Assemblage of Signaling DNA Enzymes with Intriguing Metal-Ion Specificities and pH Dependences Academic Article uri icon

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abstract

  • We report a group of new DNA enzymes that possess a synchronized RNA-cleavage/fluorescence-signaling ability and exhibit wide-ranging metal-ion and pH dependences. These DNA catalysts were derived from a random-sequence DNA pool in a two-stage process: (1) establishment of a catalytic DNA population through repetitive rounds of in vitro selection at pH 4.0, and (2) sequence-diversification and catalytic-activity optimization through five parallel paths of in vitro evolution conducted at pH 3.0, 4.0, 5.0, 6.0, and 7.0, respectively. The deoxyribozymes were evolved to cleave the phosphodiester bond of a single ribonucleotide embedded in DNA and flanked immediately by two deoxyribonucleotides modified with a fluorophore and a quencher, respectively--a setting that synchronizes catalysis with fluorescence signaling. The most dominant catalyst from each pool was examined for metal-ion specificity, catalytic efficiency, pH dependence, and fluorescence-signaling capability. Individual catalysts have different metal-ion requirements and can generate as much as a 12-fold fluorescence enhancement upon RNA cleavage. Most of the DNA enzymes have a pH optimum coinciding with the selection pH and exhibit a rate constant approximating 1 min(-)(1) under optimal reaction conditions. The demonstration of DNA enzymes that are functional under extremely high acidity (such as pH 3 and 4) indicates that DNA has the ability to perform efficient catalysis even under harsh reaction conditions. The isolation of many new signaling DNA enzymes with broad pH optima and metal-ion specificities should facilitate the development of diverse deoxyribozyme-based biosensors.

publication date

  • June 2003