A Novel Small RNA-Cleaving Deoxyribozyme With A Short Binding Arm

Nucleic acid is a biological macromolecule essential for life.In addition to storage and transmit of genetic information,nucleic acid also plays important regulatory roles in the expression of genetic information.Before the 1980s,the concept of biological enzymes was generally associated with proteins.Since the laboratories of Sidney Altman and Thomas Robert Cech have independently discovered that there are catalytically RNAs in cells,the concept of biological enzymes has expanded from proteins to include nucleic acids.In addition to naturally occurring enzymes,scientists can identify artificial biocatalysts in the laboratory by molecular evolution.These artificial enzymes play important roles in fields such as biotechnology and biomedicineDeoxyribozymes are single-stranded DNA molecules that catalyze specific chemical reactions.Many deoxyribozymes have been discovered by in vitro selection,catalyzing a wide variety of chemical reactions such as RNA cleavage,RNA ligation,DNA cleavage.Among them,DNA molecules that catalyze the sequence-specific cleavage of RNA have attracted significant attentions due to their immediate use as molecular tools in practical applications.RNA-cleaving deoxyribozymes have been incorporated into technologies that impact problems ranging from biotechnology and medicine to DNA computing and metal ion sensing Most of these utilize the general-purpose RNA-cleaving deoxyribozymes 8-17 and 10-23 isolated by Santoro and Joyce.Whether DNA molecules could fold into shapes with similar catalytic activity as 8-17 but unique structural properties is an interesting question that warrants further consideration.Here we report a novel RNA-cleaving deoxyribozyme called 10-12opt that functions with an equally small catalytic motif and an unusually short binding arm.This deoxyribozyme contains a 14-nucleotide catalytic core that preferentially catalyzes RNA cleavage at UN dinucleotide junctions(kobs=0.9h-1,for UU cleavage).Surprisingly,the left binding arm contains only three nucleotides and forms two canonical base pairs with the RNA substrate.Mutational analysis reveals that a riboguanosine residue 3-nucleotide downstream of cleavage site must not form canonical base pairing for the optimal catalysis,and this nucleobase likely participates in catalysis with its carbonyl O6 atom.Functional characterization of the 10-12opt motif addressing the divalent metal ion and pH dependence reveals that it has a observed reaction rate of 0.02 min-1 at pH 7.5 with 50 mM Mg2+.Given that deoxyribozyme 10-12opt features an unusually short left binding arm and selectively cleaves RNA substrates containing UNGRG consensus sequence,we chose three microRNA sequences with different cleavage sites that meet the above requirements as substrates.As expected,deoxyribozyme 10-12opt could effectively cleave all three microRNA substrates,while deoxyribozyme 8-17 could only cleave one RNA substrates.Futhermore,to demonstrate that deoxyribozyme 10-12opt could be used together with an aptamer to generate an analyte-responsive RNA-cleaving enzyme,we conjugated the RNA substrate to an ATP-binding DNA aptamer to build a molecular ATP detection platform.Time course experiments with varying concentration of ATP as well as other potentially interfering NTPs clearly demonstrated that the detection system could selectively respond to ATP and generate signal in a concentration-and time-dependent fashion.Together,these results suggest that this novel RNA-cleaving deoxyribozyme forms a distinct catalytic structure than 8-17 and that sequence space may contain additional examples of DNA molecules that can cleave RNA at site-specific locations.