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.

Optimizing the Chemiluminescence of a Light‐Producing Deoxyribozyme

“…Another approach could be to use a smaller, secondary structure library [29] and perform a single-step selection. [30] In this study we increased the rate enhancement of light production of Supernova by more than 1000-fold without changing its sequence. This was instead achieved by investigating the effects of a range of conditions on both the enzymatic and nonenzymatic production of light in the presence of CDP-Star.…”

“…The typical way to increase the amount of light produced by such a sensor would be to perform a standard selection experiment using a library of ∼10 15 sequences. Another approach could be to use a smaller, secondary structure library [29] and perform a single‐step selection [30] . In this study we increased the rate enhancement of light production of Supernova by more than 1000‐fold without changing its sequence.…”

Recent Advances on DNAzyme‐Based Sensing

“…These limited but exciting examples shield light on the selection of DNAzymes for a wide range of targets. Additionally, shorten the selection round also represent an interesting direction to accelerate the discovery of new catalytic DNAzymes [85] …”

High-Throughput Activity Profiling of RNA-Cleaving DNA Catalysts by Deoxyribozyme Sequencing (DZ-seq)

“…5 Next-generation sequencing (NGS) techniques have been increasingly replacing the low-throughput Sanger method in the analysis of in-vitro selection libraries of aptamers [6][7][8][9][10][11] and (deoxy)ribozymes. [12][13][14][15][16] The candidate catalysts are usually selected from the NGS data using the relative sequence abundance or sequence enrichment as proxies for the catalytic activity. However, due to PCR and other biases, [17][18] the candidate catalysts have to be validated by conventional low-throughput biochemical assays.…”

“…Although deoxyribozymes have not been yet found in nature, in vitro selection procedures allowed the discovery of artificial deoxyribozymes with a vast repertoire of catalytic activities. − A typical in vitro selection starts from a combinatorial library of up to ∼10 15 unique sequences and consists of repetitive rounds of activity screening, selection, and amplification . Next-generation sequencing (NGS) techniques have been increasingly replacing the low-throughput Sanger method in the analysis of in vitro selection libraries of aptamers − and (deoxy)­ribozymes. − The candidate catalysts are usually selected from the NGS data using the relative sequence abundance or sequence enrichment as proxies for the catalytic activity. However, due to PCR and other biases, , the candidate catalysts have to be validated by conventional low-throughput biochemical assays.…”