Chimeric Oligonucleotide Duplexes for siRNA Stability
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Solution Overview
Problem
Current siRNA technologies face challenges with poor serum stability, poor cellular uptake, and off-target effects, limiting their efficacy and specificity in gene silencing applications.
Innovation Solution
Development of oligonucleotide pairs comprising DNA-like and RNA-like residues, including 2′-substituted arabinonucleotides and locked nucleic acid nucleotides, which form duplexes that enhance serum stability and binding affinity, thereby improving gene silencing efficacy while reducing off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If native siRNA is used for gene silencing, then gene silencing activity is achieved, but serum stability is poor
Solution Approach 1:
The patent applies composite materials by creating chimeric oligonucleotide duplexes that combine DNA-like residues (2′F-ANA, 4′S-FANA) with RNA-like residues (2′F-RNA, LNA, RNA). This composite structure integrates the stability benefits of DNA-like modifications with the RNAi pathway compatibility of RNA-like structures, resolving the contradiction between gene silencing activity and serum stability.
Solution Approach 2:
The patent employs parameter changes by systematically modifying the chemical structure of oligonucleotides through various 2′-substituted arabinonucleotides and locked nucleic acid nucleotides. These parameter changes in sugar pucker, backbone flexibility, and base stacking interactions enhance serum stability while maintaining or improving gene silencing potency.
2Reliability
If native siRNA is used for gene silencing, then gene silencing activity is achieved, but cellular uptake is poor
Solution Approach 1:
The patent applies parameter changes by modifying the oligonucleotide structure with 2′-substituted arabinonucleotides and locked nucleic acid nucleotides, which alter cellular uptake parameters. The 2′-fluoro substitution and locked nucleic acid constraints change the conformational flexibility and charge distribution, facilitating improved cellular entry while maintaining gene silencing activity.
3Reliability
If native siRNA is used for gene silencing, then gene silencing activity is achieved, but off-target effects occur
Solution Approach 1:
The patent applies local quality by implementing site-specific modifications within the oligonucleotide sequence. Different positions in the sense and antisense strands receive different modifications (2′F-ANA, 4′S-FANA, 2′F-RNA, LNA), creating localized variations in stability and specificity that reduce off-target effects while preserving on-target gene silencing activity.
Solution Approach 2:
The patent uses composite materials by combining multiple types of modified nucleotides (DNA-like 2′F-ANA/4′S-FANA with RNA-like 2′F-RNA/LNA) in a chimeric structure. This composite approach allows different regions of the oligonucleotide to contribute different functional properties, enhancing specificity and reducing off-target effects.
4Stability of the object's composition
If chemically modified oligonucleotides are used to improve serum stability, then serum stability is improved, but binding affinity may be reduced
Solution Approach 1:
The patent applies parameter changes by systematically varying the 2′-substitution patterns and locked nucleic acid positions to optimize the balance between serum stability and binding affinity. The 2′-fluoro substitution maintains base pairing strength while the locked nucleic acid constraints enhance thermal stability, achieving both improved serum stability and maintained binding affinity.
Data Source
AI summary
Novel oligonucleotide pairs which can form a duplex comprising one or more DNA-like nucleotides (e.g., 2′-substituted arabinonucleotides (ANA)); in combination with one or more RNA-like nucleotides (e.g., 2′-substituted ribonucleotides (RNA) and/or locked nucleic acid nucleotides (LNA)), are disclosed. The use of such oligonucleotide duplexes, such as for silencing the expression of a nucleic acid or gene of interest using small interfering RNA (siRNA) technologies, is also disclosed.