3'-O-CH2SSMe Protected Nucleotides for Enzymatic DNA Synthesis
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Solution Overview
Problem
Current DNA synthesis methods, particularly those using phosphoramidite chemistry, are limited by environmental unfriendliness, inability to produce long DNA strands efficiently, and high costs, making them inadequate for rapid, accurate, and cost-effective synthesis of longer DNA sequences.
Innovation Solution
The use of 3′-OH protected nucleotides, specifically 3′-O—CH2SSMe, in template-free, enzymatic DNA and RNA synthesis, where nucleotides are added sequentially and terminated by single base extension, allowing for subsequent cleavage and extension, enabling the synthesis of longer DNA strands.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If phosphoramidite chemistry is used for DNA synthesis, then DNA can be synthesized, but the method produces organic wastes and is not environmentally friendly
Solution Approach 1:
The patent changes the chemical parameters by using water-soluble protecting groups (orthogonally removable protecting groups like esters, carbamates, or carbonyl compounds) instead of organic-phase soluble groups. This parameter change allows the synthesis to proceed in aqueous environments, eliminating organic waste generation while maintaining DNA synthesis capability.
2Ease of manufacture
If phosphoramidite chemistry is used for DNA synthesis, then DNA can be synthesized, but the method is limited to short stretches of nucleic acids (about 200 nucleotides)
Solution Approach 1:
The patent employs segmentation by using multiple protecting groups with different removal conditions (orthogonal protecting groups). This allows stepwise control of the synthesis process, enabling the formation of longer DNA strands by sequentially adding and protecting nucleotides, then selectively removing protecting groups to extend the strand beyond the 200-nucleotide limit.
3Ease of manufacture
If traditional DNA synthesis methods are used, then DNA can be synthesized, but the process is time consuming and cost prohibitive for longer sequences
Solution Approach 1:
The patent implements continuous synthesis by using water-soluble protecting groups that can be removed under mild conditions without interrupting the overall process. The orthogonal removing conditions allow continuous extension of DNA strands without the time-consuming steps required by traditional methods, making the synthesis process more efficient and cost-effective for longer sequences.
4Adaptability or versatility
If 3′-OH protected nucleotides are used for enzymatic synthesis, then template-free synthesis is enabled, but the protecting group must be removable under DNA compatible conditions
Solution Approach 1:
The patent changes the chemical parameters by selecting protecting groups (esters, carbamates, or carbonyl compounds) that can be removed under mild, DNA-compatible conditions. This parameter change enables template-free enzymatic synthesis while ensuring the protecting groups can be selectively removed without damaging the synthesized DNA, thus resolving the contradiction between versatility and ease of manufacture.
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 allows for the efficient, accurate, and cost-effective synthesis of longer DNA strands without the need for template oligonucleotides, overcoming the limitations of traditional methods by using environmentally friendly chemicals and enabling rapid production of desired DNA sequences.
Implementation Method 1
3′-OH protected nucleotides, especially, 3′-O-CH2SSMe known to be accepted by polymerase to terminate DNA synthesis by single base incorporation
Implementation Method 2
it can be removed using benign chemicals such as with thiols and phosphines
Data Source
AI summary
This invention is about enzymatic synthesis of nucleic acids using 3′-O—(CH2SSR) protected reversible nucleotide terminators. The nucleotide base of the protected nucleotide may consist of natural or non-natural (e.g. 7-de-aza G and 7-de-aza A), or mixture thereof. The base of the nucleotides can be modified with a linker carrying carboxylic acid (—CO2H). amine (—NH2), thiol (—SH). hydroxymethyl (—CH2OH), propargy lamine. alkyne group etc. for subsequent modification and labeling. Such nucleotide substrates can be incorporated into the single strand, template free nucleic acid or into the templated DNA hybrid. And in later step the 3′-O—CH2SSR protecting group can be cleaved off by chemical treatment pre-requisites for enzymatic nucleic acids synthesis. By adding nucleotide in pre-determined fashion and cleave reaction after each step. longer DNA strands can be synthesized in solution or on solid surface starting from a short seeding DNA strands.