In Situ Probe Inversion via Click Chemistry
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
In situ synthesized oligonucleotide arrays, particularly those fabricated using photolithography, often contain truncated probe sequences that interfere with array performance, especially in enzymatic reactions, whereas bead arrays and spotted arrays minimize such issues by immobilizing full-length probes via the 5' end, reducing 3' end truncations.
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, converting 3'-end synthesized probes to 5'-end attached probes, thereby eliminating truncated sequences and enhancing array performance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If in situ synthesized arrays are used, then manufacturing precision and array density are improved, but truncated probe sequences are generated that reduce reliability
Solution Approach 1:
The patent applies preliminary action by incorporating a cleavable linker during the synthesis process itself, rather than removing truncated probes after synthesis. The cleavable linker is integrated into the probe structure during in situ synthesis, allowing subsequent selective cleavage to remove truncated sequences. This prevents the harmful effect of truncated probes from affecting array performance in downstream applications.
2Ease of operation
If conventional probe inversion methods are used, then probe orientation is improved, but device complexity increases due to special linkers and reagents
Solution Approach 1:
The patent applies universality by designing a probe inversion method that uses standard, commercially available linkers and reagents rather than specialized components. The cleavable linker and circularization reagents are chosen from common chemical building blocks, making the inversion process compatible with existing synthesis infrastructure. This reduces device complexity while maintaining the ability to invert probes and remove truncated sequences.
3Reliability
If truncated probes are removed post-synthesis, then probe sequence completeness is improved, but loss of time and manufacturing complexity increase
Solution Approach 1:
The patent merges the probe synthesis and inversion processes into a single integrated workflow. By incorporating the cleavable linker during synthesis and performing circularization in the same processing step, the method eliminates separate post-synthesis inversion steps. This combining of operations reduces total processing time while achieving both probe synthesis and inversion in one unified 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 method effectively removes truncated sequences, inverts the oligonucleotides, and increases the percentage of full-length probes, making enzymatic reactions feasible and improving the overall performance of in situ synthesized arrays.
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
Data Source
Figure 1A
Figure 1B~1C
Figure 2A
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.