Supernova: A Deoxyribozyme that Catalyzes a Chemiluminescent Reaction

Abstract

Functional DNAm olecules are useful components in nanotechnology and synthetic biology.T oexpand the toolkit of functional DNAp arts,i nt his study we used artificial evolution to identify ag lowing deoxyribozyme called Supernova. This deoxyribozyme transfers ap hosphate from a1 ,2dioxetane substrate to its 5' hydroxylg roup,w hich triggers ac hemiluminescent reaction and af lash of blue light. An engineered version of Supernova is only catalytically active in the presence of an oligonucleotide complementary to its 3' end, demonstrating that light production can be coupled to ligand binding.W eanticipate that Supernova will be useful in awide variety of applications,i ncluding as as ignaling component in allosterically regulated sensors and in logic gates of molecular computers.

Pushing the Limits of Nucleic Acid Function

“… [74] These experiments also highlighted the importance of cofactors in artificial evolution experiments: RNA‐cleaving deoxyribozymes were about 1000 times more abundant when selections were performed in the presence of lead than when they were performed in its absence. We observed a similar phenomenon in a previous search for light‐producing deoxyribozymes, [32] which were successfully isolated in a selection performed in the presence of zinc, but not in a parallel selection performed in the absence of zinc. In principle, it should be possible to use single‐step selections to rapidly search for simple ribozymes and deoxyribozymes that catalyze reactions other than RNA cleavage, and to also explore the cofactor requirements of these motifs in a systematic way.…”

“… [31] For example, a library containing 85 mutagenized positions recently characterized in our group likely contained most possible variants within four mutations of the starting sequence. [32] Active variants are then isolated from the library by selection (usually using more stringent conditions than those utilized in the initial selection) and characterized by either Sanger sequencing or (preferably) high‐throughput sequencing.…”

“…One reason is that they typically yield variants with improved functional properties. [ 3 , 30 , 32 , 33 ] For example, in the case of ribozyme selections, variants with rates or catalytic efficiencies ≥ 10‐fold higher than that of the initial isolate are often obtained. [ 30 , 33 ]…”

“…This is an important step because functional sequences isolated from random sequence libraries often contain nonessential regions that can be deleted or replaced with short spacers or loops (Figure 1 d). [ 3 , 30 , 32 , 33 , 36 , 37 ] Although such regions can sometimes also be identified making random deletions, minimization is usually greatly facilitated by data from reselection experiments (Figure 1 d). Minimized variants of functional motifs are less expensive to synthesize, tend to be better behaved from a kinetic perspective, and are often more amenable to high‐resolution structural analysis.…”

“…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.…”