Rational Design Strategies for Stimuli‐Responsive DNAzymes Using Modified and Artificial Nucleotides

DNA, traditionally regarded as genetic material, has emerged as a versatile building block in molecular technology. Catalytic DNA oligomers known as DNAzymes, especially those capable of cleaving target RNA at specific sites, have shown great potential in DNA-based diagnostics, therapeutics, and dynamic DNA nanotechnology. For advanced applications, stimuli-responsive DNAzymes, which are activated only under specific conditions or by specific chemical stimuli, have attracted particular attention. Although such DNAzymes can be obtained by in vitro selection under strictly controlled conditions, more general strategies for their rational design are strongly needed. In this approach, stimuli-responsiveness is introduced into existing DNAzymes by sequence engineering or chemical modification. This review focuses on the rational design of stimuli-responsive RNA-cleaving DNAzymes using modified and artificial nucleotides. Here, we discuss representative strategies, including recent examples: (i) stimulus-induced reconstitution of split DNAzymes, (ii) activity control by strand cleavage or ligation, (iii) blocking of the catalytic core with removable oligonucleotides, (iv) caging with labile protecting groups, and (v) stimuli-induced conformational switching between inactive and active structures. These approaches enable externally controllable activation mechanisms, while maintaining the intrinsic catalytic activity of DNAzymes, providing a valuable toolkit for molecular sensing and DNA nanotechnology.