Enzymatic Tagging for Oligonucleotide Library Synthesis
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Solution Overview
Problem
Current split-and-mix synthesis methods for small molecules lack efficiency in identifying reactants and suffer from contamination and illegitimate hybridization issues, particularly with double-stranded identifier oligonucleotides, which affects the fidelity and robustness of the purification process.
Innovation Solution
The method employs a ligation step with a double-stranded substrate containing tags and anti-tags, where only the tags are covalently linked by a ligase, allowing for the separation of ligated tags from non-ligated anti-tags due to size and molecular weight differences, and uses a computer algorithm to maximize mismatches between tags for reduced cross-hybridization, enhancing fidelity and decoding robustness.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If double-stranded identifier oligonucleotides are used in split-and-mix synthesis, then molecule identification is achieved, but contamination and illegitimate hybridization occur reducing synthesis fidelity
Solution Approach 1:
The identifier oligonucleotide is segmented into two separate strands: tags (covalently linked to the molecule) and anti-tags (free-floating). This segmentation prevents illegitimate hybridization between identifier strands while maintaining the ability to identify synthesized molecules through tag-anti-tag binding during selection processes.
Solution Approach 2:
The anti-tag strand is extracted from the double-stranded identifier structure and separated as a free component. This extraction eliminates the problem of contamination from illegitimate hybridization between complementary identifier strands, while the anti-tags remain available to bind to their complementary tags on the molecule-attached identifiers.
2Measurement precision
If tags are covalently linked to form identifier oligonucleotides, then molecule identification is enabled, but separation from anti-tags becomes difficult
Solution Approach 1:
An asymmetric structure is created where tags are covalently linked to form the identifier oligonucleotide attached to the molecule, while anti-tags remain as separate, non-covalently-linked strands. This asymmetry in bonding structure enables easy separation during purification, as the free anti-tags can be removed without affecting the covalently-bound tag-containing identifier.
Solution Approach 2:
The tag-anti-tag interaction serves as an intermediary mechanism that enables identification without requiring permanent double-stranded structure. The anti-tags act as mediators that can transiently bind to tags for identification purposes, then be easily separated, facilitating both accurate identification and easy purification.
3Measurement precision
If tags are designed with high complementarity for specific binding, then identification specificity is improved, but cross-hybridization increases reducing decoding robustness
Solution Approach 1:
Different regions of the tag and anti-tag sequences are designed with different properties: the binding region has high complementarity for specific recognition, while other regions contain deliberate mismatches or modifications that reduce cross-hybridization potential. This local differentiation maintains binding specificity while minimizing harmful cross-reactivity.
Solution Approach 2:
The sequence parameters of tags and anti-tags are optimized to achieve high binding affinity through complementarity in critical regions, while simultaneously introducing controlled variations (mismatches, modified bases, or length differences) in non-critical regions. This parameter optimization balances binding specificity with resistance to cross-hybridization.
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 improves the fidelity and robustness of the synthesis process by reducing contamination and illegitimate hybridization, enabling the efficient identification of reactants and the generation of a library of bifunctional complexes with high accuracy.
Implementation Method 1
The substrate for the ligase is in a double stranded form and wherein the substrate comprises a plurality of tags and at least one or more anti-tags wherein tags and anti-tag(s) is/are at least partly hybridised to each other. The tags are covalently linked as a result of the action of an enzyme comprising a ligase activity on the double stranded substrate
Implementation Method 2
tags and anti-tag(s) is/are at least partly hybridised to each other
Data Source
AI summary
Disclosed is a method for obtaining a bifunctional complex comprising a molecule linked to a single stranded identifier oligonucleotide, wherein a nascent bifunctional complex comprising a chemical reaction site and a priming site for enzymatic addition of a tag is a) reacted at the chemical reaction site with one or more reactants, and b) reacted enzymatically at the priming site with one or more tag(s) identifying the reactant(s).