Differentially Cleavable Linkers for High-Throughput DNA Decoding
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Solution Overview
Problem
Current DNA and RNA decoding methods using labeled probes with stable linkers require multiple rounds of hybridization and de-hybridization steps, which are slow and can damage nucleic acid targets, limiting throughput and compatibility with certain surface chemistries.
Innovation Solution
A method using a library of DNA probes with cleavable linkers that allow sequential removal of fluorescent labels via different means, enabling faster decoding by comparing fluorescence signatures before and after cleavage steps, allowing for multiple targets to be detected with a limited number of dyes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If stable linkers are used to attach fluorescent labels to probes, then the labels remain firmly attached during hybridization, but multiple slow de-hybridization steps are required to remove labels for detecting multiple targets
Solution Approach 1:
The linker is segmented into two functional parts: a stable portion that maintains probe-label attachment during hybridization, and a cleavable portion that enables selective label removal. This segmentation allows the system to achieve both stable attachment and efficient label removal for high-throughput detection.
Solution Approach 2:
The linker's chemical structure is modified to include cleavable bonds with specific chemical sensitivity. By changing the linker's parameters (adding cleavable functional groups), the system enables selective label removal under controlled conditions without requiring full de-hybridization, thus improving throughput.
2Productivity
If harsh de-hybridization conditions are used to remove labels, then labels can be removed for detecting multiple targets, but the nucleic acid targets and substrate are damaged
Solution Approach 1:
The cleavable linker design allows selective extraction of the fluorescent label from the probe while leaving the probe itself intact and attached to the target. This extraction approach enables label removal without requiring harsh de-hybridization conditions that would damage the target or substrate.
Solution Approach 2:
The cleavable linker acts as an intermediary between the probe and the fluorescent label. It provides a controlled mechanism for label removal that bypasses the need for harsh de-hybridization conditions, protecting both the target and substrate from damage while enabling multiple detection cycles.
3Reliability
If non-cleavable linkers are used in RNA sequencing by hybridization, then labels remain stable, but throughput is limited due to reliance on de-hybridization for label removal
Solution Approach 1:
The linker's chemical parameters are modified to include cleavable bonds that respond to specific conditions (chemical, photochemical, electrochemical, or enzymatic). This parameter change enables rapid label removal without de-hybridization, significantly reducing the time required for decoding while maintaining label stability during hybridization.
4Productivity
If indiscriminately cleavable disulfide linkers are used, then labels can be removed, but stepwise cleavage and pattern generation for increased throughput is not achieved
Solution Approach 1:
The linker is segmented into functionally distinct regions with different cleavage specificities. This segmentation enables stepwise cleavage where different portions of the linker can be selectively removed under different conditions, generating distinguishable patterns that increase decoding throughput while maintaining manageable system complexity.
Solution Approach 2:
Different portions of the linker are designed with different local qualities (different cleavage specificities). This allows selective removal of label under controlled conditions, enabling stepwise cleavage and pattern generation that increases throughput without requiring indiscriminate cleavage of all linkers.
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 significantly increases the number of detectable targets per hybridization round, enhancing decoding capacity and reducing the need for repetitive hybridization-de-hybridization cycles, while minimizing damage to nucleic acid targets.
Implementation Method 1
detecting the hybridized probes via their fluorophores D
Implementation Method 2
Hybridizing a library of probes having the general formula (I) P - (CL-D) x with RNA, DNA or protein target sequences
Data Source
Figure 1~2(c)
Figure 3(A)~3(B)
Figure 4~5
AI summary
The invention is directed to a method for detecting RNA, DNA or protein target sequences by a) Hybridizing a library of probes having the general formula (I) P - (CL-D)x (I) With P: probes having at least 10 nucleotides or amino acids CL: cleavable linker D: fluorescent dye X: integer between 1 and 5 to RNA, DNA or protein target sequences wherein the library comprises probes P having different sequences of nucleotides or amino acids and cleavable linkers CL of different groups which are cleavable with different means b) Removing unhybridized probes and detecting the hybridized probes via the fluorophores D by a first image c) Cleaving sequentially by different means each group of chemical linkers CL from the hybridized probes; removing the thus cleaved fluorophores D and detecting the remaining hybridized probes via their fluorophores D by a second image d) Detecting the removed fluorophores D by comparing the first and second image. e) Obtaining a part of the sequence information of the target sequences via the sequence information of the probes P associated with the removed fluorophores D f) Repeating step c) until all groups of chemical linkers CL are cleaved.