Use of deoxyribozymes in RNA research

Abstract

Since their first identification by in vitro selection in 1994, deoxyribozymes have been developed to catalyze a variety of chemical reactions. The first DNA-catalyzed reaction was cleavage of a ribonucleotide linkage within an oligonucleotide substrate. In subsequent years, growing collections of deoxyribozymes have been developed for several reactions that have practical utility for RNA research. These deoxyribozymes are useful for site-specific RNA cleavage as well as ligation to form linear, branched, and lariat RNA products. An application related to RNA ligation is deoxyribozyme-catalyzed labeling of RNA (DECAL), which is used to attach a biophysical tag to a desired RNA sequence at a specific position. With current achievements and likely future developments, deoxyribozymes are a useful contributor to the toolbox of RNA research methods.

Figure 1

Deoxyribozyme-catalyzed RNA cleavage. (A) The cleavage reaction, which forms 2′,3′-cyclic phosphate and 5′-OH RNA termini. (B) Individual deoxyribozymes and their target sequences for efficient cleavage of all-RNA substrates. N = any nucleotide; R = purine; Y = pyrimidine. Outside of the explicitly indicated nucleotides, any RNA sequence is tolerated as long as Watson-Crick RNA:DNA covariation is maintained.

Figure 2

Reactions catalyzed by deoxyribozymes that ligate RNA to form linear products. (A) Reaction of a 2′,3′-cyclic phosphate with a 5′-OH group, leading to either a native 3′–5′ linkage or a non-native 2′–5′ linkage. (B) Reaction of a 2′,3′-diol with a 5′-triphosphate, again leading to either a 3′–5′ or 2′–5′ linkage.

Figure 3

Individual deoxyribozymes for linear 3′–5′ RNA ligation. R = purine; D = A, G, or U. Outside of the explicitly indicated nucleotides, any sequence for either the left-hand (L) or right-hand (R) RNA substrate is tolerated as long as Watson-Crick RNA:DNA covariation is maintained.

Figure 4

Synthesis of 2′,5′-branched and lariat RNA by reaction of a 2′-OH group with a 5′-triphosphate. The product is a lariat when the dashed loop is present connecting the two substrates.

Figure 5

Individual deoxyribozymes for branched RNA synthesis. (A) 7S11 deoxyribozyme. Note the four Watson-Crick paired regions denoted P1–P4. (B) 10DM24 deoxyribozyme. (C) 6CE8 deoxyribozyme. (D) 6BX22 deoxyribozyme.

Figure 6

Deoxyribozyme-catalyzed labeling (DECAL) of RNA. (A) Synthesis of the “tagging RNA”. The unlabeled tagging RNA is prepared by in vitro transcription using commercially available 5-aminoallyl-CTP and T7 RNA polymerase. The sequence is designed such that the modified C is the only such nucleotide in the entire 19 nt sequence. (B) Attachment of the tagging RNA to the target RNA by the 10DM24 deoxyribozyme.