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Computational design and validation of effective siRNAs to silence oncogenic KRAS
Prasanna Srinivasan Ramalingam1, Sivakumar Arumugam1
1Protein Engineering Lab, School of Biosciences and Technology, Vellore Institute of Technology, Vellore, India.
Abstract:
Oncogenic KRAS mutations drive cancer progression in lung, colon, breast, and pancreatic ductal adenocarcinomas. Apart from the current strategies, such as KRAS upstream inhibitors, downstream effector inhibitors, interaction inhibitors, cell cycle inhibitors, and direct KRAS inhibitors, against KRAS-mutated cancers, the therapeutic small interfering RNAs (siRNAs) represent a promising alternative strategy that directly binds with the target mRNA and inhibits protein translation via mRNA degradation. Here, in the present study, we utilized various in silico approaches to design potential siRNA candidates against KRAS mRNA. We have predicted nearly 17 siRNAs against the KRAS mRNA, and further through various criteria, such as U, R, and A rules, GC%, secondary structure formation, mRNA-siRNA duplex stability, Tm (Cp), Tm (Conc), and inhibition efficiency, they have been filtered into 4 potential siRNAs namely siRNA8, siRNA11, siRNA12, and siRNA17. Further, the molecular docking analysis revealed that the siRNA8, siRNA11, siRNA12, and siRNA17 showed higher negative binding energies, such as - 379.13 kcal/mol, - 360.19 kcal/mol, - 288.47 kcal/mol, and - 329.76 kcal/mol, toward the human Argonaute2 protein (hAgo2) respectively. In addition, the normal mode analysis of the hAgo2-siRNAs complexes indicates the structural changes and deformation of the hAgo2 protein upon the binding of siRNA molecules in the dynamic environment which suggests that these siRNAs could be effective. Finally, we conclude that these 4 siRNAs have therapeutic potential against KRAS mRNA and also have to be studied in vitro and in vivo to evaluate their specificity toward mutant KRAS (not degrading wild-type KRAS). Also, the current challenges in the use of siRNA therapeutics could be overcome by the emerging siRNA delivery methods, such as Antibody-siRNA conjugates (ARCs) and Gelatin-Antibody Delivery System (GADS), in the near future and these siRNAs could be employed as potential therapeutic agents against KRAS-mutated cancers.
Supplementary Information:
The online version contains supplementary material available at 10.1007/s13205-023-03767-w.
Insights
Researchers designed and filtered four small interfering RNAs (siRNAs) targeting KRAS mRNA for cancer therapy. These siRNAs show therapeutic potential by inhibiting KRAS protein translation and may overcome delivery challenges in KRAS-mutated cancers.
Area of Science:
- Oncology
- Molecular Biology
- Bioinformatics
Background:
- Oncogenic KRAS mutations are key drivers in various cancers, including lung, colon, breast, and pancreatic ductal adenocarcinomas.
- Current therapeutic strategies for KRAS-mutated cancers are limited, necessitating novel approaches.
- Small interfering RNAs (siRNAs) offer a promising strategy by directly targeting and degrading KRAS mRNA to inhibit protein translation.
Purpose of the Study:
- To design and identify potential siRNA candidates against KRAS mRNA using in silico methods.
- To evaluate the efficacy and binding potential of designed siRNAs against the human Argonaute2 protein (hAgo2).
- To assess the therapeutic potential of identified siRNAs for KRAS-mutated cancers.
Main Methods:
- In silico screening of numerous siRNA candidates against KRAS mRNA.
- Filtering of siRNAs based on specific criteria including U, R, and A rules, GC content, secondary structure, duplex stability, and inhibition efficiency.
- Molecular docking and normal mode analysis to assess binding affinity and structural impact on hAgo2.
Main Results:
- Nearly 17 siRNAs were predicted, filtered down to 4 potent candidates: siRNA8, siRNA11, siRNA12, and siRNA17.
- Molecular docking revealed significant negative binding energies for these siRNAs with hAgo2, indicating strong interaction.
- Normal mode analysis suggested that siRNA binding induces structural changes in hAgo2, supporting their potential effectiveness.
Conclusions:
- The four identified siRNAs (siRNA8, siRNA11, siRNA12, siRNA17) demonstrate therapeutic potential against KRAS mRNA.
- Further in vitro and in vivo studies are required to confirm specificity for mutant KRAS and therapeutic efficacy.
- Emerging delivery systems like Antibody-siRNA conjugates (ARCs) may overcome current challenges for siRNA therapeutics in KRAS-mutated cancers.
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