New DNA-hydrolyzing DNAs isolated from an ssDNA library carrying a terminal hybridization stem

DNA-hydrolyzing DNAs represent an attractive type of DNA-processing catalysts distinctive from the protein-based restriction enzymes. The innate DNA property has enabled them to readily join DNA-based manipulations to promote the development of DNA biotechnology. A major in vitro selection strategy to identify these DNA catalysts relies tightly on the isolation of linear DNAs processed from a circular single-stranded (ss) DNA sequence library by selfhydrolysis. Herein, we report that by programming a terminal hybridization stem in the library, other than the previously reported classes (I & II) of deoxyribozymes, two new classes (III & IV) were identified with the old selection strategy to site-specifically hydrolyze DNA in the presence of Zn2+. Their representatives own a catalytic core consisting of similar to 20 conserved nucleotides and a half-life of similar to 15 min at neutral pH. In a bimolecular construct, class III exhibits unique broad generality on the enzyme strand, which can be potentially harnessed to engineer DNA-responsive DNA hydrolyzers for detection of any target ssDNA sequence. Besides the new findings, this work should also provide an improved approach to select for DNA-hydrolyzing deoxyribozymes that use various molecules and ions as cofactors.

Automated summary

This study identified two new classes of DNA-hydrolyzing deoxyribozymes by programming a terminal hybridization stem, in addition to the previously reported classes, using the old selection strategy. These new deoxyribozymes can specifically hydrolyze DNA in the presence of Zn2+ and may have potential applications in engineering DNA-responsive DNA hydrolyzers for detecting target ssDNA sequences.