Structurally Designed shRNAs for DICER-Independent Gene Silencing
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Solution Overview
Problem
Conventional RNAi technology in mammals relies on the canonical microRNA pathway involving DICER processing, which can lead to off-target effects due to the incorporation of the sense strand of the duplex into the active RISC, limiting the specificity and efficiency of gene silencing.
Innovation Solution
The development of miR-451 shRNA mimics that are processed through a novel biogenesis pathway independent of DICER, requiring AGO2 catalysis, eliminating the need for DICER processing and reducing off-target effects by generating only one active strand.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional shRNA is processed through the canonical DICER pathway, then the shRNA can be efficiently processed into mature miRNA, but off-target effects occur due to incorporation of the sense strand into active RISC
Solution Approach 1:
The invention extracts and eliminates the harmful sense strand from the RNAi pathway by using a DICER-independent biogenesis pathway. The shRNA is processed directly by AGO2 without DICER involvement, preventing sense strand incorporation into RISC and thereby removing the source of off-target effects while maintaining gene silencing efficiency
Solution Approach 2:
The invention introduces AGO2 as a direct mediator in shRNA processing, bypassing the canonical DICER intermediary. This alternative mediator (AGO2) processes the shRNA hairpin directly into the active strand without generating a duplex that could incorporate the sense strand into RISC, thus resolving the contradiction between efficiency and specificity
2Ease of manufacture
If conventional shRNA design is used, then the structure is simple and easy to manufacture, but specificity and efficiency of gene silencing are limited
Solution Approach 1:
The invention changes the biogenesis pathway parameter from DICER-dependent to DICER-independent processing. This parameter change maintains the simplicity of shRNA design and manufacture while dramatically improving gene silencing specificity by eliminating off-target effects through direct AGO2 processing of the hairpin structure
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 enhances the specificity and efficiency of gene silencing by minimizing off-target effects, allowing for targeted attenuation of gene expression without the limitations of conventional shRNAs.
Implementation Method 1
The structure of Ago proteins is well conserved, consisting of an amino-terminal domain, the mid domain, and their signature PAZ and Piwi domains. The Piwi domain contains an RNAse H motif that was cryptic in the primary sequence but easily recognizable in the tertiary structure. Loading of a highly complementary target into an Ago brings the scissile phosphate, opposite nucleotides 10 and 11 of the small RNA guide, into the enzyme active site, allowing cleavage of the RNA
Implementation Method 2
Starting with PolII transcribed precursor RNAs, the biogenesis pathway involves two steps: DROSHA/DGCR8 cleaves the precursor transcript into a short hairpin RNA that is exported into the cytoplasm
Implementation Method 3
This invention relates in part to improvements directed to use of RNA interference (RNAi) technology that exploits a newly identified small RNA biogeneisis pathway
Data Source
AI summary
Provided is an improved design of shRNA based on structural mimics of miR-451 precursors. These miR-451 shRNA mimics are channeled through a novel small RNA biogenesis pathway, require AGO2 catalysis and are processed by Drosha but are independent of DICER processing. This miRNA pathway feeds active elements only into Ago2 because of its unique catalytic activity. These data demonstrate that this newly identified small RNA biogenesis pathway can be exploited in vivo to produce active molecules.