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 (N20 through N60) for two particular deoxyribozyme catalytic activities, DNA cleavage and tyrosine-RNA nucleopeptide linkage formation. For both activities, we previously identified deoxyribozymes using only N40 regions. In the case of DNA cleavage, here we found that shorter N20 and N30 regions allowed robust catalytic function, either by DNA hydrolysis or by DNA deglycosylation and strand scission via β-elimination, whereas longer N50 and N60 regions did not lead to catalytically active DNA sequences. Follow-up selections with N20, N30, and N40 regions revealed an interesting interplay of metal ion cofactors and random region length. Separately, for Tyr-RNA linkage formation, N30 and N60 regions provided catalytically active sequences, whereas N20 was unsuccessful, and the N40 deoxyribozymes were functionally superior (in terms of rate and yield) to N30 and N60. 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.

Small, Highly Active DNAs That Hydrolyze DNA

“…Similarly, Zn 2+ - and Mn 2+ -dependent self-hydrolyzing deoxyribozymes were isolated 16 by selecting for the cleavage of DNA sequences displayed in pre-organized bulges that are formed when a substrate domain interacts with the deoxyribozyme. For example, deoxyribozymes selected to cleave within a six-nucleotide bulge exhibit rate constants of ~1 min −1 .…”

DNA Catalysts with Tyrosine Kinase Activity

“…13 Therefore, here the initial pool of DNA sequences included either 30, 40, or 50 random nucleotides (N 30 , N 40 , or N 50 ) embedded between two fixed-sequence binding arms that enable PCR amplification during each selection round (Figure S2). The selection process, after extensive optimization of the experimental details (see Supporting Information), led to numerous DNA catalysts of all three random-region lengths that transfer the γ-phosphoryl group of the pppRNA donor to the Tyr acceptor within the DNA-anchored hexapeptide substrate.…”

DNA-Catalyzed DNA Cleavage by a Radical Pathway with Well-Defined Products

“…26 This selection experiment used the amide substrate of Figure 1 along with solely unmodified DNA and resulted in one new deoxyribozyme, named RadDz6, that cleaves a simple DNA oligonucleotide substrate. Under single-turnover conditions and in the absence of H 2 O 2 , RadDz6 has only 6% yield in 24 h, whereas, in the presence of 100 µ M H 2 O 2 , k obs is 1.0 h −1 with 39% yield in 24 h; both k obs and yield are even higher at 1 mM H 2 O 2 (Figure 8A).…”