Modified Nucleotides for Controlled Enzymatic Synthesis
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Solution Overview
Problem
Current nucleic acid synthesis methods, both chemical and enzymatic, face challenges in efficiently synthesizing very long nucleic acid fragments due to uncontrolled polymerization, use of harmful reagents, and compatibility issues with elongation enzymes, leading to inefficient and unreliable results.
Innovation Solution
Development of modified nucleotides with specific modifier groups that allow controlled polymerization and interaction with enzymes, enabling enzymatic synthesis of long nucleic acids by blocking polymerization and facilitating interaction with proteins or solid supports, while maintaining compatibility with elongation enzymes and allowing for efficient deprotection steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If natural nucleotides are used for enzymatic synthesis, then the polymerization reaction can proceed, but uncontrolled polymerization occurs resulting in heterogeneous mixture of nucleic acid molecules
Solution Approach 1:
The patent applies preliminary anti-action by introducing modified nucleotides with blocking groups that prevent uncontrolled polymerization before it occurs. The blocking groups are strategically placed on the nucleotide structures to inhibit premature phosphodiester bond formation, allowing controlled sequential addition of nucleotides during enzymatic synthesis while maintaining the desired sequence fidelity.
2Reliability
If protected nucleotides are used to prevent uncontrolled polymerization, then sequence control is improved, but compatibility with elongation enzymes is reduced
Solution Approach 1:
The patent applies local quality by introducing modifications at specific, localized positions on the nucleotide structure rather than comprehensive protection. The blocking groups are placed selectively on functional groups that would cause uncontrolled polymerization, while leaving other critical regions of the nucleotide unchanged to maintain enzyme recognition and catalytic activity. This selective modification approach preserves enzyme compatibility while achieving sequence control.
3Productivity
If chemical synthesis methods are used, then nucleic acid fragments can be produced, but harmful reagents are required and synthesis of long fragments is inefficient
Solution Approach 1:
The patent applies mechanics substitution by replacing the chemical synthesis mechanism with an enzymatic mechanism. Instead of using chemical reagents to form phosphodiester bonds, the invention employs DNA polymerases or terminal deoxynucleotidyl transferase enzymes to catalyze the polymerization reaction. This substitution eliminates the need for harmful chemical reagents while enabling efficient synthesis of long nucleic acid fragments through the biocatalytic process.
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 modified nucleotides enable reliable and efficient enzymatic synthesis of very long nucleic acids, improving synthesis performance by ensuring controlled addition of nucleotides and reducing the need for multiple deprotection steps, thus enhancing the quality and yield of synthetic nucleic acid sequences.
Implementation Method 1
comprising at least one R or R' group, called modifier group, borne by said natural nitrogenous base or analog, and/or by the oxygen in position 3' of the ribose or deoxyribose molecule, making it possible to block the polymerization of said nucleotide
Implementation Method 2
making it possible to enable the interaction of said nucleotide with another molecule, different than another nucleotide, such as a protein, during nucleic acid synthesis
Implementation Method 3
T represents a cleavable group... The cleavage of all the modifier groups is carried out entirely and simultaneously, that is to say during the same deprotection step
Data Source
AI summary
A modified nucleotide, intended for the synthesis of long chain nucleic acids by enzymatic processes, comprising a “natural” nitrogenous base or a natural nitrogenous base analogue, a ribose or deoxyribose carbohydrate, and at least one phosphate group, characterized in that said nucleotide comprises at least one R group, termed the modifier group, carried by said nitrogenous base or analogue and/or by the oxygen in position 3′ of the ribose or deoxyribose molecule, making it possible to block the polymerization of said nucleotide and/or to allow the interaction of said nucleotide with another molecule, such as a protein, during the nucleic acid synthesis, R comprising at least one functional terminal group.