Introduction. Testicular germ cell tumors (TGCTs) represent the most common solid malignancy in young men. MicroRNA-371a-3p (miR371) has been suggested as a sensitive biomarker in TGCTs since it increases in the plasma in the early stages. There are five other miRNAs that have a high similarity to the miR371 sequence, which makes it difficult to detect using PCR. The binary deoxyribozyme Dz contributes to the DNA nanomachine's function by serving as a molecular tool for selectively cleaving fluorophore-labeled substrates (F-sub) and can be developed to cleave F-sub in response, selectively, to miR371 recognition for TGCT detection.

Methods. A DNA nanomachine able to recognize miR371 selectively was designed, equipped with a reporter substrate delivery system for high sensitivity. Native polyacrylamide gel electrophoresis was performed to study the assembly and interactions among the designed DNA nanomachine. A fluorescence assay using a spectrophotometer was applied to investigate the selectivity and sensitivity of the designed DNA nanomachine using synthetic miRNA analytes, followed by the examination of its capability to detect miR371 in patient samples.

Results. The designed DNA nanomachine was successfully assembled. The suggested design is selective for miR371 in the presence of the other five similar miRNAs and can induce increasing fluorescence in response to increasing concentrations of miR371, with a limit of detection (LOD) of ~28.9 pM.

Conclusion. Due to the suggested design, we developed a DNA nanomachine based on binary Dz that is highly selective and sensitive for miR371. DNA nanomachines based on binary Dz can be considered as a simple, inexpensive, sequence-specific, and sensitive tool for early TGCT diagnoses.