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Abstract <p><b>Objective:</b> This study aimed to improve the DNA-nanomachine (DNM) platform based on the 10– 23 RNA-cleaving DNAzyme for efficient recognition of dsDNA at near-physiological temperatures. Two DNM variants with distinct scaffold architectures were designed and compared with the binary deoxyribozyme (BiDz) probe in terms of sensitivity and selectivity. <b>Methods:</b> <i>In vitro</i> fluorescence measurements and gel shift assays were used to assess the secondary structures, sensitivity, and selectivity of the designs. <b>Results and Discussion:</b> All three sensors successfully detected a synthetic HPV-16 ssDNA analyte, with BiDz demonstrating the highest sensitivity and the lowest limit of detection (10 pM). DNM-I exhibited higher background fluorescence due to partial self-activation, while DNM-II showed improved background control but slightly reduced sensitivity. Both DNMs retained excellent single-nucleotide selectivity (99.9%). <b>Conclusions:</b> The scaffold topology was found to strongly influence sensor performance, affecting catalytic activity and background fluorescence. The introduction of displaced strand-binding elements resulted in poorer performance compared to multiple-binding armed DNMs. Although both DNMs retained high selectivity, further optimization is required to achieve efficient dsDNA recognition at physiological temperatures.</p>

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Optimizing Scaffold Structures to Enhance DNAzyme-Based Nanomachine Activity in Double-Stranded DNA

  • 期刊论文

  • 发表日期：

    2025年12月1日

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  • M. Ateiah,
  • M. S. Rubel

摘要

Abstract

Objective: This study aimed to improve the DNA-nanomachine (DNM) platform based on the 10– 23 RNA-cleaving DNAzyme for efficient recognition of dsDNA at near-physiological temperatures. Two DNM variants with distinct scaffold architectures were designed and compared with the binary deoxyribozyme (BiDz) probe in terms of sensitivity and selectivity. Methods: In vitro fluorescence measurements and gel shift assays were used to assess the secondary structures, sensitivity, and selectivity of the designs. Results and Discussion: All three sensors successfully detected a synthetic HPV-16 ssDNA analyte, with BiDz demonstrating the highest sensitivity and the lowest limit of detection (10 pM). DNM-I exhibited higher background fluorescence due to partial self-activation, while DNM-II showed improved background control but slightly reduced sensitivity. Both DNMs retained excellent single-nucleotide selectivity (99.9%). Conclusions: The scaffold topology was found to strongly influence sensor performance, affecting catalytic activity and background fluorescence. The introduction of displaced strand-binding elements resulted in poorer performance compared to multiple-binding armed DNMs. Although both DNMs retained high selectivity, further optimization is required to achieve efficient dsDNA recognition at physiological temperatures.