source of catalysis by these nanosystems (nanozymes). The results have been compared with those obtained with two Zn(II) dinuclear catalysts for which the mechanism is fairly understood. Binding to the Zn(II) ions by the substrate and the uracil of uridine was observed. The latter leads to inhibition of the process and formation of less productive binding complexes than in the absence of the nucleobase. The nanozyme operates with these substrates mostly via a nucleophilic mechanism with little stabilization of the pentacoordinated phosphorane and moderate assistance in leaving group departure. This is attributed to a decrease of binding strength of the substrate to the catalytic site in reaching the transition state due to an unfavorable binding mode with the uracil. The nanozyme favors substrates with better leaving groups than the less acidic ones.

A Gold Nanoparticle Nanonuclease Relying on a Zn(II) Mononuclear Complex.

Similarly to enzymes, functionalized gold nanoparticles efficiently catalyze chemical reactions, hence the term nanozymes. Herein, we present our results showing how surface-passivated gold nanoparticles behave as synthetic nanonucleases, able to cleave pBR322 plasmid DNA with the highest efficiency reported so far for catalysts based on a single metal ion mechanism. Experimental and computational data indicate that we have been successful in creating a catalytic site precisely mimicking that suggested for natural metallonucleases relying on a single metal ion for their activity. It comprises one Zn(II) ion to which a phosphate diester of DNA is coordinated. Importantly, as in nucleic acids-processing enzymes, a positively charged arginine plays a key role by assisting with transition state stabilization and by reducing the pKa of the nucleophilic alcohol of a serine. Our results also show how designing a catalyst for a model substrate (bis-p-nitrophenylphosphate) may provide wrong indications as for its efficiency when it is tested against the real target (plasmid DNA).

Host-Guest Allosteric Control of an Artificial Phosphatase.

The activity of many enzymes is regulated by associative processes. To model this mechanism, we report here that the conformation of an unstructured bimetallic Zn(II) complex can be controlled by its inclusion in the cavity of a γ-cyclodextrin. This results in the formation of a catalytic bimetallic site for the hydrolytic cleavage of the RNA model substrate HPNP, whose reactivity is 30-fold larger with respect to the unstructured complex. Competitive inhibition with 1-adamantanecarboxylate displaces the metal complex from the cyclodextrin decreasing the reactivity.

Factors Influencing the Activity of Nanozymes in the Cleavage of an RNA Model Substrate.

A series of 2-nm gold nanoparticles passivated with different thiols all featuring at least one triazacyclonanone-Zn(II) complex and different flanking units (a second Zn(II) complex, a triethyleneoxymethyl derivative or a guanidinium of arginine of a peptide) were prepared and studied for their efficiency in the cleavage of the RNA-model substrate 2-hydroxypropyl-p-nitrophenyl phosphate. The source of catalysis for each of them was elucidated from the kinetic analysis (Michaelis-Menten profiles, pH dependence and kinetic isotope effect). The data indicated that two different mechanisms were operative: One involving two Zn(II) complexes and the other one involving a single Zn(II) complex and a flanking guanidinium cation. The mechanism based on a dinuclear catalytic site appeared more efficient than the one based on the cooperativity between a metal complex and a guanidinium.