Computationally Guided Design and Experimental Validation of ssDNA Aptamers Targeting the SARS-CoV-2 Spike Protein
- 1Centro de Bioinformática, Simulación y Modelado (CBSM), Universidad de Talca, Talca, Chile
- 2Centro de Nanomedicina, Diagnóstico y Desarrollo de Fármacos (ND3), Laboratorio de Fisiología Molecular, Escuela de Medicina, Universidad de Talca, Talca, Chile
- 3Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28660 Boadilla del Monte, Spain
Abstract
Aptamer development has traditionally relied on iterative, labor-intensive experimental approaches. Recent advances in bioinformatics and computational power now enable the rational in silico design of high-affinity nucleic acid ligands, drastically reducing time and cost. Here, we present an integrated computational pipeline for designing ssDNA aptamers targeting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike (S) protein. A library of 4000 randomly generated 20-nucleotide sequences with defined base ratios (C:G:A:T = 3:3:2:2) was screened based on secondary structure and free energy. The 100 most stable sequences (ΔG ≤ −4.1 kcal/mol) were modeled in three dimensions, yielding 1000 conformations. The 100 structures with the lowest potential energies (≤−78,051.7 kcal/mol) were selected for molecular docking with the RBD in the closed, open, and transitional states. Two aptamers—model-2-seq_2714 (seq_2714) and model-5-seq_1397 (seq_1397)—were selected for experimental assessment based on their favorable predicted docking scores against the RBD. Western blot and dot blot assays showed detectable recognition of the recombinant RBD by both aptamers under the experimental conditions tested. Exploratory testing in a limited number of clinical and environmental samples further showed that detectable signals could be obtained in selected complex matrices. These findings provide an initial proof-of-principle assessment of a computational-first strategy for prioritizing candidate aptamers for subsequent experimental testing.
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BibTeX
@article{montesinos2026computationally,
title = {{Computationally Guided Design and Experimental Validation of ssDNA Aptamers Targeting the SARS-CoV-2 Spike Protein}},
author = {José C. E. Márquez Montesinos and Ana Maria Bernardez and Yuliet Mazola and Lucianna Vaccaro and Yanira Sáez-Álvarez and Rubén Agudo and Elizabeth Valdivieso and Sergio Llorens-Berzosa and Soledad Fenoy and Wendy González and Fernando Izquierdo and Dolores Ollero and Carmen del Aguila and Carolina Hurtado-Marcos},
journal = {Biomolecules},
year = {2026},
month = oct,
doi = {10.3390/biom16101476},
url = {https://www.mdpi.com/2218-273X/16/10/1476},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Computationally Guided Design and Experimental Validation of ssDNA Aptamers Targeting the SARS-CoV-2 Spike Protein
AU - José C. E. Márquez Montesinos
AU - Ana Maria Bernardez
AU - Yuliet Mazola
AU - Lucianna Vaccaro
AU - Yanira Sáez-Álvarez
AU - Rubén Agudo
AU - Elizabeth Valdivieso
AU - Sergio Llorens-Berzosa
AU - Soledad Fenoy
AU - Wendy González
AU - Fernando Izquierdo
AU - Dolores Ollero
AU - Carmen del Aguila
AU - Carolina Hurtado-Marcos
PY - 2026
DA - 2026/10/01
JO - Biomolecules
DO - 10.3390/biom16101476
UR - https://www.mdpi.com/2218-273X/16/10/1476
AB - Aptamer development has traditionally relied on iterative, labor-intensive experimental approaches. Recent advances in bioinformatics and computational power now enable the rational in silico design of high-affinity nucleic acid ligands, drastically reducing time and cost. Here, we present an integrated computational pipeline for designing ssDNA aptamers targeting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike (S) protein. A library of 4000 randomly generated 20-nucleotide sequences with defined base ratios (C:G:A:T = 3:3:2:2) was screened based on secondary structure and free energy. The 100 most stable sequences (ΔG ≤ −4.1 kcal/mol) were modeled in three dimensions, yielding 1000 conformations. The 100 structures with the lowest potential energies (≤−78,051.7 kcal/mol) were selected for molecular docking with the RBD in the closed, open, and transitional states. Two aptamers—model-2-seq_2714 (seq_2714) and model-5-seq_1397 (seq_1397)—were selected for experimental assessment based on their favorable predicted docking scores against the RBD. Western blot and dot blot assays showed detectable recognition of the recombinant RBD by both aptamers under the experimental conditions tested. Exploratory testing in a limited number of clinical and environmental samples further showed that detectable signals could be obtained in selected complex matrices. These findings provide an initial proof-of-principle assessment of a computational-first strategy for prioritizing candidate aptamers for subsequent experimental testing.
SN - 2218-273X
ER -