title: Systematic evaluation of the dependence of deoxyribozyme catalysis on random region length.
abstract: Functional nucleic acids are DNA and RNA aptamers that bind targets, or they are deoxyribozymes and ribozymes that have catalytic activity. These functional DNA and RNA sequences can be identified from random-sequence pools by in vitro selection, which requires choosing the length of the random region. Shorter random regions allow more complete coverage of sequence space but may not permit the structural complexity necessary for binding or catalysis. In contrast, longer random regions are sampled incompletely but may allow adoption of more complicated structures that enable function. In this study, we systematically examined random region length (N(20) through N(60)) for two particular deoxyribozyme catalytic activities, DNA cleavage and tyrosine-RNA nucleopeptide linkage formation. For both activities, we previously identified deoxyribozymes using only N(40) regions. In the case of DNA cleavage, here we found that shorter N(20) and N(30) regions allowed robust catalytic function, either by DNA hydrolysis or by DNA deglycosylation and strand scission via β-elimination, whereas longer N(50) and N(60) regions did not lead to catalytically active DNA sequences. Follow-up selections with N(20), N(30), and N(40) regions revealed an interesting interplay of metal ion cofactors and random region length. Separately, for Tyr-RNA linkage formation, N(30) and N(60) regions provided catalytically active sequences, whereas N(20) was unsuccessful, and the N(40) deoxyribozymes were functionally superior (in terms of rate and yield) to N(30) and N(60). Collectively, the results indicate that with future in vitro selection experiments for DNA and RNA catalysts, and by extension for aptamers, random region length should be an important experimental variable.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, 61801, United States.
authors.forename: Tania E
authors.initials: TE
authors.lastname: Velez
authors.forename: Jaydeep
authors.initials: J
authors.lastname: Singh
authors.forename: Ying
authors.initials: Y
authors.lastname: Xiao
authors.forename: Emily C
authors.initials: EC
authors.lastname: Allen
authors.forename: On Yi
authors.initials: OY
authors.lastname: Wong
authors.forename: Madhavaiah
authors.initials: M
authors.lastname: Chandra
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Kwon
authors.forename: Scott K
authors.initials: SK
authors.lastname: Silverman
mesh_terms: D055162:Biocatalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053837:DNA Cleavage; D021881:DNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D021881:DNA, Catalytic; D014443:Tyrosine; D012313:RNA; D004247:DNA