Fusion Protein Enhancing mRNA Translation via PPR Binding

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Solution Overview

Problem

Current methods lack effective techniques for specifically regulating target RNAs using protein factors, despite advancements in DNA-targeting technologies like ZFN, TALEN, and Crispr-cas9, with limited development in RNA-targeting approaches.

Innovation Solution

A fusion protein comprising a predetermined functional domain and a PPR protein with specific RNA-binding properties, featuring PPR motifs that selectively bind to target mRNA sequences, enhancing protein expression levels by guiding ribosomes, promoting translation, or regulating mRNA localization.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If DNA-targeting techniques (ZFN, TALEN, Crispr-cas9) are used, then DNA sequence-specific binding is achieved, but RNA-targeting capability is limited

Engineering Contradiction:

ImproveRNA-targeting capabilityVSAvoidbinding specificity

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent merges the RNA-binding capability of PPR proteins with the functional domains of DNA-targeting techniques (ZFN, TALEN, or Crispr-cas9) to create a fusion protein that can specifically target and regulate RNA sequences. This combination enables the system to achieve both RNA adaptability and reliable sequence-specific binding by integrating the strengths of different molecular recognition systems.

Inventive Principle:

Principle #5Merging (Combining)

2Measurement precision

If PPR motifs are used for RNA binding, then RNA sequence-specific binding is enabled, but protein expression level from target mRNA is insufficient

Engineering Contradiction:

ImproveRNA binding specificityVSAvoidprotein expression level

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The fusion protein combines PPR motifs responsible for specific RNA recognition with functional domains that enhance protein expression from target mRNA. This merging allows the system to maintain high RNA binding specificity while simultaneously improving protein production efficiency through the coordinated action of both functional elements.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The fusion protein achieves multi-functionality by integrating RNA-binding PPR motifs with expression-enhancing domains. This single protein entity performs multiple functions: specific RNA recognition, recruitment of translation machinery, and enhancement of protein expression, thereby resolving the contradiction between specificity and productivity.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Productivity

If fusion protein with functional domain and PPR protein is constructed, then protein expression level is improved, but device complexity increases

Engineering Contradiction:

Improveprotein expression levelVSAvoidprotein structure complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The fusion protein is constructed by segmenting different functional elements (PPR motifs and expression-enhancing domains) that can be independently designed and optimized. This segmentation allows for modular assembly of the fusion protein, making the complex structure more manageable and enabling systematic optimization of each functional component without redesigning the entire system.

Inventive Principle:

Principle #1Segmentation

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 fusion protein significantly improves protein expression levels by specifically binding to target mRNA sequences, demonstrating enhanced translation activity and regulatory capabilities, as shown in experimental assays.

Implementation Method 1

a polypeptide moiety which can bind to a target mRNA in an RNA base-selective or RNA base sequence-specific manner

Methodology Applied

Scientific EffectRNA base-selective binding:

Implementation Method 2

a polypeptide moiety which can bind to a target mRNA in an RNA base-selective or RNA base sequence-specific manner

Methodology Applied

Scientific EffectRNA base sequence-specific binding:

Implementation Method 3

one or more functional domains which improve a protein expression level from an mRNA

Methodology Applied

Scientific EffectRibosome guidance:

Implementation Method 4

one or more functional domains which improve a protein expression level from an mRNA

Methodology Applied

Scientific EffectTranslation promotion:

Data Source

PatentUS11136361B2Fusion protein for improving protein expression from target mRNA

Publication Date: 2021.10.05 KYUSHU UNIV

AI summary

[Problem to be Solved] The object of the present invention is to develop a method of regulating a target RNA.[Solution] There is provided a fusion protein comprising a functional domain which improves the protein expression level from mRNA and a PPR protein which can bind to a target mRNA in an RNA base-selective or RNA base sequence-specific manner.