Single-Stranded siRNA With 5' Phosphate for RNA Interference

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Solution Overview

Problem

Current RNA interference (RNAi) methods rely on double-stranded siRNAs, which are inefficient and costly to produce, and require specific conditions, whereas single-stranded siRNAs with a 5' phosphate are effective in mediating target-specific RNAi in both Drosophila and mammalian cells without the need for RNA-dependent RNA polymerases.

Innovation Solution

Development of single-stranded small interfering RNA (ss-siRNA) molecules with a 5' phosphate, which are capable of directing target-specific RNA interference in vitro and in vivo, eliminating the need for 3' hydroxyl groups and RNA-dependent RNA polymerases, and are more economical to produce.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If double-stranded siRNAs are used for RNA interference, then RNAi function is achieved, but production efficiency is low and cost is high

Engineering Contradiction:

ImproveRNAi functionVSAvoidproduction efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent extracts and utilizes only the essential 5' phosphate group from the siRNA molecule, eliminating the need for the 3' hydroxyl group and double-stranded structure. This extraction of critical functional elements enables the use of simpler single-stranded siRNAs that are more efficient to produce while maintaining RNAi activity

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent changes the structural parameters of siRNA from double-stranded to single-stranded, and from requiring both 5' phosphate and 3' hydroxyl to requiring only 5' phosphate. These parameter changes result in molecules that are easier and more cost-effective to produce while retaining functional efficacy

Inventive Principle:

Principle #35Parameter changes

2Reliability

If double-stranded siRNAs with 3' hydroxyl groups are used, then RNAi function is achieved, but molecular complexity increases

Engineering Contradiction:

ImproveRNAi functionVSAvoidmolecular structure complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent removes the 3' hydroxyl group requirement from siRNA structure, extracting this element as non-essential for RNAi function. This simplification reduces molecular complexity while the 5' phosphate group remains essential for maintaining RNAi activity

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent inverts the conventional understanding of siRNA structure by demonstrating that the 5' phosphate group is the critical element for function, rather than the 3' hydroxyl group. This inversion leads to simpler molecular designs that achieve the same biological effect

Inventive Principle:

Principle #13The other way round (Inversion)

3Reliability

If traditional siRNA production methods are used, then RNAi is achieved, but cost increases

Engineering Contradiction:

ImproveRNAi functionVSAvoidproduction cost

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The patent employs simplified single-stranded siRNA molecules that are more economical to produce compared to traditional double-stranded siRNAs. The reduced structural requirements and simplified synthesis pathways lower production costs while maintaining adequate functional performance for RNAi applications

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

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 ss-siRNA molecules demonstrate improved efficacy in mediating RNAi, with a 5' phosphate being essential for function, allowing for efficient target-specific RNAi in both Drosophila and mammalian cells, and are more stable and cost-effective compared to traditional double-stranded siRNAs.

Implementation Method 1

In diverse eukaryotes, double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the double-strand. In animals and basal eukaryotes, this process is called RNA interference (RNAi).

Methodology Applied

Scientific EffectRNA interference:

Implementation Method 2

each siRNA directs endonucleolytic cleavage of the target RNA at a single site

Methodology Applied

Scientific EffectEndonucleolytic cleavage:

Implementation Method 3

Dicer also cleaves ̃70 nt precursor RNA stem-loop structures into single-stranded 21-23 nt RNAs known as microRNAs (miRNAs; Grishok et al. (supra))

Methodology Applied

Scientific EffectRNase III cleavage:

Implementation Method 4

the multi-domain RNase III enzyme, Dicer (Bernstein et al. (2001) Nature 409:363-366; Billy et al. (2001) Proc. Natl. Acad. Sci. USA 98:14428-14433)

Methodology Applied

Scientific EffectRNase III enzyme activity: Enzyme

Implementation Method 5

a 5′ phosphate is required for siRNA function. In contrast, there is no evidence in flies or humans for a role in RNAi for the siRNA 3′ hydroxyl group

Methodology Applied

Scientific EffectPhosphate-dependent RNA interference:

Data Source

PatentUS8729036B2Compositions for RNA interference and methods of use thereof

Publication Date: 2014.05.20 MASSACHUSETTS UNIV OF

AI summary

The present invention provides compositions for RNA interference and methods of use thereof. In particular, the invention provides single-stranded small interfering RNAs. Functional and genomic and proteomic methods are featured. Therapeutic methods are also featured.