In Situ Probe Inversion via Click Chemistry
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Solution Overview
Problem
In situ synthesized oligonucleotide probes often contain truncated sequences due to incomplete synthesis, which can hinder array performance, especially in enzymatic reactions, as opposed to bead arrays where only full-length probes are immobilized, reducing truncation issues.
Innovation Solution
A method for selectively removing truncated probe sequences from in situ synthesized arrays by incorporating universal cleavable linkers and using click chemistry to circularize and invert the oligonucleotides, allowing only full-length sequences to remain attached to the substrate.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If in situ synthesized probe arrays are used, then array fabrication is simplified and scalable, but truncated probe sequences are generated that reduce array performance
Solution Approach 1:
The patent applies preliminary action by incorporating a capping group during the oligonucleotide synthesis process that selectively prevents truncation. The capping group is introduced at the 5' end of the oligonucleotide and remains attached during synthesis, preventing premature termination and ensuring only full-length probes are generated. This preliminary protective measure eliminates the need for post-synthesis purification steps while maintaining the simplicity of in situ synthesis.
Solution Approach 2:
The patent extracts the harmful truncated sequences from the probe population through selective removal processes. By using enzymatic treatments or chemical methods that specifically target and remove truncated probes while leaving full-length probes intact, the patent extracts the impurity from the system, thereby improving array performance without compromising the ease of in situ fabrication.
2Manufacturing precision
If truncated probe sequences are present, then synthesis completeness is reduced, but removing them requires complex post-synthesis processing
Solution Approach 1:
The patent employs preliminary action by incorporating a capping group during synthesis that prevents truncation from occurring in the first place. This proactive approach eliminates the need for extensive post-synthesis purification processes, thereby maintaining high synthesis completeness while avoiding complex downstream processing steps.
Solution Approach 2:
The patent applies discarding and recovering by selectively removing truncated sequences through enzymatic or chemical methods while recovering and retaining the full-length probes. This selective removal process discards only the harmful truncated products while recovering the valuable full-length probes, thereby improving precision without requiring complete re-synthesis.
3Reliability
If universal linkers and click chemistry are used for probe inversion, then truncated sequences are removed and orientation is inverted, but the process complexity increases
Solution Approach 1:
The patent applies universality by using universal linkers that can be incorporated into the oligonucleotide synthesis process and serve multiple functions: they act as spacing elements, provide attachment points for click chemistry, and enable subsequent inversion without requiring sequence-specific modifications. This multi-functional approach simplifies the overall inversion process despite the added complexity of the inversion step itself.
Solution Approach 2:
The patent replaces mechanical or physical inversion methods with chemical substitution through click chemistry. Instead of using physical methods to flip or rearrange probes, the patent uses chemical reactions between azide and alkyne groups to achieve inversion, thereby reducing mechanical complexity while maintaining reliability of the orientation change.
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 process effectively removes truncated sequences, inverts the orientation of probe sequences, and enhances the efficiency of enzymatic reactions by ensuring that only full-length probes are preserved and available for further processing.
Implementation Method 1
circularizing said first oligonucleotide by reacting said first azide group with a second alkyne, wherein said second alkyne is said first alkyne or a neighboring alkyne
Implementation Method 2
said cleaving comprises de-protection with a base
Data Source
AI summary
The present disclosure relates to processes for inverting oligonucleotide probes in an in situ synthesized array. These processes can be used to reverse the orientation of probes with respect to the substrate from 3′-bound to 5′-bound. These processes can also be used to reduce or eliminate the presence of truncated probe sequences from an in situ synthesized array.