Single-round deoxyribozyme discovery

0544488 - ÚOCHB 2022 RIV GB eng J - Journal Article
Streckerová, Tereza - Kurfürst, Jaroslav - Curtis, Edward A.
Single-round deoxyribozyme discovery.
Nucleic Acids Research. Roč. 49, č. 12 (2021), s. 6971-6981. ISSN 0305-1048. E-ISSN 1362-4962
R&D Projects: GA ČR(CZ) GA19-20989S; GA MŠMT(CZ) EF16_019/0000729
Institutional support: RVO:61388963
Keywords : in-vitro selection * lead-dependent ribozyme * RNA aptamer
OECD category: Biochemistry and molecular biology
Impact factor: 19.160, year: 2021
Method of publishing: Open access
https://doi.org/10.1093/nar/gkab504

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.
Permanent Link: http://hdl.handle.net/11104/0321332