Linear DNA With Internal Phosphorothioate Modifications

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Solution Overview

Problem

Existing linear double-stranded DNA products are susceptible to nuclease digestion, particularly exonuclease digestion, which limits their molecular lifetime and suitability for applications such as RNA production, protein expression, nanoparticle production, and vector assembly.

Innovation Solution

The development of a linear double-stranded DNA product with nuclease-resistant nucleotides, specifically phosphorothioated nucleotides, incorporated at internal positions in each strand, enhancing resistance to exonuclease digestion and prolonging the DNA product's life in both cellular and cell-free systems.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If phosphorothioated nucleotides are incorporated at the ends of polynucleotide chains to cap them, then resistance to exonuclease digestion is improved, but the molecular lifetime is still limited due to susceptibility at internal positions

Engineering Contradiction:

Improveresistance to exonuclease digestionVSAvoidmolecular lifetime

Core Design Contradiction:

ReliabilityVSDuration of action of moving object

Solution Approach 1:

The patent applies local quality by incorporating phosphorothioated nucleotides at specific internal positions within the polynucleotide chain rather than uniformly throughout. This targeted approach places protective modifications precisely where exonuclease attack is most likely to occur internally, while maintaining natural phosphodiester bonds elsewhere to preserve overall chain integrity and function.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent employs preliminary action by pre-incorporating phosphorothioated nucleotides at internal positions during DNA synthesis before the molecule is exposed to nuclease environments. This proactive placement of protective groups ensures that when the molecule encounters exonucleases, the internal positions are already fortified against cleavage, extending molecular lifetime without requiring post-synthesis modification.

Inventive Principle:

Principle #10Preliminary action

2Reliability

If phosphorothioated nucleotides are used to protect DNA ends, then exonuclease resistance is enhanced, but the DNA product remains susceptible to digestion at internal positions

Engineering Contradiction:

Improveexonuclease resistanceVSAvoidsusceptibility to nuclease digestion

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent addresses this contradiction by applying local quality through selective placement of phosphorothioated nucleotides at specific internal positions. This creates a gradient of protection where high-risk internal positions are fortified with nuclease-resistant modifications, while lower-risk regions maintain natural bonding, achieving comprehensive protection without uniform over-modification.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent utilizes composite materials by creating a hybrid nucleic acid structure that combines both phosphorothioated nucleotides (for nuclease resistance) and natural phosphodiester bonds (for structural integrity and function). This composite approach allows the DNA molecule to exhibit both resistance to exonuclease digestion at critical positions and maintained biological functionality throughout the chain.

Inventive Principle:

Principle #40Composite materials

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 incorporation of phosphorothioated nucleotides at internal positions in the linear double-stranded DNA product significantly enhances its resistance to exonuclease digestion, leading to prolonged in vivo expression and improved stability for applications in RNA production, protein expression, and nanoparticle production.

Implementation Method 1

Phosphorothioated nucleotides comprise a sulphur atom instead of a non-bridging oxygen atom. These modified nucleotides show comparable physical and chemical characteristics to corresponding unmodified nucleotides, but are resistant to exonuclease digestion.

Methodology Applied

Scientific EffectPhosphorothioate modification:

Data Source

PatentUS20250171781A1Linear DNA with Enhanced Resistance Against Exonucleases

Publication Date: 2025.05.29 4BASEBIO S L U

AI summary

The present invention relates to linear double-stranded DNA products comprising nuclease-resistant nucleotides at internal positions in each strand. In addition, the present invention relates to complex molecules, nanoparticles, compositions and libraries comprising the linear double-stranded DNA products. Also provided are methods for producing and using the linear double-stranded DNA products.