A DNA triangular prism scaffold-guided directionally-controlled derailment-free DNAzyme walker

INTRODUCTION: Deoxyribozyme (DNAzyme) with RNA cleavage activity, including a DNAzyme walker, has been extensively explored as the tool for cancer diagnosis and therapy due to its excellent programmability, stability, and activity. However, the precise control over the directionality and progressivity of molecular movement towards its predetermined destination remains a formidable challenge. OBJECTIVES: We aim to construct a three-dimensional DNA walker capable of addressing the uncertainty of motion direction because of the deviation from the predesignated track in uniform solutions. METHODS: The cleavage sequence of substrates by DNAzyme was explored through gel electrophoresis analysis . RESULTS: Gel electrophoresis analyses show that DNAzyme cleaves the substrates in a sequential manner and preferentially digests the ones in the shortest spatial distance regardless of their nature, indicating a distance-dependent cleavage process. Moreover, the substrate stands freely moving in a homogenous solution (not confined onto DNA scaffold) cannot be cleaved, implying that the derailment of DNAzmye walker from the tri-prism scaffold is completely circumvented. When the adjacent substrates were removed to leave the vacant sites, the cleavage order of substrates is changed, indicating that the controllability of cleavage behavior of DNAzyme is realized. CONCLUSION: DNA triangular prism-controlled DNAzyme walker circumvents the intractable derailment event and has several advantages, such as low cost, programmability, high cleavage specificity and directional controllability, showing great potential applications in biosensing, cancer, diagnosis and therapy.