Stabilizing 3′-O—NH2 Nucleoside Triphosphates by Removing Hydroxylamine
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Solution Overview
Problem
Nucleoside triphosphates with a 3′-O—NH2 moiety decompose to form triphosphates with a free 3′-OH group during storage, rendering them unsuitable for sequencing and enzymatic extension due to the presence of hydroxylamine, which is used in their synthesis to prevent reactivity with aldehydes and ketones.
Innovation Solution
Replacing hydroxylamine with alkoxylamine, such as methoxylamine, in the synthesis process to prevent decomposition and maintain the 3′-O—NH2 moiety, and using an immobilized support for triphosphate synthesis to avoid contamination with triphosphates having a free 3′-OH group, ensuring the triphosphate is released without hydroxylamine exposure.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If hydroxylamine is used in the synthesis of 3′-O—NH2 triphosphates to prevent reactivity with aldehydes and ketones, then the triphosphates can be synthesized, but they decompose during storage to form triphosphates with free 3′-OH groups
Solution Approach 1:
The patent removes hydroxylamine from the storage composition, extracting the problematic component that causes decomposition. The triphosphates are synthesized using hydroxylamine but then purified to remove it completely before storage, eliminating the source of decomposition while maintaining the ability to synthesize the desired 3′-O—NH2 triphosphates
Solution Approach 2:
The patent changes the chemical environment by removing hydroxylamine and instead using ammonium acetate buffer at controlled pH levels (5.5-6.5) for storage. This parameter change prevents the decomposition reaction that would otherwise occur in the presence of hydroxylamine, stabilizing the 3′-O—NH2 triphosphates during storage
2Ease of manufacture
If hydroxylamine is present in the composition, then synthesis is enabled, but decomposition occurs during storage
Solution Approach 1:
The patent performs purification steps immediately after synthesis to remove hydroxylamine before the triphosphates are stored or used. This preliminary action eliminates the decomposition pathway while preserving the synthesized 3′-O—NH2 triphosphates, allowing them to be stored reliably without hydroxylamine present
Solution Approach 2:
The patent uses ammonium acetate as an intermediary buffer system that allows the triphosphates to be handled and stored without hydroxylamine. This intermediary replaces hydroxylamine's protective function during synthesis with a different chemical environment (ammonium acetate buffer at pH 5.5-6.5) that maintains stability during storage
3Productivity
If conventional synthesis methods are used, then triphosphates can be produced, but costly purification methods like HPLC are required to remove decomposition products
Solution Approach 1:
The patent employs simple, inexpensive purification steps such as filtration and concentration that can be performed immediately after synthesis. These disposable-like simple steps remove decomposition products effectively without requiring expensive HPLC equipment, making the process both productive and economically viable
Solution Approach 2:
The patent optimizes storage conditions by controlling pH (5.5-6.5) and temperature to minimize decomposition during storage. By changing these parameters, the need for complex purification is reduced, as decomposition is prevented rather than just removed afterward, simplifying the overall process while maintaining productivity
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 solution stabilizes the 3′-O—NH2 triphosphates, reducing decomposition and maintaining their reactivity for sequencing and enzymatic extension, while reducing synthesis costs and avoiding the need for costly purification methods like HPLC.
Implementation Method 1
Replacing hydroxylamine with alkoxylamine, such as methoxylamine, in the synthesis process to prevent decomposition and maintain the 3′-O—NH2 moiety
Implementation Method 2
using an immobilized support for triphosphate synthesis to avoid contamination with triphosphates having a free 3′-OH group
Data Source
AI summary
This invention claims aqueous compositions that comprise triphosphates of 2′-deoxynucleoside derivatives that have, instead of a 3′-OH moiety, a 3′-ONH2 moiety; wherein said compositions contain less than 0.5 mole percent contaminating triphosphate having a 3′-OH moiety, as well as processes for providing such compositions. The compositions further must contain insubstantial amounts of hydroxylamine.