Optimizing the Chemiluminescence of a Light‐Producing Deoxyribozyme

Abstract

Supernova is a chemiluminescent deoxyribozyme recently discovered in our group. It transfers the phosphate group from the 1,2-dioxetane substrate CDP-Star to its 5' hydroxyl group, which triggers a decomposition reaction and the production of light. Here we investigated the effects of reaction conditions on the ability of Supernova to generate a chemiluminescent signal (using a plate reader assay) and to phosphorylate itself (using a ligation assay). Our experiments indicate that multiple zinc ions are required for catalytic function, suggesting links between Supernova and protein enzymes that catalyze similar reactions. They also show how factors such as pH, potassium concentration, CDP-Star concentration, and DNA concentration affect the reaction. By combining information from different experiments, the rate enhancement of light production was increased by more than 1000-fold. These results should be useful for applications in which Supernova is used as a sensor.

Recent progress on DNAzyme-based biosensors for pathogen detection

“…Moreover, it should be noted that the design or optimization of Supernova DNAzyme allows for a substantial increase in the rate of enhancing light generation, surpassing a >1000-fold improvement. 162 Using this DNAzyme, Qin et al created a biosensor to identify synthetic DNA associated with the SARS-CoV-2 N gene and the monkeypox virus (MPXV) F3L gene with LODs of 1.5 and 2.4 pM, respectively. 161 They did this by integrating the Supernova DNAzyme with CRISPR/Cas12a, which can be activated in the presence of a target to rapidly cleave the ssDNA domain of Supernova, leading to an attenuation of the blue light signal.…”

Pushing the Limits of Nucleic Acid Function

“…One catalytic motif, which we named Supernova, was further optimized using a combination of random mutagenesis, selection, and high-throughput sequencing (as described in Figure 1b). Comparative sequence analysis revealed a 46-nucleotide catalytic core consisting of a purine Adapted from [32] and [49].…”

“…Our group recently developed a deoxyribozyme that generates light using a completely different mechanism (Figure 3 ). [ 32 , 49 ] The starting point was a previously described 1,2‐dioxetane substrate called CDP‐Star (Figure 3 c). [ 50 , 51 ] This substrate is stabilized by a phosphate group, and decomposes to generate a chemiluminescent product when the phosphate is removed.…”

“…(B) Secondary structure model of Supernova, a light-producing deoxyribozyme discovered in our group. (C) Chemical structure of CDP-Star, the substrate used by Supernova.Adapted from[32] and[49].…”