Cleavable-Site DNA-Encoded Library Evaluation for Binder Recovery
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Solution Overview
Problem
Existing DNA-encoded library (DEL) screening methods face challenges such as protein immobilization-induced structural changes, limited target adaptation range, and difficulty in recovering binders with medium affinity, particularly in cross linker-modified DELs, which do not effectively utilize the merits of hairpin- and double-stranded DNA structures.
Innovation Solution
Introduce a cleavable site, such as deoxyuridine, into the DNA strand of a cross linker-modified double-stranded DEL to facilitate conversion between hairpin and double-stranded forms, allowing for efficient screening by cleaving, separating, and identifying bound library molecules, thereby enhancing DEL synthesis and evaluation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If a target protein is immobilized on an immobilization carrier for screening, then the screening process can be performed, but the steric structure of the protein changes and the target adaptation range is limited
Solution Approach 1:
The invention extracts the target protein from the immobilized state and performs screening in solution phase instead. The cross-linker is introduced to covalently bind the target protein with library molecules without requiring immobilization on a carrier, thereby maintaining the protein's native steric structure and expanding target adaptation range while still enabling systematic screening.
Solution Approach 2:
The cross-linker serves as an intermediary between the target protein and library molecules. It enables covalent bonding and stable complex formation without requiring protein immobilization, thus resolving the contradiction between facilitating screening operation and preserving protein structural integrity for broad target adaptation.
2Reliability
If conventional screening methods are used to recover binders, then high affinity binders can be recovered, but binders with medium affinity have low kinetic stability and are difficult to recover
Solution Approach 1:
The cross-linker is introduced in advance to enable covalent bonding between the target protein and library molecules during the screening process. This preliminary action of covalent cross-linking stabilizes medium affinity binders that would otherwise have low kinetic stability, allowing their reliable recovery and identification alongside high affinity binders.
3Ease of manufacture
If hairpin-stranded DNA is used in DEL, then short DNA tags can be used and chemical stability is high, but conversion to cross-linker modified DEL is difficult
Solution Approach 1:
The invention changes the DNA strand configuration from a closed hairpin structure to an open single-stranded or double-stranded structure by controlling denaturation conditions. This parameter change enables the DNA to be converted into a cross-linker modified DEL format while retaining the advantages of short DNA tags and chemical stability, thus resolving the contradiction between ease of manufacture and adaptability.
4Adaptability or versatility
If double-stranded DNA is used in DEL, then conversion to cross-linker modified DEL is possible, but DNA tags must be longer and chemical stability is reduced
Solution Approach 1:
The invention introduces dynamic control over DNA strand configuration, allowing reversible transition between hairpin and double-stranded forms through controlled denaturation and renaturation. This enables the system to adopt the optimal structure at different stages: hairpin form during synthesis for stability and compactness, and double-stranded form during cross-linker modification for versatility.
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 enables both the merits of hairpin- and double-stranded DELs to be utilized, expanding the opportunity to acquire hit compounds useful in drug, agrochemical, and medical material development by simplifying DEL synthesis and improving evaluation methods.
Implementation Method 1
when deoxyuridine is introduced into a DNA strand, it can be selectively cleaved by USER® enzyme
Implementation Method 2
crosslinking a cross linker of the library molecule bound to the biological target with a biological target
Data Source
AI summary
The invention provides a method of inducing a DNA-encoded library (DEL) comprising cleavable site(s) in the DNA strand to a cross linker-modified double-stranded DEL and evaluating the DEL. Both the merits of the hairpin-stranded DEL and the double-stranded DEL are achieved in the invention by introducing the cleavable site(s) such as deoxyuridine into the DNA strand. The invention further provides a technique for screening a compound in which both “simple DEL synthesis method” and “expansion and improvement of the DEL evaluation method” are achieved by easily inducing the compound into a cross linker-modified DEL.