Zn²⁺‐Dependent DNAzymes: From Solution Chemistry to Analytical, Materials and Therapeutic Applications

Abstract

Since 1994, deoxyribozymes or DNAzymes have been in vitro selected to catalyze various types of reactions. Metal ions play a critical role in DNAzyme catalysis, and Zn2+ is a very important one among them. Zn2+ has good biocompatibility and can be used for intracellular applications. Chemically, Zn2+ is a Lewis acid and it can bind to both the phosphate backbone and the nucleobases of DNA. Zn2+ undergoes hydrolysis even at neutral pH, and the partially hydrolyzed polynuclear complexes can affect the interactions with DNA. These features have made Zn2+ a unique cofactor for DNAzyme reactions. This review summarizes Zn2+‐dependent DNAzymes with an emphasis on RNA‐/DNA‐cleaving reactions. A key feature is the sharp Zn2+ concentration and pH‐dependent activity for many of the DNAzymes. The applications of these DNAzymes as biosensors for Zn2+, as therapeutic agents to cleave intracellular RNA, and as chemical biology tools to manipulate DNA are discussed. Future studies can focus on the selection of new DNAzymes with improved performance and detailed biochemical characterizations to understand the role of Zn2+, which can facilitate practical applications of Zn2+‐dependent DNAzymes.

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

“…The two classes of deoxyribozymes (Zn-III & Zn-IV) identified here behave very similar to the two previously reported classes (Zn-I & Zn-II) ( 10 ) in several aspects, including the sharp pH range (∼7) and Zn 2+ concentration range (∼2 mM) (Figure 6 ), which is near the critical point of forming Zn(OH) 2 /ZnO precipitation. At these conditions, it is believed that the polynuclear Zn 2+ complexes generated in the solution act as the active species to promote DNA hydrolysis ( 13 ). Also all four classes favor high reaction temperatures, with improved k obs beyond 37°C.…”

“…This method relies on an enzyme named CircLigase to circularize DNAs twice in one round of selective amplification: the first (step i) is to turn a linear DNA library (145 nt) into circular, thus enabling the separation of self-hydrolyzed DNAs (linear) from un-cleaved ones (circular) in the following steps by denaturing polyacrylamide gel electrophoresis (dPAGE); the second (step v) is to re-ligate the collected hydrolyzed DNAs (linear) back into circles as effective templates for PCR amplification to rebuild the library for next round of selection (Figure 1 ). Based on this strategy, two classes (I & II) of highly active deoxyribozymes had been identified to selectively hydrolyze DNA in the presence of Zn 2+ ( 10 ), with a robust member (I-R3) of class I prevailing in several DNA-based manipulations ( 13–28 ). Due to their robustness, neatness, steadiness, and easiness-to-operation, more types of DNA-hydrolyzing DNAs are urgently needed by technology developers in order to satisfy various demands ( 13 , 22 , 24 ).…”

“…Based on this strategy, two classes (I & II) of highly active deoxyribozymes had been identified to selectively hydrolyze DNA in the presence of Zn 2+ ( 10 ), with a robust member (I-R3) of class I prevailing in several DNA-based manipulations ( 13–28 ). Due to their robustness, neatness, steadiness, and easiness-to-operation, more types of DNA-hydrolyzing DNAs are urgently needed by technology developers in order to satisfy various demands ( 13 , 22 , 24 ).…”

Zn2+‐Coordination‐Driven RNA Assembly with Retained Integrity and Biological Functions

“…Thes trong Zn 2+ /phosphate interactions have been well documented. [32,33] In both Fe 2+ /G20 and Zn 2+ /rG20, G20 formed the gel-like network structures,w hich can be attributed to the strong inter-guanine interactions. [34] To investigate the tolerance of consecutive Gb ases,R NA sequences with serial Aa nd G base compositions were tested (Figure S9).…”

Intelligent demethylase-driven DNAzyme sensor for highly reliable metal-ion imaging in living cells

“…Toward this goal, DNAzymes have aroused wide interest due to their specific requirement for metal ion cofactors that contribute to the high catalytic activities of DNAzymes. [23][24][25][26] Accordingly, a series of DNAzyme-powered fluorescence sensors have been developed for imaging metal ions, such as Zn 2+ , 27,28 Mg 2+ , 29 Pb 2+ , 30 Na + , 31 etc. Despite these rapid progresses, the extensive biosensing application of DNAzymes in live cells is challenged by the annoying signal leakage (offtarget activation), originating from their invariably Catalytic ON state that can non-specifically react with the surrounding metal ions.…”