mRNA Poly-A Tail Encoding with Non-Adenine Anchors for Plasmid Stability

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Solution Overview

Problem

Existing methods for encoding poly-A tails in mRNA are unstable during DNA replication, leading to variations in tail length and reduced mRNA stability and efficiency.

Innovation Solution

Incorporating non-adenine nucleotides as anchors within the poly-A tail to stabilize the DNA encoding and the resulting mRNA, preventing the loss of adenine nucleotides during replication.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If poly-A tail is encoded on plasmid, then poly-A tail length homogeneity is improved, but plasmid stability deteriorates

Engineering Contradiction:

Improvepoly-A tail length homogeneityVSAvoidplasmid stability

Core Design Contradiction:

Manufacturing precisionVSReliability

Solution Approach 1:

The patent introduces non-adenine nucleotides as intermediary elements (anchors) within the poly-A tail sequence. These anchors act as stabilizing mediators that prevent the loss of adenine nucleotides during DNA replication, thereby maintaining both poly-A tail length homogeneity and plasmid stability simultaneously.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent modifies the compositional parameter of the poly-A tail by incorporating non-adenine nucleotides at specific positions. This parameter change transforms the purely adenine-based sequence into a stabilized structure that resists nucleotide loss during replication, resolving the contradiction between homogeneity and stability.

Inventive Principle:

Principle #35Parameter changes

2Manufacturing precision

If poly-A tail is added enzymatically, then poly-A tail length control is improved, but process complexity increases

Engineering Contradiction:

Improvepoly-A tail length controlVSAvoidprocess complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent extracts the poly-A tail encoding function from the complex enzymatic addition process and integrates it directly into the plasmid DNA sequence. This allows the poly-A tail to be replicated passively during DNA replication, eliminating the need for separate enzymatic steps and reducing process complexity while maintaining length control.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent incorporates the poly-A tail sequence (with stabilizing anchors) into the plasmid DNA before replication begins. This preliminary inclusion ensures that the poly-A tail is automatically replicated with the plasmid, avoiding the need for subsequent enzymatic addition steps and simplifying the overall process.

Inventive Principle:

Principle #10Preliminary action

3Productivity

If poly-A tail is encoded on plasmid, then mRNA production efficiency is improved, but poly-A tail length variability increases

Engineering Contradiction:

ImprovemRNA production efficiencyVSAvoidpoly-A tail length variability

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The non-adenine nucleotide anchors serve as mediators that prevent stochastic variation in poly-A tail length during plasmid replication. By stabilizing the sequence against nucleotide loss, these anchors ensure consistent poly-A tail length across all mRNA molecules produced from the plasmid, thereby reducing length variability while maintaining high production efficiency.

Inventive Principle:

Principle #24Intermediary (Mediator)

Data Source

PatentUS12391722B2Stabilized nucleic acids encoding messenger ribonucleic acid (mRNA)

Publication Date: 2025.08.19 INTELLIA THERAPEUTICS INC

AI summary

This disclosure relates to the field of poly-adenylated (poly-A) tails. In some embodiments, a DNA encodes a poly-A tail located 3′ to nucleotides encoding a protein of interest, wherein the poly-A tail comprises one or more non-adenine nucleotide.