Reversible Biomolecule Labeling via Cleavable Linker
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Solution Overview
Problem
Current bioconjugation methods primarily focus on forming stable linkages, limiting biomolecules to a single analytical process and hindering integrative approaches in cell biology, where multiple analyses are often necessary.
Innovation Solution
A method involving a linker molecule with a reactive centre, a cleavable hydrolysable moiety, and a second functional group allows for reversible labelling and unlabelling of biomolecules, enabling multiple sequential analyses by forming and breaking covalent bonds, facilitating site-selective modification, erasure, and re-writing of functionality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If stable linkages are formed between biomolecule and label, then reliability of conjugation is improved, but adaptability for multiple analytical processes deteriorates
Solution Approach 1:
The patent applies dynamics by introducing a cleavable moiety into the linker molecule, transforming the static stable linkage into a dynamic system that can transition between bound and unbound states. The linker comprises a first stable portion for reliable conjugation and a second cleavable portion that can be hydrolyzed under specific conditions, enabling the linkage to change from stable to unstable state as needed for different analytical processes
Solution Approach 2:
The patent employs parameter changes by utilizing chemical hydrolysis to alter the stability parameter of the linkage. The cleavable moiety is designed to be stable under physiological conditions but can be hydrolyzed by changing pH or other chemical parameters, thereby controlling the stability of the biomolecule-label conjugation to enable sequential analytical procedures
2Ease of operation
If single analytical process is used, then simplicity of procedure is improved, but loss of information about integrative cell behaviors increases
Solution Approach 1:
The patent applies periodic action by enabling sequential cycles of labeling and unlabeling of the biomolecule. The cleavable linkage allows the biomolecule to be labeled for one analytical process, then unlabeled and relabeled for subsequent analyses, creating periodic cycles of modification that facilitate multiple measurements without permanent alteration, thereby preserving integrative cell behavior information
3Reliability
If label is permanently attached, then reliability of detection is improved, but loss of substance through inability to recover biomolecule increases
Solution Approach 1:
The patent applies discarding and recovering by designing a system where the label can be selectively discarded from the biomolecule through hydrolysis of the cleavable moiety. The first stable portion of the linker remains attached to the biomolecule while the second cleavable portion with the label is hydrolyzed and removed, allowing recovery of the biomolecule for further use while maintaining detection reliability during the labeling period
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
Enables holistic understanding of cell behavior through multiple analyses on a single sample, allowing for the recovery of the biomolecule and label, and supports bio-orthogonal, reversible, and rewritable modifications without interfering with native biochemical processes.
Implementation Method 1
d. cleaving the cleavable, e.g. hydrolysable, moiety, of the linker molecule to remove the first label and to form a third functional group comprising a reactive centre
Data Source
AI summary
A method of reversibly labeling a biomolecule including providing a linker molecule (11) having a first functional group (LG) with a reactive center, a second functional group (FG) with a reactive center, and a cleavable, e.g. hydrolyzable, moiety (A-B-C). The method further includes forming a covalent bond between the biomolecule (10) and the reactive center of the first functional group (LG), forming a covalent bond between a first label (L1) and the reactive center of the second functional group (FG), cleaving the cleavable moiety (A-B-C), e.g. hydrolyzing the hydrolyzable moiety, of the linker molecule (11) to remove the first label (L1) and to form a third functional group (W) with a reactive center, and forming a covalent bond between a further molecule and the reactive center of the third functional group (W) to reform the cleavable moiety, e.g. hydrolyzable moiety (A-B-C).