Nucleic Acid Enzyme-Based DNA Nanomachine for Biosensing - DNA Nanotechnology: From Structure to Function - page 306

PCR-mimicking DNA machine could be also developed for the detection of cocaine
[ 29 ], Hg 2C [ 30 ], thrombin [ 31 ], and methyltransferase activity [ 32 ] by rational
encoded aptamers of the respective analytes in the amplification template.

14.3.3 RNA-Cleavage DNAzyme-Assisted DNA Machine

As mentioned in the above section, some artificial RNA, for example, the Mg 2C -
dependent E6 DNAzyme, or metal ion-dependent DNAzyme (MNAzyme), possess
the ability to cleavage RNA, thereby, were extensively studied with regard to
their structure and substrate specificity. The organization of the Mg 2C -dependent
DNAzyme was started by cleaving the parent DNAzyme sequence into two subunits.
Each subunit contained a portion of the substrate binding sequence, as well as,
a part of the catalytic core of the DNAzyme. The subunits on their own usually
show no catalytic activity at all. In order to apply the MNAzyme as an amplifying
label for sensing events, “sensor arms” consisting of nucleic acids with recognition
properties to the target analyte were tethered to the MNAzyme units. The sensor
arms are used to recognize and bind a specific input or target sequence. This binding
event acts cooperatively in the assembly of the catalytic MNAzyme units into a
supramolecular structure that reveals the catalytic function of the DNAzyme [ 33 ].
Thus, the analyte triggers the MNAzyme activity which transduces and amplifies
the sensing event. This approach was successfully applied to sense DNA, using
the Mg 2C -dependent E6 DNAzyme sequence as the base element to construct the
recognition site, as well as the biocatalytic amplifying sensing unit of the target
DNA. The DNAzyme sequence was cleaved into two subunits, and two nucleic acid
sequences complementary to the target DNA were tethered to the 3 0 and 5 0 ends
of the subunits. The substrate of the E6 DNAzyme included a ribonucleobase and
was cleaved in the presence of Mg 2C ions after hybridization to the DNAzyme. In
order to apply the DNAzyme subunits as biocatalytic sensing elements, the 3 0 and
5 0 ends of the substrate were modified with a fluorophore-quencher pair. The close
proximity of the components resulted in the quenching of the fluorophore units.
In the absence of the target analyte, the base-pairing complementarity between the
DNAzyme subunits and the substrate was too weak to attain stable duplex structures.
Once the added target hybridized to the complementary “sensor arms” tethered
to the DNAzyme subunits, it triggered a process that acted cooperatively towards
the hybridization of the substrate to the respective complementary domains of the
subunits. Thus, the interaction of the target DNA with the mixture of DNAzyme
subunits and the substrate resulted in the cooperative self-assembly of the active
Mg 2C -dependent DNAzyme that cleaved the substrate. The cleavage of the substrate
switched on the fluorescence of the fluorophore, which provided the optical read-out
signal for the sensing process. This approach allowed the sensitive detection of the
target nucleic acid and even the discrimination of single-base mismatches within the
target sequence.