Supernova: A Deoxyribozyme that Catalyzes a Chemiluminescent Reaction

Abstract

Functional DNA molecules are useful components in nanotechnology and synthetic biology. To expand the toolkit of functional DNA parts, in this study we used artificial evolution to identify a glowing deoxyribozyme called Supernova. This deoxyribozyme transfers a phosphate from a 1,2-dioxetane substrate to its 5' hydroxyl group, which triggers a chemiluminescent reaction and a flash 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. We anticipate that Supernova will be useful in a wide variety of applications, including as a signaling component in allosterically regulated sensors and in logic gates of molecular computers.

Keywords: aptazyme; catalytic DNA; chemiluminescence; deoxyribozyme; in vitro selection; luminescence; sensor.

Figure 1

Design and progress of the in vitro selection experiment. (a) Reaction Scheme of light production using CDP‐Star. (b) Selection Scheme used to isolate deoxyribozymes that produce light by dephosphorylation of CDP‐Star. The random sequence library was first incubated with CDP‐Star. Library members that transferred a phosphate from CDP‐Star to their 5′ hydroxyl group during the incubation were tagged using a ligation reaction that requires a 5′ phosphate. These molecules were then purified by PAGE, excised from the gel, and amplified by PCR using one primer with a 5′ phosphate and one with a single ribonucleotide at the 3′ end. Single‐stranded library was then generated by incubating with lambda exonuclease (to selectively degrade one DNA strand) and base (to cleave the other DNA strand at the ribonucleotide and regenerate the 5′ end of the library). See the Supplementary Methods and Tables S1–S2 for more information. (c) Progress of the initial selection, with a time course of self‐phosphorylation of the most active deoxyribozyme identified shown in the inset.

Figure 2

Correlation network and secondary structure of a minimized light‐producing deoxyribozyme. Correlations are ranked by mutual information value. Those corresponding to base triples (T) are shown in green, and those corresponding to other interactions (O) are shown in orange. Nucleotides in variable regions 1 and 2 are shown in lower‐case type.

Figure 3

The rate of Supernova and comparison to other systems. (a) Michaelis–Menten plot showing the initial velocity of Supernova at different concentrations of substrate. Velocities were measured at a Supernova concentration of 1 μM. (b) Cumulative light production by Supernova under optimal conditions. The rate enhancement peaks at ≈6,500‐fold after 10 minutes. Reactions contained 30 μM Supernova and 62.5 μM CDP‐Star. Note that the rate at which CDP‐Star decomposes after dephosphorylation is slower than the rate of the dephosphorylation reaction catalyzed by Supernova under these conditions. For this reason, the light‐producing reaction takes longer to reach a plateau (Figure S9). (c) Comparison of Supernova to existing nucleic acid‐based methods to generate colorimetric, fluorescent, and chemiluminescent signals. G4=the DNA G‐quadruplex‐peroxidase system to generate light (in the presence of luminol) or color (in the presence of ABTS); MG=the malachite green RNA aptamer; Broc=the Red Broccoli RNA aptamer; Mng=the Mango RNA aptamer; SN=the Supernova deoxyribozyme described in this work.

Figure 4

A programable light‐producing deoxyribozyme sensor that detects oligonucleotides. (a) Schematic representation of the sensor in its OFF and ON conformations. (b) Rate enhancement of light production as a function of concentration of the target oligonucleotide. (c) Rate enhancement of light production of five deoxyribozyme sensors in the presence of either the target oligonucleotide (for example, sensor 1 and oligonucleotide 1) or non‐target oligonucleotides (for example sensor 1 and oligonucleotides 2–5). The shade of each square indicates the rate enhancement of light production for a particular combination of sensor and oligonucleotide.