Design of DNAzyme Based Fluorescent Sensors

To convert the metal-dependent substrate cleavage into a fluorescent signal, the DNAzyme is labeled with a fluorophore and a quencher. There are several ways of positioning fluorophores and quenchers in a DNAzyme. In the first method, a fluorophore and a quencher can be placed on the ends of the substrate strand, as shown in Fig. 2A. Because the labeling is on both ends of the substrate, perturbations to the global structure, and especially to the catalytic core of the DNAzyme should be minimal; both the activity and metal selectivity of the DNAzyme are maintained. However, the distance between the fluorophore and the quencher can be relatively large in this design, and the quenching efficiency may be low, resulting in high background fluorescence. For the purpose of enzyme activity assays, high background fluorescence does not significantly affect the kinetic results. However, for sensor applications, a lower background fluorescence is desirable. Examples of such design can be found in literature (18). In the second method of labeling, the fluorophore and the quencher can be positioned right next to the cleavage site on the substrate strand to enhance quenching efficiency and decrease background fluorescence (Fig. 2B). However, placing of fluorophores close to the cleavage site may strongly affect the structure and property of the DNAzyme, resulting in decreased or inhibited enzyme activity. Recently, Li and co-workers reported a method to employ a fluorophore- and quencher-labeled DNA library for the in vitro selection, in which a fluorophore and a quencher are placed as shown in Fig. 2B. With the fluorescently labeled DNA library, the selected DNAzymes showed up to a 16-fold increase in fluorescence upon cleavage of the substrate (9). The third method of labeling is shown in Fig. 2C (12). One end of the substrate strand is labeled with a fluorophore, and the complementary end of the enzyme strand is labeled with a quencher. This design incorporates the advantages of both previously described designs. Similar to the first design, the fluorophore and the quencher are attached to the end of each strand; therefore, their effect on the activity of the DNAzyme is minimal. The distance between the fluorophore and the quencher is similar to that of the second

Fig. 2. Positions of labels of fluorophore and quencher on a typical DNAzyme.

design; therefore, the background fluorescence is low. We focus our discussion on this design because it does not complicate the in vitro selection process and allows for low background fluorescence (12).

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