Single-round deoxyribozyme discovery

Abstract

Artificial evolution experiments typically use libraries of ∼1015 sequences and require multiple rounds of selection to identify rare variants with a desired activity. Based on the simple structures of some aptamers and nucleic acid enzymes, we hypothesized that functional motifs could be isolated from significantly smaller libraries in a single round of selection followed by high-throughput sequencing. To test this idea, we investigated the catalytic potential of DNA architectures in which twelve or fifteen randomized positions were embedded in a scaffold present in all library members. After incubating in either the presence or absence of lead (which promotes the nonenzymatic cleavage of RNA), library members that cleaved themselves at an RNA linkage were purified by PAGE and characterized by high-throughput sequencing. These selections yielded deoxyribozymes with activities 8- to 30-fold lower than those previously isolated under similar conditions from libraries containing 1014 different sequences, indicating that the disadvantage of using a less diverse pool can be surprisingly small. It was also possible to elucidate the sequence requirements and secondary structures of deoxyribozymes without performing additional experiments. Due to its relative simplicity, we anticipate that this approach will accelerate the discovery of new catalytic DNA and RNA motifs.

Figure 1.

A DNA architecture for single-round selections. The ribonucleotide at the expected site of cleavage is indicated by rA. Bases shown in a pale background (‘possible primer binding site’) were deleted in most experiments (see also Table 1).

Figure 2.

Single-round discovery of RNA-cleaving deoxyribozymes. PAGE, polyacrylamide gel electrophoresis; HTS = high-throughput sequencing.

Figure 3.

Identification of deoxyribozymes that cleave RNA in a single round of selection. Sequences from the unselected library are shown in yellow and those from the selected library in dark blue. The library contained 12 randomized positions and 1.7 × 10⁷ different sequences, and was incubated in a buffer containing potassium, magnesium, and lead. A time course showing the catalytic activity of one of the sequences with the highest read number (indicated by an arrow in the graph) is shown in the inset.

Figure 4.

Effect of metal ions and library size on the number of enriched sequences in selections for RNA-cleaving deoxyribozymes. (A) Distribution of read numbers in an unselected N₁₂ library, in an N₁₂ library selected for the ability to cleave RNA in the presence of lead, and in an N₁₂ library selected for the ability to cleave RNA in the absence of lead. (B) Same, but for an N₁₅ library.

Figure 5.

Diversity of catalytic motifs isolated in single-round selections. Sequence logos were derived from the read numbers of all single-mutation variants of deoxyribozymes isolated in single-round selections. Panels A and B show motifs isolated from the N₁₂ library incubated in the presence of lead. Panels C and D show motifs from the N₁₂ library incubated in the absence of lead. Panels E-G show motifs from the N₁₅ library incubated in the presence of lead. See Table 1 for the catalytic rates of these deoxyribozymes.

Figure 6.

Rapid characterization of deoxyribozyme sequence requirements. (A) Sequence logo generated from read numbers of all possible single mutant variants of Dvanactka. (B) Pairwise correlations based on read numbers of single and double mutants of Dvanactka. Blue squares indicate pairs at which a double mutant occurred more often than expected based on the read numbers of the corresponding single mutants. (C) Secondary structure model of Dvanactka. Nucleotides from the randomized region are shown in green, and positions 1 and 7 with a green background. (D, E) Evidence for base pairing between positions 1 and 7 based on read numbers (panel D) and catalytic activity (panel E). Error bars indicate the standard deviation from three experiments. (F) Correlation between read number and catalytic activity for variants of Dvanactka. R² = 0.72. See Supplementary Table S3 for more information about these sequences.