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Expanding DNAzyme Functionality through Enzyme Cascades with Applications in Single Nucleotide Repair and Tunable DNA-Directed Assembly of Nanomaterials.

ABSTRACT: Many biological functions require two or more enzymes working together in cascades. While many examples of protein and RNA enzyme cascades are known, few enzyme cascades containing solely DNAzymes have been reported. Herein we demonstrate the combination of an 8-17 DNAzyme with RNA cleavage activity and an E47 DNAzyme with DNA ligation activity to achieve a new function of single ribonucleotide repair in DNA while maintaining the integrity of the original DNA sequence, which is difficult for a single DNAzyme to achieve. In addition, this method is applied to modify the sequences of DNA strands immobilized on the surface of nanoparticles to control the DNA-directed assembly selectively and sequentially. Such an approach can be applied to other DNAzymes with different activities to expand the functions of DNAzymes and the scope of their applications.

SUBMITTER: Xiang Y

PROVIDER: S-EPMC3524592 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Expanding DNAzyme Functionality through Enzyme Cascades with Applications in Single Nucleotide Repair and Tunable DNA-Directed Assembly of Nanomaterials.

Xiang Yu Y Wang Zidong Z Xing Hang H Lu Yi Y

Chemical science 20120921 1

Many biological functions require two or more enzymes working together in cascades. While many examples of protein and RNA enzyme cascades are known, few enzyme cascades containing solely DNAzymes have been reported. Herein we demonstrate the combination of an 8-17 DNAzyme with RNA cleavage activity and an E47 DNAzyme with DNA ligation activity to achieve a new function of single ribonucleotide repair in DNA while maintaining the integrity of the original DNA sequence, which is difficult for a s ...[more]

PMID: 23264874

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Project description:Biocatalysis is a useful strategy for sustainable green synthesis of fine chemicals due to its high catalytic rate, reaction specificity, and operation under ambient conditions. Addressable immobilization of enzymes onto solid supports for one-pot multistep biocatalysis, however, remains a major challenge. In natural pathways, enzymes are spatially coupled to prevent side reactions, eradicate inhibitory products, and channel metabolites sequentially from one enzyme to another. Construction of a modular immobilization platform enabling spatially directed assembly of multiple biocatalysts would, therefore, not only allow the development of high-efficiency bioreactors but also provide novel synthetic routes for chemical synthesis. In this study, we developed a modular cascade flow reactor using a generalizable solid-binding peptide-directed immobilization strategy that allows selective immobilization of fusion enzymes on anodic aluminum oxide (AAO) monoliths with high positional precision. Here, the lactate dehydrogenase and formate dehydrogenase enzymes were fused with substrate-specific peptides to facilitate their self-immobilization through the membrane channels in cascade geometry. Using this cascade model, two-step biocatalytic production of l-lactate is demonstrated with concomitant regeneration of soluble nicotinamide adenine dinucleotide (NADH). Both fusion enzymes retained their catalytic activity upon immobilization, suggesting their optimal display on the support surface. The 85% cascading reaction efficiency was achieved at a flow rate that kinetically matches the residence time of the slowest enzyme. In addition, 84% of initial catalytic activity was preserved after 10 days of continuous operation at room temperature. The peptide-directed modular approach described herein is a highly effective strategy to control surface orientation, spatial localization, and loading of multiple enzymes on solid supports. The implications of this work provide insight for the single-step construction of high-power cascadic devices by enabling co-expression, purification, and immobilization of a variety of engineered fusion enzymes on patterned surfaces.

Dataset Information

Expanding DNAzyme Functionality through Enzyme Cascades with Applications in Single Nucleotide Repair and Tunable DNA-Directed Assembly of Nanomaterials.

Publications

Expanding DNAzyme Functionality through Enzyme Cascades with Applications in Single Nucleotide Repair and Tunable DNA-Directed Assembly of Nanomaterials.

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