Publication:

In silico selex in designing novel dna aptamer hairpin for in vitro detection of he4 : an ovarian cancer biomarker

Ovarian cancer (OC) poses a significant risk as it usually remains asymptomatic until advanced stages, resulting in delayed diagnosis and lowering the chances of survival. This fifth most common cancer among women globally lacks efficient screening approaches for early stages OC (stage I and II), increasing the threat of OC progression and mortality. In this study, the in silico method was applied to design DNA aptamer hairpins for the detection of human epididymis protein 4 (HE4), an OC biomarker. The in silico work outcome was supported by an in vitro assay. The work began with the HE4 protein modelling using AlphaFold, I-TASSER, and Robetta protein structure prediction servers. Subsequently, molecular dynamics (MD) simulation was conducted on each predicted model for 100 ns using the OPLS force field, for structure refinement. The tertiary structure quality was validated by PROCHECK and ERRAT, showing the refined model from AlphaFold, RF1, was the highest-quality HE4 tertiary conformation. All amino acids were located in the favoured regions of Ramachandran plot with ERRAT overall quality score of 97.701. Next, this HE4 structure was docked using AutoDock Vina with four HE4 aptamer candidates (A1, A2, A3, and A4). The HE4-A4 binding energy was -6.0 kcal/mol, and the complex formed 24 hydrogen bonds (H-bonds); 5 identified at the aptamer hairpin loop region. A4 was chosen as the most suitable candidate to be utilised in the designing of the DNA hairpin, as it exhibited good affinity with highest number of H-bonds at the hairpin loop. To initiate the in silico design of new hairpins, the 25-mer A4 aptamer was truncated at the hairpin region, 5’-CGCAAG-3’ and the stem was extended, forming the 5’-GCGCAAGC-3’ sequence. The loop nucleotides, -GCAA-, were substitutionally mutated, producing 256 sequences. These 256 hairpins were docked with HE4 individually, and H16, H101, and H256 have shown good binding affinities with binding energies ranging between -10.6 and -11.6 kcal/mol. Consequently, 100 ns MD simulation using CHARMM27 force field was applied to the HE4-H16, HE4-H101, and HE4-H256 complexes. Based on the RMSD, radius of gyration, number of H-bonds, H-bond occupancy, and the overall total energy, H256 was deduced as the most promising DNA hairpin against HE4 marker with great affinity and stability throughout the simulation. This H256 hairpin was synthesised and its binding with HE4 was analysed via in vitro electrophoretic mobility shift assay (EMSA). Based on the DNA band intensities, the designed H256 (3.27 %) bound four times better to HE4 than the A4 aptamer (0.84 %). Finally, a preliminary study for future diagnostic potential was carried out by conjugating gold nanoparticle (GNP) with H256. The FTIR and Raman spectra confirmed the presence of amide group, formed by the successful conjugation of the carboxylated GNP with the aminated-H256. The GNP solution changed from red to purple-red, indicating the size increment after conjugation, that was confirmed by particle size analyser. In conclusion, H256 is a promising DNA hairpin in HE4 screening and is recommended for future development of a fully functional OC diagnostic kit, suitable to be used in routine screening for all women, with or without symptoms. This potentially improves the detection among early stages (I and II) patients, enhancing patients’ outcome.