3'-Aminoxy Ribonucleoside Synthesis for Reversible Termination
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Solution Overview
Problem
Current methods for enzymatic RNA synthesis face challenges due to the difficulty in synthesizing ribonucleoside triphosphates with a 3′-O amino group, which are essential for reversible termination processes, as RNA is more expensive and prone to degradation compared to DNA, and existing synthetic routes have been unsuccessful.
Innovation Solution
Development of synthetic routes to produce ribonucleoside derivatives with a 3′-ONH2 moiety, enabling the creation of ribonucleoside triphosphates that can be used in enzymatic RNA synthesis, including aqueous compositions of nucleosides and their phosphorylated derivatives, allowing for reversible termination and subsequent extension.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If phosphoramidite-based chemical synthesis is used for RNA, then DNA synthesis is cheaper and easier, but RNA synthesis becomes considerably more difficult and expensive due to the 2′-hydroxyl group
Solution Approach 1:
The patent extracts the problematic 2′-hydroxyl group functionality by replacing it with a 2′-amino group in the ribonucleoside structure. This substitution removes the source of degradation and synthesis difficulty while preserving the essential ribose backbone structure needed for RNA function.
Solution Approach 2:
The patent changes the chemical parameter at the 2′-position from hydroxyl to amino group. This parameter change fundamentally alters the chemical reactivity and stability profile, making the molecule amenable to standard phosphoramidite synthesis while improving stability.
2Ease of manufacture
If standard phosphoramidite chemistry is used for RNA synthesis, then nucleobase protecting groups can be removed, but RNA is easily degraded under the alkaline conditions required for deprotection
Solution Approach 1:
The patent employs acid-labile protecting groups that can be removed under mild acidic conditions rather than harsh alkaline conditions. These protecting groups are designed to be stable during synthesis but easily removable under specific deprotection conditions, allowing the RNA to survive the synthesis process.
Solution Approach 2:
The patent changes the deprotection condition parameter from alkaline to acidic. By using acid-labile protecting groups instead of base-labile ones, the synthesis conditions become compatible with RNA stability, preventing degradation during the deprotection step.
3Adaptability or versatility
If 3′-O-amino blocked ribonucleoside triphosphates are synthesized using existing routes, then reversible termination can be achieved, but all attempts to make these compounds have failed
Solution Approach 1:
The patent introduces a 3′-O-amino blocking group as a preliminary protective measure during synthesis. This blocking group prevents unwanted reactions at the 3′-position and can be selectively removed later to enable chain extension, facilitating controlled enzymatic RNA synthesis.
Solution Approach 2:
The patent uses a 3′-O-amino group as an intermediary blocking moiety. This group serves as a temporary placeholder that controls the enzymatic reaction by blocking the 3′-position during synthesis, then can be removed to allow continuation of the chain, enabling reversible termination methodology.
4Productivity
If RNA is synthesized using traditional methods, then DNA oligonucleotide synthesis is straightforward, but RNA is much more expensive and prone to degradation
Solution Approach 1:
The patent extracts the degradation-prone 2′-hydroxyl group and replaces it with a 2′-amino group. This removal of the problematic functional group eliminates the primary source of RNA instability while maintaining the molecule's ability to function in enzymatic synthesis.
Solution Approach 2:
The patent creates a composite nucleoside structure that combines elements of both DNA and RNA. The 2′-amino ribonucleoside has characteristics of both DNA (stability) and RNA (enzymatic recognition), creating a hybrid molecule that benefits from the advantages of both.
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 approach provides a viable method for enzymatic RNA synthesis, enabling the use of 3′-O-amino blocked ribonucleosides in RNA synthesis, overcoming the challenges of RNA degradation and cost associated with traditional methods, and allowing for the formation of oximes with aldehydes and ketones.
Implementation Method 1
ribonucleosides, their analogs, and their derivatives whose 3′-oxygen atoms are covalently bonded to a moiety that is stable under standard conditions where oligonucleotides are used, but can be removed by mild chemical treatments
Data Source
AI summary
This invention claims ribonucleosides and their derivatives, including triphosphates, that have a 3′-ONH2 moiety instead of a 3′-OH moiety.