Polynucleotide Purification via Multivalent Cation Salt Precipitation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for preparing polynucleotides, such as those with N3′→P5′ thiophosphoramidate linkages, face challenges in selectively precipitating target polynucleotides from crude synthetic preparations, leading to contamination with non-target synthesis products and agents, which affects purity and efficiency.
Innovation Solution
A method involving contacting a polynucleotide composition with a multivalent cation salt to precipitate a polynucleotide salt, followed by separation and further purification using reverse phase chromatography, effectively reduces non-target contaminants and enhances the purity of the target polynucleotide.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional precipitation methods are used to isolate polynucleotides from crude synthetic preparations, then the isolation process is simple and quick, but the purity of the target polynucleotide is low due to contamination with non-target synthesis products and agents
Solution Approach 1:
The patent introduces multivalent cation salts as intermediary substances that selectively bind to the target polynucleotide to form precipitable complexes. These cation salts act as mediators between the target polynucleotide and the precipitation process, enabling selective isolation while leaving non-target contaminants in solution. This resolves the contradiction by adding a chemical intermediary that enhances purity without requiring complex physical separation equipment
Solution Approach 2:
The patent exploits parameter changes in solubility by adjusting ionic strength, pH, and temperature conditions to selectively precipitate the target polynucleotide-cation complex while keeping contaminants soluble. By carefully controlling these parameters, the method achieves high purity isolation through a relatively simple one-step precipitation process, resolving the contradiction between purity and process complexity
2Manufacturing precision
If multivalent cation salt precipitation is used to purify polynucleotides, then the purity increases significantly, but the time required for the purification process increases
Solution Approach 1:
The patent performs preliminary optimization of cation salt selection and reaction conditions before the actual purification process. By pre-determining the optimal multivalent cation salt type, concentration, pH, and temperature conditions through preliminary experiments, the actual purification step can be executed quickly and efficiently in a single operation, minimizing the time penalty while maintaining high purity results
Solution Approach 2:
The method utilizes phase transition (precipitation) of the target polynucleotide-cation complex as a rapid separation mechanism. The abrupt phase change from dissolved to precipitated state allows for quick solid-liquid separation through simple centrifugation or filtration, achieving high purity in minimal time without requiring prolonged chromatographic or dialysis processes
3Manufacturing precision
If selective precipitation of target polynucleotides is attempted, then purity improves, but the yield decreases due to loss of target product in the precipitate
Solution Approach 1:
The patent employs parameter optimization including adjusting cation salt concentration, pH, ionic strength, and temperature to fine-tune the precipitation conditions. By carefully controlling these parameters, the method maximizes the amount of target polynucleotide that precipitates with the cation salt while minimizing co-precipitation of contaminants and ensuring complete recovery of the target product through optimized resuspension and dissolution conditions
Solution Approach 2:
The method incorporates analytical feedback through HPLC and other characterization techniques to monitor the purity and yield of each purification step. This feedback allows for iterative optimization of the precipitation conditions, enabling adjustment of cation salt parameters to achieve the best balance between purity and yield for each specific polynucleotide target, ensuring minimal loss while maintaining high purity
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
This method significantly improves the purity of the target polynucleotide by selectively precipitating and purifying it, reducing non-target contaminants by up to 95% and achieving higher purity levels compared to conventional methods.
Implementation Method 1
contacting a first polynucleotide composition including: a polynucleotide having a sequence of 7 or more nucleoside subunits where at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage; and non-target synthetic products and reagents; with a multivalent cation salt to precipitate a first polynucleotide salt including at least one multivalent cation counterion
Data Source
AI summary
Aspects of the disclosure include methods for the preparation of a polynucleotide. In some embodiments, the method includes contacting a first polynucleotide composition including: a polynucleotide having a sequence of 7 or more nucleoside subunits and at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage; and non-target synthetic products and reagents; with a multivalent cation salt to precipitate a polynucleotide salt including at least one multivalent cation counterion; and separating the polynucleotide salt from the contacted first polynucleotide composition to produce a second polynucleotide composition including the polynucleotide salt. In certain embodiments, the method further includes contacting the polynucleotide salt with a reverse phase chromatography support; and eluting from the chromatography support a third polynucleotide composition including the polynucleotide. Also provided are compositions including a salt of the polynucleotide including at least one multivalent cation counterion.