Multiplexed Analysis of Genes and of Metal Ions Using
Enzyme/DNAzyme Amplification Machineries
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Abstract
The
progressive development of amplified DNA sensors using nucleic acid-based
machineries, involving the isothermal autonomous synthesis of the
Mg<sup>2+</sup>-dependent DNAzyme, is used for the amplified, multiplexed
analysis of genes (Smallpox, TP53) and metal ions (Ag<sup>+</sup>,
Hg<sup>2+</sup>). The DNA sensing machineries are based on the assembly
of two sensing modules consisting of two nucleic acid scaffolds that
include recognition sites for the two genes and replication tracks
that yield the nicking domains for Nt.BbvCI and two different Mg<sup>2+</sup>-dependent DNAzyme sequences. In the presence of any of the
genes or the genes together, their binding to the respective recognition
sequences triggers the nicking/polymerization machineries, leading
to the synthesis of two different Mg<sup>2+</sup>-dependent DNAzyme
sequences. The cleavage of two different fluorophore/quencher-modified
substrates by the respective DNAzymes leads to the fluorescence of
F<sub>1</sub> and/or F<sub>2</sub> as readout signals for the detection
of the genes. The detection limits for analyzing the Smallpox and
TP53 genes correspond to 0.1 nM. Similarly, two different nucleic
acid scaffolds that include Ag<sup>+</sup>-ions or Hg<sup>2+</sup>-ions recognition sequences and the replication tracks that yield
the Nt.BbvCI nicking domains and the respective Mg<sup>2+</sup>-dependent
DNAzyme sequences are implemented as nicking/replication machineries
for the amplified, multiplexed analysis of the two ions, with detection
limits corresponding to 1 nM. The ions sensing modules reveal selectivities
dominated by the respective recognition sequences associated with
the scaffolds