Engineering fluorescent RNase aptazymes by rational design. (a) The use of a regulatory oligonucleotide. The DNAzyme (black line) is joined to an ATP-binding DNA aptamer (blue line) and the combined sequence can form a duplex structure with a regulatory oligonucleotide (purple strand), which prevents the DNAzyme domain from binding the fluorogenic substrate. Binding of ATP to the aptamer domain causes the release of the regulatory oligonucleotide, freeing up the DNAzyme for binding and cleaving the substrate. (b) An ATP-responsive DNAzyme with internal regulatory nucleotides. The DNAzyme is designed from pH6DZ1 and the same ATP-binding aptamer. Nucleotides in the sensor sequence are color-coded for easy tracking of their relative positions in the two alternative structures. In the absence of ATP, the aptamer is designed to form an inhibitory duplex with the catalytic core; in the presence of ATP, the aptamer pulls away from the DNAzyme, which activates the DNAzyme leading to the generation of a fluorescent signal.
