2.4. PNA:DNA and DGL:DNA heteroduplex formation and DCL reaction in human plasma

PNA:DNA hybridisation. Hybridisation assays were performed by incubating ssDNA (1 μM) and PNA probes (1 μM) in SCD buffer (pH 6) in a 1.5 mL tube placed in a Thermo-Shaker at 80 °C for 5 min, with shaking at 1400 rpm. Then, the reaction was cooled to 41 °C at 3 °C/min followed by incubation for 1 h.

DCL reactions. DCL reactions were performed by incubating ssDNA (1 μM) and DGL probe (1 μM) together with SMART-C-biotin (5 μM) under the same conditions described above for PNA:DNA heteroduplex formation. Once the temperature reached 41 °C, sodium cyanoborohydride was added to a final concentration of 1 mM, and the reaction was incubated at 41 °C for 1 h while shaking at 1400 rpm.

To assess the DCL reaction occurs in a complex biological matrix, a calibration curve was performed in diluted human plasma. Five quantities of target (ssDNA-KRAS-wt-Cy5) were spiked in (2.5 pmol, 12.5 pmol, 25 pmol, 37.5 pmol and 50 pmol), followed by the addition of SCD buffer (pH 6; 1:4) containing 1 μM of DGL probe in a total volume of 50 μL. Then, the reaction was mixed and incubated at 80 °C for 5 min, shaken at 1400 rpm, cooled to 41 °C, and incubated for 1 h. The human plasma samples from healthy donors were provided by the Biobank of the Andalusian Public Health System (agreement number S1900297) approved by the Committee of Ethics of Biomedical Research of Andalusia (study code: 32,160,041).

In all cases, after incubation at 41 °C, 5 × 10⁸ Strep-FAM-NPs (13) were added and incubated at 25 °C for 1 h with shaking at 1400 rpm. For more details about the determination of the number of NPs, see Supporting Information 2.7. Finally, the NPs were washed with PBS 0.1% tween (3 × 200 μL; 5 min at 12,000 rpm)), resuspended in 200 μL of PBS, and analysed by flow cytometry.