Cleavable Linker Probes for High-Throughput DNA Decoding
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Solution Overview
Problem
Current DNA and RNA decoding methods using labeled probes with non-cleavable or indiscriminately cleavable linkers are limited by slow hybridization and de-hybridization steps, harsh conditions, and limited throughput, which can damage nucleic acid targets and substrates, restricting the number of iterative cycles and compatibility with certain surface chemistries.
Innovation Solution
A method utilizing a library of DNA probes with different cleavable linkers that allow sequential and predictable removal of fluorescent labels, enabling faster decoding by hybridizing probes with cleavable linkers that can be removed using various chemical, photochemical, or enzymatic means, allowing for multiple targets to be detected in each round of hybridization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If labeled DNA probes with stable linkers are used for detecting multiple targets, then the probes can be removed only under de-hybridizing conditions, but this requires multiple rounds of hybridization and de-hybridization steps which are slow and time-consuming
Solution Approach 1:
The linker is segmented into different cleavable groups (disulfide, photocleavable, enzymatically cleavable linkers) that can be selectively removed under different conditions. This allows sequential detection of multiple targets without requiring complete de-hybridization, thereby increasing throughput while maintaining probe stability during hybridization
Solution Approach 2:
The invention changes the chemical parameters of the linker to create different cleavable groups with distinct removal conditions. By varying the linker chemistry (disulfide for reducing agents, photocleavable for light, enzymatically cleavable for specific enzymes), the system enables selective label removal without affecting probe hybridization stability
2Productivity
If multiple rounds of hybridization and de-hybridization steps are performed to detect multiple targets, then all targets can be detected, but the harsh conditions used to perform these steps can be damaging to nucleic acid targets and substrate
Solution Approach 1:
The fluorescent label is extracted from the probe through selective cleavage of the linker, allowing the label to be removed while the probe remains hybridized to the target. This eliminates the need for harsh de-hybridization conditions, protecting both the nucleic acid targets and substrate from damage while enabling detection of multiple targets
Solution Approach 2:
The cleavable linker acts as an intermediary between the probe and fluorescent label. This intermediary allows selective removal of the label under mild conditions (chemical, photochemical, or enzymatic treatment) without requiring harsh de-hybridization, thereby protecting the target and substrate from damage
3Productivity
If the number of iterative cycles of hybridization-de-hybridization steps is increased to detect more targets, then higher throughput is achieved, but the time required for each step increases the total processing time
Solution Approach 1:
Multiple detection functions are merged into a single hybridization round by using probes with different cleavable linkers. Instead of performing separate hybridization-de-hybridization cycles for each target, the invention combines multiple targets in one hybridization step and distinguishes them through selective label removal, dramatically reducing total processing time while maintaining high throughput
4Device complexity
If non-cleavable or indiscriminately cleavable linkers are used, then the decoding process can be simplified, but the throughput is limited and the method is not compatible with certain surface chemistries
Solution Approach 1:
Different regions of the probe (different linkers) are given different local qualities - disulfide linkers, photocleavable linkers, and enzymatically cleavable linkers - each responsive to specific stimuli. This local differentiation enables selective label removal for different targets within the same hybridization round, increasing throughput while maintaining manageable process complexity through standardized probe designs
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, reduces the need for iterative hybridization and de-hybridization steps, and minimizes damage to samples, enabling the decoding of a large number of DNA and RNA targets with improved efficiency and compatibility across different surface chemistries.
Implementation Method 1
detecting the hybridized probes via their fluorophores D by a first image
Implementation Method 2
Cleaving sequentially by different means each group of chemical linkers CL from the hybridized probes
Implementation Method 3
cleavable linkers that can be removed using various chemical, photochemical, or enzymatic means
Implementation Method 4
cleavable linkers that can be removed using various chemical, photochemical, or enzymatic means
Implementation Method 5
Hybridizing a library of probes having the general formula (I) P—(CL-D)x with P: probes having at least 10 nucleotides or amino acids
Data Source
AI summary
The invention is directed to a method for detecting RNA, DNA or protein target sequences bya) 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 acidsCL: cleavable linkerD: fluorescent dyeX: 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 meansb) Removing unhybridized probes and detecting the hybridized probes via the fluorophores D by a first imagec) 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 imaged) 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 Df) Repeating step c) until all groups of chemical linkers CL are cleaved.