shRNA Loop Motif Redirects Nuclear Export Pathway

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Solution Overview

Problem

The microRNA processing pathway can be saturated by over-expression of shRNAs, leading to cellular toxicity, primarily due to saturation of the nuclear export factor exportin-5, which affects the native microRNA processing pathway.

Innovation Solution

Incorporating a repeated sequence motif derived from the ORF2 transcript of an L1 retrotransposon, specifically the 32 nt L1 motif, into the loop of shRNAs to redirect nuclear export through the Nxf-1 pathway, thereby circumventing exportin-5 mediated export.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If shRNAs are over-expressed to achieve efficient gene silencing, then gene silencing efficiency is improved, but cellular toxicity increases due to saturation of exportin-5

Engineering Contradiction:

Improvegene silencing efficiencyVSAvoidcellular toxicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent introduces a loop sequence as an intermediary element that redirects the export pathway. This loop sequence acts as a mediator to switch from exportin-5 mediated export to Nxf-1 mediated export, thereby resolving the saturation issue without reducing silencing efficiency

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent changes the structural parameter of the shRNA by incorporating a specific loop sequence (12-50 nucleotides long) that alters the export mechanism. This parameter change enables use of an alternative export pathway that is not saturable, thus maintaining high productivity while eliminating toxicity

Inventive Principle:

Principle #35Parameter changes

2Reliability

If exportin-5 mediated export is used for shRNA, then RNAi pathway activation is achieved, but native microRNA processing pathway is disrupted

Engineering Contradiction:

ImproveRNAi pathway activationVSAvoiddisruption of native microRNA processing

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The patent segments the export function by separating shRNA export from native microRNA processing. By using a distinct export pathway (Nxf-1 instead of exportin-5), the system achieves reliable RNAi activation without interfering with or disrupting the native microRNA processing pathway

Inventive Principle:

Principle #1Segmentation

Data Source

PatentUS10006028B2Alternative export pathways for vector expressed RNA interference

Publication Date: 2018.06.26 THE UNIVERSITY OF IOWA RESEARCH

AI summary

The present invention is directed to nucleic acid molecules containing a loop sequence designed to circumvent exportin-5 mediated export, and methods using these novel molecules.