Structure-Based Design of RNA Aptamers Targeting the Oncogenic KRAS G12D Variant

Background: Mutations in the KRAS gene, particularly the G12D variant, play a critical role in colorectal cancer (CRC) by driving oncogenic signaling and conferring resistance to targeted therapies. Despite recent progress in KRAS inhibition, G12D remains a challenging target with limited therapeutic options. Objective: This study aimed to identify and characterize an RNA aptamer capable of selectively binding to and inhibiting the KRAS G12D protein through a comprehensive in silico approach. Methods: A pool of 1000 randomized 45-mer RNA sequences was computationally generated and screened using secondary structure analysis, thermodynamic profiling, and 3D structure prediction. Top candidates were docked against KRAS G12D (PDB ID: 6ULI) using the HDOCK server. The most promising aptamer, RNA-106, was further evaluated through 200-nanosecond molecular dynamics (MD) simulations and MM-PBSA free energy calculations. Results: RNA-106 exhibited the most favorable binding profile, with a docking score of –324.11 and a confidence score of 0.9702. It formed stable hydrogen bonds with residues in the Switch I (ARG-21, GLN-35) and Switch II (GLY-237) domains of KRAS—key regions for effector protein binding. MD simulations confirmed the long-term stability of the KRAS–RNA-106 complex, maintaining 7–9 hydrogen bonds throughout the trajectory. MM-PBSA analysis estimated a binding free energy of –33.76 kcal/mol. Structural modeling suggests RNA-106 may interfere with downstream signaling via steric blocking and allosteric modulation. Conclusion: This study identifies RNA-106 as a promising RNA aptamer that stably and specifically targets KRAS G12D, potentially inhibiting its oncogenic function. These findings provide a foundation for experimental validation and further development of RNA-based therapeutics for KRAS-driven cancers.