siNA Molecules with 2′ Internucleoside Linkage for RISC Loading
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Solution Overview
Problem
Current RNAi technologies face limitations in using 2′-5′ linked ribonucleotides within siRNA oligonucleotides for effective target gene expression degradation due to potential conformational issues with the 5′ end of the antisense strand, which is crucial for loading into RISC and subsequent cleavage.
Innovation Solution
Development of siNA molecules with a 5′ modified nucleotide at the 5′ end featuring a 2′ internucleoside linkage, allowing for improved interaction with the RNA-induced silencing complex (RISC) and enhanced gene silencing activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If 2'-5' linked ribonucleotides are used in siRNA oligonucleotides, then nuclease resistance and binding affinity are improved, but conformational issues at the 5' end of the antisense strand reduce RISC loading efficiency
Solution Approach 1:
The patent applies local quality by using 2'-5' linked ribonucleotides specifically at positions 2-4 of the antisense strand while maintaining conventional 3'-5' linkages at other positions. This localized modification approach preserves nuclease resistance benefits in the internal regions while avoiding conformational issues at the critical 5' end, thereby maintaining RISC loading efficiency.
Solution Approach 2:
The siRNA oligonucleotide is segmented into different regions with different linkage types: the 5' end region uses conventional 3'-5' linkages to ensure proper RISC loading, while internal regions (positions 2-4) use 2'-5' linkages to provide nuclease resistance. This segmentation allows each region to optimize its function independently.
2Strength
If 2'-5' linked ribonucleotides are incorporated into siRNA, then binding affinity to target mRNA is enhanced, but gene silencing activity is reduced due to conformational issues
Solution Approach 1:
The patent enhances binding affinity locally at positions 2-4 of the antisense strand using 2'-5' linked ribonucleotides, which provide stronger binding to target mRNA. Meanwhile, the 5' end region maintains conventional linkages to ensure proper RISC loading and gene silencing activity, thus achieving both enhanced binding and maintained productivity.
Solution Approach 2:
The oligonucleotide is divided into functional segments: the 5' end segment ensures proper RISC loading and gene silencing, while the internal segment (positions 2-4) provides enhanced binding affinity through 2'-5' linkages. This segmentation allows simultaneous optimization of both binding strength and gene silencing activity.
3Productivity
If conventional 3'-5' phosphodiester linkages are used, then RISC loading and gene silencing proceed efficiently, but nuclease stability is reduced
Solution Approach 1:
The patent applies 2'-5' linked ribonucleotides locally at positions 2-4 of the antisense strand to enhance nuclease stability, while maintaining conventional 3'-5' linkages at the 5' end to ensure efficient RISC loading and gene silencing. This localized approach allows simultaneous achievement of both stability and efficiency.
Solution Approach 2:
The siRNA oligonucleotide is segmented into stability-enhancing regions (positions 2-4 with 2'-5' linkages) and functionally critical regions (5' end with 3'-5' linkages). This segmentation allows each segment to optimize its specific function: internal segments provide nuclease stability while the 5' end segment ensures efficient gene silencing.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The siNA molecules demonstrate improved activity in inhibiting gene expression by effectively mediating RNA interference, offering potential therapeutic applications through enhanced binding and stability.
Implementation Method 1
the siNA molecules of the present invention comprise an antisense strand having a 5′ modified nucleotide having a 2′ internucleoside linkage
Implementation Method 2
RNA interference (RNAi) is an evolutionarily conserved cellular mechanism of post-transcriptional gene silencing found in fungi, plants and animals that uses small RNA molecules to inhibit gene expression in a sequence-specific manner
Data Source
AI summary
The present invention relates to RNAi molecules, and compositions thereof, comprising a 2′ internucleoside linkage connecting the nucleotide at position 1 and the nucleotide at position 2 at the 5′ end of the antisense strand. Specifically, the invention relates to single- and double-stranded short interfering nucleic acid (siNA) molecules that are capable of mediating RNA interference comprising 5′ modified nucleotides that comprise, among other potential modifications, a 2′ internucleoside linkage. The invention further relates to 5′ modified nucleotides used as reagents to generate the RNAi molecules of the invention and methods of using the disclosed RNAi molecules.