Cyclic Phosphonate siRNA Modification for Stable Ago2 Binding
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Solution Overview
Problem
Existing phosphonate modifications at the 5′ end of siRNA, such as 5′-E-VP, allow for free rotation, leading to unfavorable conformations that reduce binding to the Ago2 protein, thereby decreasing siRNA activity and stability.
Innovation Solution
A rigid loop structure is introduced to lock the phosphonate group in the Ago2 binding conformation, using a four-membered carbocyclic ring to stabilize the binding to the Ago2 protein, enhancing siRNA activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a phosphonate modification is introduced at the 5′ end of siRNA to replace 5′-phosphate, then resistance to endogenous phosphatases is improved, but the C—C bond can freely rotate leading to unfavorable conformations that reduce binding to Ago2 protein
Solution Approach 1:
The patent changes the conformational parameter of the phosphonate group by introducing a rigid loop structure that locks the C—C bond rotation, fixing the phosphonate group in a specific conformation that is favorable for Ago2 binding while maintaining phosphatase resistance
Solution Approach 2:
The patent transitions from a dynamic system where the C—C bond can freely rotate to a static system where the rigid loop structure constrains the phosphonate group in a fixed conformation, ensuring consistent and favorable binding to Ago2 protein
2Ease of operation
If the C—C bond is made rigid to lock the phosphonate group in Ago2 binding conformation, then binding to Ago2 protein is improved, but the structural complexity increases
Solution Approach 1:
The patent embeds the rigid loop structure within the phosphonate modification, creating a nested configuration where the loop is integrated into the existing phosphonate group structure rather than adding a separate complex element, thus minimizing overall structural complexity
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 rigid loop structure improves siRNA activity by forming multiple salt bridges and hydrogen bonds with Ago2, mimicking the natural 5′-phosphate binding, thereby stabilizing the siRNA complex and enhancing its functional efficacy.
Implementation Method 1
forming multiple salt bridges and hydrogen bonds, as well as two hydrogen bonds with Tyr529 and Cys526, respectively
Implementation Method 2
forming multiple salt bridges and hydrogen bonds, as well as two hydrogen bonds with Tyr529 and Cys526, respectively
Implementation Method 3
The 5′-phosphate of the guide strand (antisense strand) of siRNA stabilizes the complex formed by the guide strand and Ago2 through electrostatic interactions with cationic amino acid residues near the interface between the MID and PIWI domains of the Ago2 protein
Data Source
AI summary
The present invention provides an oligonucleotide having a cyclic phosphonate modification. The oligonucleotide of the present invention exhibits one or more among enhanced stability, reduced off-target toxicity, and enhanced effectiveness.