Ago2 Binding Assay for RNAi Potency Determination
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for determining the potency of chemically synthesized RNAi agents are inadequate, as they do not effectively account for variations in cellular Ago2 levels and their impact on siRNA competition and activity.
Innovation Solution
Measuring the binding efficiencies of RNAi agents to human eukaryotic translation initiation factor 2C (eIF2C, or Ago2), using techniques such as binding assays and in silico screening, to assess their potency and develop structure-activity relationships for chemically synthesized RNAi agents, including antisense oligonucleotides with sugar-modified nucleotides and phosphorothioate internucleoside linkages.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional potency determination methods are used, then the assessment process is simple, but the measurement precision is insufficient due to not accounting for cellular Ago2 levels and siRNA competition
Solution Approach 1:
The patent uses Ago2 protein as an intermediary component to mediate between the siRNA agent and the potency measurement. By incorporating Ago2 into the in vitro assay system, the method captures the competitive binding interactions that occur in cellular environments, thereby improving potency determination accuracy without requiring complex cellular systems
Solution Approach 2:
The patent extracts the critical Ago2 binding step from the complex cellular RNAi pathway and isolates it in an in vitro assay system. This extraction allows for simplified measurement of siRNA potency based on Ago2 binding affinity, eliminating the need for complex cellular competition studies while maintaining measurement precision
2Measurement precision
If in vitro binding assays to Ago2 are performed, then the measurement precision of potency is improved, but the ease of operation decreases due to requiring purified proteins and specialized techniques
Solution Approach 1:
The patent performs preliminary purification of Ago2 protein and preparation of binding buffers before conducting the actual potency assay. This preliminary action allows the main assay step to be simpler and more straightforward, as the complex protein purification and buffer optimization are completed in advance
Solution Approach 2:
The patent creates an in vitro copy of the cellular Ago2-siRNA binding interaction, replacing the complex cellular environment with a simplified in vitro system that replicates the essential binding event. This copying approach maintains measurement precision while significantly improving ease of operation compared to cellular assays
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
This approach allows for the accurate determination of RNAi agent potency by evaluating their ability to activate RISC and reduce target RNA, providing insights into their kinetic interactions and relative potency, thereby improving the development of RNAi-based therapies.
Implementation Method 1
measuring the binding efficiencies of one or more chemically synthesized RNAi agents to human eukaryotic translation initiation factor 2C, 2 (eIF2C2) (Argonaut 2 or Ago2)
Data Source
AI summary
Provided herein are methods for determining potency of RNAi agents. Such methods include, but are not limited to, cell-based and cell-free assays that measure binding of an RNAi agent with Ago2 or that measure Ago2 activity in the presence of such RNAi agents. Also provided are assays that determine potency of RNAi agents by assessing their ability to compete with other RNAi agents, including control RNAi agents, for binding and/or activation of Ago2.