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

Abstract

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 self-hydrolysis. 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 ∼20 conserved nucleotides and a half-life of ∼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.

Small DNAs that Bind Nickel(II) Specifically and Tightly

“…The collected L-DNA was religated back into circles to serve as templates for PCR amplification to rebuild the sequence library for iteration. 23,24 After six rounds of selective amplification, the C-DNA pool (G6) displayed a weak but visible Ni 2+ -induced "cleavage" signal (L-DNA band) in the gel stained by SYBR gold (Figure 1B, I). Strangely, after elution, this signal migrated with a much enhanced intensity to a position nearby the C-DNA but not the L-DNA reference (indication of non DNA cleavers), when it was subjected to electrophoresis by mistake (Figure 1B, II).…”

“…We aim to break the bottleneck by selecting for DNA-cleaving deoxyribozymes that can specifically respond to Ni 2+ , using a strategy that has been previously demonstrated on Zn 2+ by us. , The identified Zn 2+ -dependent deoxyribozymes exhibit great specificity to Zn 2+ . With the same strategy, we separated potentially Ni 2+ -triggered linear (L) cleavage DNAs from a circular (C) DNA sequence library by denaturing polyacrylamide gel electrophoresis (dPAGE) (Figure A).…”

“…With 2 mM Ni 2+ , the effective pH range falls into 7.75–8.25 (Figure S5B); at pH 8.0, the effective Ni 2+ range is around 0.3–2 mM (Figure S5C). These conditions actually approach the critical point (CP) of forming Ni­(OH) 2 /NiO precipitation, under which we speculate that the generated polynuclear Ni 2+ complexes are indeed recognized by TX1b, a phenomenon observed also in the Zn 2+ -dependent deoxyribozymes. ,,, The speculation was supported by the experiment that at pH 8.0 a decrease of the Ni 2+ level from 2.0 to 0.2 mM (away from the CP) abolished the band shifting, while with 0.2 mM Ni 2+ , a readjustment of pH from 8.0 to 8.8 (close to the CP) had the shifting restored (Figure S6). Among Ni 2+ complexes, we ruled out NiO nanoparticles and Ni 2+ -NTA (nitrilotriacetic acid) as the ligands (Figures S7 and S8), despite that they can adsorb DNA. − Construct TX2b displayed similar characteristics to TX1b (Figure S9).…”

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.…”