Polynucleotide Synthesis via Closed-Loop Verification
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Solution Overview
Problem
Current methods for large-scale de novo DNA synthesis, such as solid phase phosphoramidite chemistry, face limitations in length and error rates due to sub-unity stepwise yields and destructive side reactions, particularly acidic depurination, which restrict the synthesis of long oligonucleotides and introduce errors.
Innovation Solution
The use of error-prone or template-independent DNA polymerases, like terminal deoxynucleotidyl transferase (TdT), in conjunction with closed-loop verification of nucleotide addition, ensures accurate incorporation of nucleotides through the application of selected nucleotides with labile protecting groups, monitored by fluorescent signals or other means, to prevent incorrect additions and maintain high fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If solid phase phosphoramidite chemistry is used for large-scale de novo DNA synthesis, then high productivity and large-scale synthesis are achieved, but length limitations and high error rates occur due to exponentially decaying full length oligo fractions
Solution Approach 1:
The patent implements closed-loop verification by monitoring fluorescent signals after each nucleotide addition step. The system detects whether the correct nucleotide has been incorporated and can identify incorrect additions in real-time, providing feedback that allows for error correction during synthesis rather than only after completion.
Solution Approach 2:
The patent uses labile protecting groups on nucleotides that are removed in advance of incorporation. This preliminary deprotection ensures that only the correct nucleotide can be added at each position, preventing errors before they occur. The protecting groups are designed to be removed under specific conditions that precede the coupling step.
2Ease of manufacture
If iterative pH-mediated deprotection and coupling is performed to synthesize oligos, then polynucleotides can be produced, but destructive side reactions such as acidic depurination occur
Solution Approach 1:
The patent employs enzymatic synthesis using terminal deoxynucleotidyl transferase (TdT) instead of chemical phosphoramidite chemistry. This fundamental parameter change from chemical to biological catalysis eliminates the need for harsh pH-mediated deprotection steps that cause depurination. The enzymatic process occurs under physiological conditions that are gentle on the nucleotide bases.
Solution Approach 2:
The patent replaces the mechanical/chemical synthesis system (phosphoramidite chemistry requiring pH changes and organic solvents) with a biological enzymatic system. The TdT enzyme catalyzes nucleotide addition without requiring acidic or basic conditions, substituting a biochemical mechanism for a chemical one and thereby eliminating depurination side reactions.
3Manufacturing precision
If closed-loop verification is implemented to ensure accurate nucleotide incorporation, then high fidelity polynucleotides are produced, but device complexity and monitoring requirements increase
Solution Approach 1:
The patent uses fluorescently labeled nucleotides that emit light at specific wavelengths when incorporated into the growing polynucleotide chain. This optical signal provides direct, real-time verification of correct nucleotide incorporation without requiring complex analytical instrumentation. The fluorescent signal acts as a simple binary indicator of successful incorporation.
Solution Approach 2:
The patent introduces fluorescent labels as intermediary molecules attached to the nucleotides. These labels serve as mediators that translate the chemical event of nucleotide incorporation into an easily detectable optical signal. The fluorescent intermediary simplifies the detection process by converting a chemical bonding event into a photonic signal that can be monitored with relatively simple fluorescence detection equipment.
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 enables the synthesis of polynucleotides with high fidelity and accuracy, overcoming the limitations of existing methods by ensuring correct nucleotide incorporation and minimizing errors, thus producing longer sequences with improved reliability.
Implementation Method 1
delivering one or more reaction reagents including an error prone or template independent DNA polymerase... to a reaction site including an initiator sequence having a terminal nucleotide... such that the selected nucleotide becomes a terminal nucleotide
Implementation Method 2
whether the selected nucleotide has been added to the terminal nucleotide is determined by monitoring of a fluorescent signal
Implementation Method 3
The disclosure provides that the selected nucleotide includes a photolabile protecting group
Data Source
AI summary
A method for making a polynucleotide is provided including (a) delivering one or more reaction reagents including an error prone or template independent DNA polymerase, cations and a selected nucleotide to a reaction site including an initiator sequence having a terminal nucleotide for a time period and under conditions capable of covalently adding one or more of the selected nucleotide to the terminal nucleotide at the 3′ end of the initiator such that the selected nucleotide becomes a terminal nucleotide, and (b) determining whether the selected nucleotide has been added to the terminal nucleotide, wherein if the selected nucleotide has not been added to the terminal nucleotide, then repeating step (a) until the selected nucleotide has been added, and (c) repeating steps (a) and (b) until the polynucleotide is formed.