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A new concept for the design of antisense oligonucleotides based on nucleic acid thermostability
1Department of Chemistry, Faculty of Science and Engineering and High Technology Research Center, Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan. sugimoto@konan-u.ac.jp
Abstract:
The antisense method is one of the most promising anti-cancer methods, however, the design of antisense oligonucleotides is difficult because many factors affecting their activitiy and stability must be considered. Recently, the oligonucleotide stabilities related to the antisense effects were quantitatively investigated based on nearest-neighbor parameters. We demonstrated that DeltaG(o) (37, hyb), a free energy change for the hybridization of antisense oligodeoxynucleotides (ODNs) with target RNAs is related to the RNase H cleavage of TAg (SV40 large T antigen) mRNA, the expression of a rabbit globin mRNA, and the protein function encoded by hMDR1 (human multidrug resistance-1) mRNA, while DeltaG(o) (37, hp), a free-energy change for hairpin formations of the antisense ODNs significantly affected the arrest efficiency of the DHFR (dihydrofolate reductase) mRNA transcription, the expression of the proalpha1(I) chain of human, and the hybridization extent for HIV-1 alpha-1. For ras RNA (Ha-ras mRNA), DeltaG(o) (37, sc), a free energy change for the conformational change of the mRNA required for antisense ODN binding showed the best correlation with the equilibrium constants for the hybridization with their target RNA. On the other hand, the antisense effects ifor the HSV-1 IE5 (herpes simplex virus type 1 immediate early pre-mRNA5) showed less of a relationship to the hybridization stability of the antisense ODNs with the target pre-mRNA, because the antisense ODNs targeting the pre-mRNA must collapse its secondary structure around the splicing site to cancel out the expected antisense effects. Based on these results, we illustrate a new concept for the design of antisense ODNs based on DeltaG(o) (37, hyb), DeltaG(o) (37, hp), and DeltaG(o) (37, sc).
Insights
Designing effective antisense oligonucleotides (ODNs) for cancer therapy is challenging. This study quantitatively links thermodynamic parameters like hybridization (DeltaG(o) (37, hyb)), hairpin formation (DeltaG(o) (37, hp)), and conformational change (DeltaG(o) (37, sc)) to antisense activity, offering a new design strategy.
Area of Science:
- Biochemistry
- Molecular Biology
- Antisense Technology
Background:
- Antisense oligonucleotides (ODNs) show promise for cancer treatment, but their design is complex due to numerous factors influencing activity and stability.
- Quantitative investigations of oligonucleotide stability and antisense effects are crucial for optimizing therapeutic design.
Purpose of the Study:
- To quantitatively investigate the relationship between thermodynamic parameters of antisense ODNs and their biological activity.
- To establish a novel concept for designing antisense ODNs based on thermodynamic stability parameters.
Main Methods:
- Utilized nearest-neighbor parameters to quantitatively assess oligonucleotide stabilities.
- Correlated free energy changes for hybridization (DeltaG(o) (37, hyb)), hairpin formation (DeltaG(o) (37, hp)), and conformational change (DeltaG(o) (37, sc)) with various antisense effects.
- Evaluated effects on mRNA cleavage, protein expression, transcription arrest, and hybridization extent across different target mRNAs (TAg, globin, hMDR1, DHFR, proalpha1(I), HIV-1, ras, HSV-1 IE5).
Main Results:
- DeltaG(o) (37, hyb) correlated with RNase H cleavage, mRNA expression, and protein function.
- DeltaG(o) (37, hp) significantly affected transcription arrest and hybridization extent.
- DeltaG(o) (37, sc) showed the best correlation for ras RNA hybridization; however, secondary structure disruption was key for HSV-1 IE5 pre-mRNA.
- Established distinct correlations between thermodynamic parameters and specific antisense outcomes.
Conclusions:
- A new concept for antisense ODN design integrating DeltaG(o) (37, hyb), DeltaG(o) (37, hp), and DeltaG(o) (37, sc) is proposed.
- Understanding these thermodynamic parameters is essential for predicting and optimizing antisense oligonucleotide efficacy.
- The findings provide a framework for rational design of more effective antisense-based therapeutics.