Figure 1.

The proposed secondary structure of the 8–17 DNAzyme and its fluorophore attachments for the single molecule FRET studies. (a) The original 8–17 DNAzyme with enzyme (17E) and substrate (17S) strands. For the bulk activity assays described here, the enzyme strand was labeled with a dabcyl (Dy) quencher at the 3′ end, and the substrate strand was labeled with a Alexa fluoro 488 flurophore (Alx) at the 5′ end and a dabcyl quencher at the 3′ end. The arrow indicates a cleavage site. (b) Modified 8–17 DNAzyme and its variants for the single molecule experiments described here. The enzyme strand was extended by 2 nts at the 5′ end and by 2 nts and (dT)₅ at the 3′ end, and modified with Cy5 at the 5′ end and a biotin at the 3′ end. The substrate is extended by 2 nts at the 3′ end, and labeled with Cy3 at the 5′ end. For control experiments, two mutant enzymes and two mutant substrates were used. An inactive enzyme has a GC base pair instead of GT wobble pair right next to the cleavage site and a less active 17E(sda5) enzyme has one more T base inserted on the hairpin loop. Two more substrates were used. A non–cleavable substrate has a ribonucleotide A (rA) at the cleavage site instead of a deoxyribonucleotide A, rendering it non–cleavable. Another substrate is extended by 2 more nts at the 5′ end for further thermal stability, and is called long substrate.