Protected ssDNA Cassettes With Nuclease-Resistant End Protection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for producing long single-stranded DNA molecules are inefficient and prone to degradation by nucleases, limiting their stability and effectiveness for in vivo applications.
Innovation Solution
A protected DNA comprising a single-stranded DNA cassette with nuclease-resistant nucleotides at the 5′ and 3′ ends, enhancing resistance to exonuclease digestion and allowing for prolonged in vivo expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If chemical synthesis is used to produce single-stranded DNA, then the DNA can be generated with controlled sequence, but the yield becomes limited and side reactions increase when the DNA length exceeds 100 nucleotides
Solution Approach 1:
The patent divides the long single-stranded DNA production process into segments: first synthesizing shorter protected DNA fragments (each under 100 nucleotides) with controlled sequences, then assembling these fragments through ligation to form the complete long ssDNA molecule. This segmentation allows each synthesis step to maintain high precision while the overall process achieves high productivity for long molecules.
2Ease of manufacture
If standard DNA molecules are used, then they can be easily synthesized and manipulated, but they are susceptible to degradation by nucleases which limits their effective molecular lifetime
Solution Approach 1:
The patent modifies the chemical parameters of the DNA molecule by incorporating phosphorothioated nucleotides, which replace non-bridging oxygen atoms with sulfur atoms in the phosphate backbone. This parameter change maintains the DNA's synthesizability and manipulability while dramatically increasing its resistance to nuclease degradation, thereby extending its effective molecular lifetime in biological systems.
3Duration of action of stationary object
If phosphorothioated nucleotides are incorporated to protect DNA from exonuclease digestion, then the half-life of the nucleic acid molecule is prolonged, but the chemical synthesis of longer single-stranded polynucleotides becomes limited due to side reactions
Solution Approach 1:
The patent segments the protected DNA into shorter fragments that can be efficiently synthesized with phosphorothioated nucleotides, then ligates these fragments together. This approach maintains the protective benefits of phosphorothioation for each fragment while avoiding the cumulative side reactions that would occur in synthesizing one continuous long protected strand.
Solution Approach 2:
The patent applies phosphorothioate protection to the ends of DNA fragments before ligation, creating preliminarily protected fragments that can then be assembled into longer molecules. This preliminary protection ensures that the final long ssDNA molecule has protected ends resistant to exonuclease digestion, while the synthesis of each fragment remains efficient.
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 protected DNA exhibits enhanced resistance to exonucleases, extending its lifespan in both cellular and cell-free systems, and is suitable for pharmaceutical compositions without bacterial backbones or antibiotic resistance genes, making it suitable for cell delivery systems like viral vectors or nanoparticles.
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.
Implementation Method 2
DNA is susceptible to degradation by nucleases which are naturally occurring enzymes within organisms and which have a vital role in the regulation of many cellular processes, while also protecting against foreign DNA species.
Data Source
AI summary
Protected DNA comprising a single-stranded DNA (ssDNA) cassette is provided. Further provided are uses of the protected DNA, methods for producing protected DNA, products generated in performing such methods (including intermediate and final products), and kits for use in such methods.