In vitro selection, characterization, and application of deoxyribozymes that cleave RNA

Abstract

Over the last decade, many catalytically active DNA molecules (deoxyribozymes; DNA enzymes) have been identified by in vitro selection from random-sequence DNA pools. This article focuses on deoxyribozymes that cleave RNA substrates. The first DNA enzyme was reported in 1994 and cleaves an RNA linkage. Since that time, many other RNA-cleaving deoxyribozymes have been identified. Most but not all of these deoxyribozymes require a divalent metal ion cofactor such as Mg2+ to catalyze attack by a specific RNA 2'-hydroxyl group on the adjacent phosphodiester linkage, forming a 2',3'-cyclic phosphate and a 5'-hydroxyl group. Several deoxyribozymes that cleave RNA have utility for in vitro RNA biochemistry. Some DNA enzymes have been applied in vivo to degrade mRNAs, and others have been engineered into sensors. The practical impact of RNA-cleaving deoxyribozymes should continue to increase as additional applications are developed.

Figure 1

RNA cleavage with formation of 2′,3′-cyclic phosphate and 5′-hydroxyl termini. This reaction can occur alone or with a catalyst such as a protein enzyme, ribozyme or deoxyribozyme. In most but not all cases, a divalent metal ion cofactor (M²⁺) is required to achieve an appreciable reaction rate.

Figure 2

In vitro selection strategies for identifying RNA-cleaving deoxyribozymes. (A) The general four-step selection strategy, which involves iterated rounds of PCR to incorporate biotin; streptavidin chromatography to isolate the single-stranded nucleic acid; M²⁺-catalyzed RNA cleavage; and PCR to regenerate the pool, which has been enriched in deoxyribozymes capable of cleaving RNA. (B) Simplified depictions of the three strategy variations used in all selection experiments for RNA-cleaving DNA enzymes reported to date. In strategy 1, a single ribonucleotide linkage (rA) is the cleavage target, and interactions between the substrate strand and the deoxyribozyme are not pre-programmed. This strategy was used for the first in vitro selection effort that identified catalytic DNA, which resulted in a Pb²⁺-dependent RNA-cleaving deoxyribozyme (26), and it has been employed in other studies as well (28,33). In strategy 2, the single ribonucleotide cleavage site was placed between two Watson–Crick binding arms, as first reported by Breaker and Joyce (27) and used in several subsequent experiments (30,32,33,36,40). In strategy 3, a 12 nt stretch of RNA rather than a single ribonucleotide linkage was the cleavage target, giving rise to the 10–23 and 8–17 deoxyribozymes (29). The biotinylated RNA–DNA chimera was generated during the first step of each selection round by primer extension using reverse transcriptase instead of PCR (not depicted explicitly).

Figure 3

Gallery of RNA-cleaving deoxyribozymes identified by in vitro selection. Most of these deoxyribozymes function in the intermolecular format for in trans cleavage of an RNA linkage. Thick lines and lowercase letters denote RNA; thin lines and uppercase letters denote DNA, and the green arrow marks the cleavage site. The DNA enzyme regions are green; DNA nucleotides involved in Watson–Crick base pairing are brown; nucleotides of the substrate to the 5′-side of the cleavage site are red; and nucleotides to the 3′-side are blue. For clarity, not all nucleotides are shown explicitly, but this does not necessarily imply a lack of sequence requirements. In panel D, the 8–17 deoxyribozyme is shown with the RNA cleavage site requirement a↓g. However, the closely related 17E deoxyribozyme (32) can cleave n↓g (although a↓g and g↓g are preferred). Furthermore, a comprehensive study showed that 8–17 variants can be identified that collectively cleave almost any RNA dinucleotide linkage (36). In panel H, the letters F and Q denote a fluorophore and quencher that are separated upon cleavage by the signaling deoxyribozyme.

Figure 4

RNA-cleaving deoxyribozymes as sensors. (A) Transducing DNA-catalyzed RNA cleavage into a fluorescence signal by separation of a fluorophore and a quencher. These are denoted F and Q, respectively (e.g., TAMRA and Dabcyl, although other combinations may be used). The fluorophore and quencher may be placed in various places on the RNA substrate and deoxyribozyme, as long as RNA cleavage results in physical separation of F and Q (125). (B) Using gold nanoparticles to induce a colorimetric signal upon RNA cleavage, which separates the nucleic acid strands that hold the nanoparticles together in an aggregate. The RNA substrates are colored brown to avoid confusion with the aggregated (blue) and separated (red) gold nanoparticles. The gold nanparticles are shown in a ‘head-to-tail’ orientation (128,129,134), but subsequent efforts showed that a ‘tail-to-tail’ orientation is optimal (126,135). (C) An allosteric adenosine sensor that was engineered by combining an aptamer with a deoxyribozyme (129). The deoxyribozyme binds well to the RNA substrate only when adenosine (orange) is bound with the aptamer (pink), whereupon RNA cleavage is transduced to a color change via separation of the gold nanoparticles.