Zn2+-dependent DNAzymes that cleave all combinations of ribonucleotides

Although several DNAzymes are known, their utility is limited by a narrow range of substrate specificity. Here, we report the isolation of two zinc-dependent DNAzymes, ZincDz1 and ZincDz2, which exhibit compact catalytic core sequences with highly versatile hydrolysis activity. They were selected through in vitro selection followed by deep sequencing analysis. Despite their sequence similarity, each DNAzyme showed different Zn2+-concentration and pH-dependent reaction profiles, and cleaved the target RNA sequences at different sites. Using various substrate RNA sequences, we found that the cleavage sequence specificity of ZincDz2 and its highly active mutant ZincDz2-v2 to be 5′-rN↓rNrPu-3′. Furthermore, we demonstrated that the designed ZincDz2 could cut microRNA miR-155 at three different sites. These DNAzymes could be useful in a broad range of applications in the fields of medicine and biotechnology. Inomata et al. isolate zinc-dependent DNAzymes with compact catalytic core sequences displaying highly versatile hydrolysis activity. The cleavable substrate variation for the DNAzymes (ZincDz2 and ZincDz2-v2) is very broad and the authors further demonstrate their utility by showing ZincDz2 can hydrolyze miR-155 in vitro at three different sites. With their broad substrate tolerance, this study advances the DNAzyme field for RNA cleavage applications.


Results and Discussion
Screening of DNAzymes that cleave rPy-rPy junctions. To obtain RNA-cleaving DNAzymes that can cleave between any sequence junctions, we used in vitro selection experiments using the substrate 5′-rCrC-3′ (Fig. 1a), because it is considered difficult to cleave among the rPy-rPy junctions. The DNA library with 16nt randomized sequence was chemically synthesized and annealed with the 3′-biotinylated substrate bound to streptavidincoated magnetic beads. After washing out the unbound strands, the DNA library/substrate complexes were incubated in buffer containing 1 mM Zn 2+ at 37°C for 30 min. DNA sequences that have RNA-cleaving activity were released into the supernatant and recovered (Fig. 1b). PCR amplification of the selected DNA library was done with a primer set corresponding to the fixed-arm sequences of the DNA library. After 3 cycles of the selection, approximately 13,000-read sequences were determined by a nextgeneration sequencing. We analyzed the sequences using MEME software to extract concentrated motifs.
Two previously unreported motifs, ZincDz1 and ZincDz2, composed of 6 and 7 sequences were identified in the analysis (Fig. 1c). To verify their RNA-cleaving activities, we performed an in vitro cleavage assay with representative DNAzyme candidates of each motif (ZincDz1 and ZincDz2 in Fig. 1d) using fluorescence-labeled substrate. As shown in Fig. 1d, both DNAzyme candidates efficiently cleaved the RNA-substrate within 30 min under the buffer conditions used in the selection (50 mM HEPES, pH 7.5, 150 mM NaCl, and 1 mM ZnCl 2 ).
Despite their sequence similarity, the sizes of the cleaved substrates were different. This surprising observation led us to consider the possibility that each DNAzyme cuts the substrate at different sites. To determine the cleavage site of each DNAzyme, we compared the size of substrates cleaved by DNAzymes using substrate markers that is partially alkaline-hydrolyzed at three sites on the substrate RNA region, 5′-rC↓rC↓rA↓-3′ (Fig. 1e). As the result, it was found that the cleavage sites of ZincDz1 and ZincDz2 were 5′-rCrCrA↓-3′ and 5′-rC↓rCrA-3′, respectively. We confirmed the cleavage sites of ZincDz1 and ZincDz2 using mass spectroscopic analysis. Further, the molecular weights of the cleaved products and the predicted chemical structure at the cleaved ends are shown in Supplementary Fig. 1.
Enzymatic properties of ZincDz1 and ZincDz2. Next, we examined the enzymatic properties of the two DNAzymes. From the time-course cleavage experiments (Fig. 2a), the observed cleavage rates (k obs ) of ZincDz1 and ZincDz2 were determined as 0.199 ± 0.007 min −1 and 0.47 ± 0.08 min −1 , respectively. These values were comparable to those of typical DNAzymes, such as 10-23 DNAzyme 47 (k obs =~0.28 min −1 ) and 8-17 DNAzyme 48,49 (k obs =~0.5-0.9 min −1 ). It was also found that ZincDz1 and ZincDz2 cleaved substrates with multiple turnovers in a temperature-dependent manner ( Supplementary Fig. 2). Zinc concentration dependencies of enzymatic activities of both DNAzymes exhibited bell-shape profiles with peaks at 0.3-1 mM (ZincDz1) and 1 mM (ZincDz2) (Fig. 2b). The decrease in DNAzyme activity at high concentrations of zinc ion might be because of disruption of the active site structure due to nonspecific interactions with Zn 2+ . The pH profile of both DNAzymes also showed bell shapes with peaks at pH 7.0-7.5 (ZincDz1) and pH 7.5 (ZincDz2) (Fig. 2c). The zinc concentration-and pH-profiles of ZincDz2 tended to be narrower than those of ZincDz1, possibly indicating that the active structure of ZincDz2 might be more rigid than that of ZincDz1. The number of zinc ions bound to ZincDz1 and ZincDz2 was examined by the method reported by Liu et al. 42 and were estimated as around 1 and 3, respectively ( Supplementary Fig. 3). However, the broad spectrum of cleavage activity of ZincDz1 on zinc concentration (Fig. 2b) suggests that multiple zinc ions might be involved in the cleavage reaction. More detailed analyses will be needed to determine the exact number of zinc ion involved in structural stability and/or enzymatic activity.
A single point mutation analysis of ZincDz2. Focusing on ZincDz2 that cleaves between ribocytidines, we undertook a single point mutation analysis on conserved bases in the catalytic core region. For highly conserved bases (A3, G4, G10, G11, T12, T13, G14, G15, and G16 in Fig. 1b, c), purines (pyrimidines) were replaced by another base of purines (pyrimidines) (for example, G → A, A → G, C → T, T → C). As shown in Fig. 3a, the point mutations of G4A, G10A, G11A, T13C, G14A, G15A, and G16A drastically decreased the cleavage activity. While the mutation A3G showed moderate reduction, the mutation T12C exhibited no inhibitory effect on the enzymatic activity in spite of their high conservation in the motif. The alternative point mutations of T1A, T2C, T6C, and T9C were fully tolerated. However, the mutation A8T moderately reduced the activity. We have summarized the results of mutation experiments in Fig. 3b.
Cleavage of miR-155 at three different sites with designed ZincDz2. Finally, to demonstrate the utility of the DNAzyme, we performed cleavage experiments targeting miRNA 50 , which are functional RNAs of 21-25 bases in length present in living cells. They are known to be involved in various physiological phenomena such as development and regulation of gene expression. It has also been reported that they are useful as diagnostic markers for various diseases. ZincDz2 DNAzyme was designed to target miR-155 at three different sites (Fig. 5a). As shown in Fig. 5b, all designed DNAzymes could efficiently cleave miR-155 in the reaction buffer containing 1 mM Zn 2+ , demonstrating that the DNAzyme can be designed to cleave various RNA substrate sequences with "5′-↓rNrPu-3′" by changing the arm sequences.
Recently, gene silencing has been shown using Zn 2+ -dependent DNAzyme using a pH-responsive nanoparticles that release Zn 2+ into the cytoplasm to knock down cancer-related mRNA in both cells and animal experiments 24 . The DNAzyme that we developed here can also be used for gene silencing in cells and for therapeutics as well. Using our versatile DNAzyme, more numbers of DNAzyme can be designed for an mRNA, it allows the selection of more potent DNAzymes targeting various sites in specific mRNAs in the cells. The in vitro selection procedure used in this study. In each cycle, the DNA library sequences that exhibited cleavage activity were released into the solution. The reaction was carried out in a reaction buffer, 50 mM HEPES, pH 7.5, 150 mM NaCl, and 1 mM ZnCl 2 at 37°C for 30 min. The catalytic core region and the core substrate RNA region are shown in blue and red, respectively. c Two consensus sequences, motif 1 and motif 2, enriched by the in vitro selection, as identified with the MEME suite software. d The proposed secondary structures of ZnicDz1 (motif 1) and ZincDz2 (motif2). e The 3′-FAM labeled substrates were cleaved by ZincDz1 or ZincDz2 and analyzed by denaturing PAGE gel (20% polyacrylamide/7 M urea) with an alkaline-hydrolyzed substrate maker (lane 1). ZincDz1 and ZincDz2 cleave the substrate at the sites indicated by the arrows depicted in d.
One specific feature of the ZincDz2 is the narrow working range for zinc concentration and pH. This feature might be useful for applications, such as molecule switching or DNA computing 51 , where strict control of enzyme activity is required. Moreover, based on the ZincDz2 backbone, it should be possible to develop allosteric DNAzymes that can be controlled more tightly by a ligand. For these applications, it was considered more practical to use a single-nucleotide RNA substrate (Supplementary Fig. 6).
Future structural analysis of the DNAzyme by NMR spectrum analysis or X-ray crystallography might give clues as to why the DNAzyme is able to cleave various sequence junctions and why they have unique features for high zinc-selectivity and narrow optimal zinc-concentration-dependencies and pH-dependencies. We hope the future progress of applied researches on various applications along with fundamental analysis will benefit from the compact and versatile DNAzymes reported here.  Table 2 were supplied from Eurofins Genomics (Tokyo, Japan).

Methods
In vitro selection. In vitro selection was basically performed using the methods reported by Lockett et al. 52

Cleavage site
ZincDz2-v2 Fig. 4 Substrate sequence specificity of ZincDz2. a Twelve substrates that have different core RNA sequences were cleaved by ZincDz2. b ZincDz2-G16T mutant, which was designed to form a base pair between the 1st rA in the core substrate region and 16th T in the core catalytic region, can recover the cleavage activities for the substrates with "5′-rArCrA-3′" and "5′-rArArG3′". The error bars show the SD of three independent experiments. c A diagram summarizing the results of the sequence specificity of ZincDz2. The redefined catalytic core region and core substrate region are represented in blue and red, respectively. d The predicted secondary structure of highly active mutant, ZincDz2-v2. The mutated bases are represented in green. Kinetic experiments. The RNA-cleavage reaction was carried out under a single turnover condition with a 10-fold excess of the DNAzyme (1 μM) to the fluorescein (FAM)-labeled substrate (0.1 μM). The FAM-labeled substrate and DNAzymes were heated at 95°C for 5 min and allowed to cool at 4°C. The cofactor (zinc ion) was added to initiate the reaction. The DNAzymes annealed with the substrate were incubated at 37°C for designated periods of time (t: 0, 5, 20, 60, and 180 min) in the reaction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, and 1 mM ZnCl 2 ). The reaction was terminated by an addition of a stop buffer (25 mM EDTA, 8 M urea, and 0.025% bromophenol blue). The cleaved products were separated by electrophoresis on a denaturing gel (20% polyacrylamide/7 M urea). The amount of cleavage products was quantified using a ChemiDocTM XRS + imaging system (Bio-Rad, Hercules, CA, USA) from the gel images. The average and the standard deviation were calculated from three parallel experiments for each experiment. The cleavage percentage was calculated based on the amount analyzed by the imaging system as follows Eq. (1): The data was fit to exponential function [Eq. (2)] using nonlinear regression in the SigmaPlot 12 software (Systat Software, Inc., CA, USA).
where f and is the fraction cleaved, f max is the amplitude, t is the time, and k obs is the reaction rate constant (min −1 ).  A   T  C   G  T T T AC G  G  T  T  G  G   A  U  A   -3'  5'-GGGGUU  G  U  G  C  U  A  A  CU  AAUG  UU  A  A  T  A  C  G  T  A  T C  T AC A  G T  CCCC   5 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/.