Fig 1: Binding curve of anti-HE4 aptamers to ovarian cancer biomarker HE4 in 1/125 urine. The binding curve was constructed by plotting HE4 protein concentration with negative derivative fluorescence signal -d(RFU)/dT at the Tm corresponding to bound aptamer state. The non-linear regression model was used to calculate Kd value, showing good affinity in nanomolar range of anti-HE4 aptamers for HE4 in urine, with Kd (AHE1) = 87 ± 9 nM and Kd (AHE3) aptamer of 127 ± 28 nM. The results showed the average of the minimum 3 independent experiments. The results suggest that the described aptamers could have diagnostic potential as detection probes for HE4 in urine tests for ovarian cancer.
Fig 2: Thermofluorimetic analysis of the anti-HE4 aptamer binding to ovarian cancer biomarker HE4 in urine. The constant aptamer concentration of 100 nM was subjected to an increasing concentration of HE4 protein in urine ranging from 0 to 800 nM. The melting profile was analyzed from 4 °C to 90 °C. The binding thermal curves (melting DNA profile) were constructed by plotting temperature with negative derivative fluorescent signal -d(RFU)/dT. The results showed the average of the minimum 3 independent experiments. (A) The raw melting profile. The raw melting profile obtained for aptamers showed two distant peaks corresponding to free aptamer (no HE4) state and bound aptamer state (with HE4 protein). Upon binding to HE4 protein, more thermally stable species were present, with a shift to higher Tm. (B) The melting profile after subtraction of signal from aptamer only. After subtraction of the blank (aptamer only, no HE4), peaks of the aptamers bound to HE4 were visible, with a shift to higher Tm values, corresponding to 60 °C for AHE1 and 63 °C for AHE3, suggesting binding to target protein HE4.
Fig 3: Digital droplet PCR analysis of aptamers recovered during Hi-Fi SELEX to ovarian cancer biomarker HE4. (A) 1-D plot of Hi-Fi SELEX positive selection to target HE4 protein and counter selection to 6xhistidine peptide. Many sequences were detected in all cycles of selection. In the plot, each droplet from a sample in each SELEX cycle is plotted on the graph of the fluorescence intensity vs. droplet number. All positive droplets with anti-HE4 aptamer present (in blue), are those above the pink threshold line and are scored as positive. All negative droplets are those without target aptamers present (in dark gray) below the red threshold line and scored as negative. (B) ddPCR droplet enumeration throughout Hi-Fi SELEX positive and negative selection. The droplets were classified and quantified as positive (target anti-HE4 aptamers present), negative (no target aptamers) and total (positive + negative droplets). In the first few selection cycles, all droplets were positive (saturation) due to the excess of aptamer sequences present. After cycle 5, repartition was optimal, with positive and negative droplets present, due to the decrease of aptamers present. Decrease of aptamer sequences present equaled increase of sequence specificity to HE4 protein. As SELEX evolved, many specific sequences were being enriched to target HE4 protein. (C) Quantification of the anti-HE4 aptamers throughout Hi-Fi SELEX positive and negative selection. Numerous sequences were detected and quantified in each cycle. At the beginning of selection, a maximum number of sequences were observed (saturation). After cycle 5, sequence number and diversity decreased and specificity to HE4 increased. Most importantly, a higher number of sequences were observed as enriched in positive selection to target 6xhistidine-HE4 protein compared to counter-selection to 6xhistidine peptide (sample matrix) in both cycle 9 and 10, suggesting that enrichment of specific sequences to HE4 occurred.
Fig 4: Evolution of the top 10 most enriched DNA aptamers targeting ovarian cancer biomarker HE4 obtained with DNA sequencing. The results show clear enrichment of certain families (especially AHE1, AHE2 and AHE3) after positive selection to target HE4 protein in urine (from cycles C5+ to C10+), but not in counter-selection (C9- and C10-). C0 represents native library, C5+ to C10+ represent positive selection to target protein 6xhistidine-HE4 protein and C9- and C10- represent counter-selection to 6xhistidine peptide and beads (sample matrix). Green color = no enrichment to HE4, yellow = intermediate enrichment, orange = mild enrichment, red = intense enrichment to HE4 protein.
Fig 5: Predicted secondary structures of anti-HE4 aptamers. The secondary structures of DNA aptamers (AHE1 have three potential structures, AHE3 has one potential structure predicted) including full length 70-mer sequences were created using online DNA folding software Unafold (http://www.unafold.org) at temperature 25 °C and urine concentration of [Na+] = 55.4 mM and [Mg2+] = 4.4 mM.
Supplier Page from Abcam for Recombinant Human HE4 protein (His tag)