Non-saturated nucleic acid probes with a broad dynamic range

Molecular probes are essential for analytical detection but encounter major limitations, such as a limited dynamic range, the need for probe concentrations to exceed target concentrations, and restricted reusability. These issues stem from the irreversible binding of probes to targets, resulting in signal saturation when the target concentration surpasses that of the probe. To overcome these challenges, we developed a non-saturating nucleic acid probe (NSNAP) inspired by dynamic DNA nanotechnology and non-equilibrium chemistry. The NSNAP combines an affinity probe with an enzyme that degrades the target within the probe-target complex, allowing the probe to reset and produce continuous signals before complete target degradation. This design dramatically expands the dynamic range by up to 5000-fold, compared to the typical 81-fold range of conventional probes, and enables the NSNAP to detect targets at concentrations up to 250 times greater than that of the probe. Moreover, the NSNAP is reusable, with experimental validation demonstrating at least seven cycles of reuse, enhancing both cost-effectiveness and sustainability. Exonuclease III or λ Exonucleases can support NSNAP operation, and these enzymes can be pre-loaded with DNA probes. Using the NSNAP, we successfully quantified both viral genes (HIV, HHV, HPV) and bacterial marker genes (oprL, dnaJ, ddl) across concentrations from 1 to 1000 fM in complex biological matrices, achieving strong linear correlations. This innovative approach offers great potential for advancing DNA nanotechnology-based diagnostic tools for scientific research and clinical applications.