Encoded Library Synthesis via Split-Pool and Oligonucleotide Tagging
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Solution Overview
Problem
Current methods for producing combinatorial libraries, especially those with high complexity, face challenges in synthesizing vast numbers of distinct compounds efficiently and identifying active molecules due to the dilution of library members, which complicates the screening process.
Innovation Solution
A method utilizing a 'split and pool' strategy for synthesizing libraries with encoding oligonucleotide tags, where a solution is divided into fractions, reacted with unique building blocks, and then pooled, allowing for the production of libraries with a high number of distinct members by repeating the process, enabling efficient identification of active compounds through oligonucleotide amplification and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If the number of distinct members within the library is increased to achieve higher complexity, then the probability of containing active molecules is improved, but the concentration of any particular library member is lowered which complicates identification
Solution Approach 1:
An oligonucleotide tag is introduced as an intermediary component that is coupled to each library member. This tag serves as a mediator that enables identification of the library member through amplification and sequencing, solving the problem of identifying active molecules in high complexity libraries where individual member concentrations are low.
Solution Approach 2:
The oligonucleotide tag acts as a copyable identifier that can be amplified using PCR techniques. Instead of directly detecting and identifying library members, the method creates multiple copies of the oligonucleotide tag associated with each library member, enabling sensitive detection and identification even when the original library member concentration is low.
2Quantity of substance
If combinatorial syntheses are used to produce vast numbers of compounds, then library complexity is improved, but the time frame for synthesis must remain reasonable which creates a challenge
Solution Approach 1:
The synthesis process is segmented into discrete cycles, with each cycle adding a specific number of building blocks to the library members. This segmentation allows for systematic production of vast numbers of compounds through repeated cycles, making the overall synthesis process more manageable and time-efficient while achieving high library complexity.
Solution Approach 2:
The combinatorial synthesis employs periodic cycles of building block addition, where each cycle consists of controlled steps to incorporate specific building blocks. This periodic action enables the systematic generation of diverse compound libraries over time, balancing the production of vast numbers of compounds with reasonable synthesis timeframes through efficient cyclic processing.
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 enables the synthesis of large libraries with a high number of distinct members, facilitating the identification of active molecules by amplifying and sequencing the oligonucleotide tags, thereby simplifying the screening process and allowing multiple rounds of selection while maintaining sufficient quantities for analysis.
Implementation Method 1
a DNA tag identifying a library member can be amplified using techniques of molecular biology, such as the polymerase chain reaction
Implementation Method 2
reacting the initial oligonucleotide with an incoming oligonucleotide which identifies the building block of step (b) in the presence of an enzyme which catalyzes ligation of the initial oligonucleotide and the incoming oligonucleotide
Data Source
AI summary
The present invention provides methods of synthesizing libraries of molecules comprising a functional moiety which is operatively linked to an encoding oligonucleotide, wherein the encoding oligonucleotide comprises a capping sequence containing degenerate nucleotides. The methods generally include providing a solution comprising initiator compounds comprising an initial functional moiety comprising n building blocks which is operatively linked to an initial oligonucleotide; dividing the solution into reaction vessels; reacting the initiator compounds in each reaction vessel with a building block comprising a complementary reactive group to form a covalent bond; and reacting the initial oligonucleotide in each aliquot with a distinct incoming oligonucleotide in the presence of an enzyme which catalyzes the ligation of the incoming oligonucleotide and the initial oligonucleotide, under conditions suitable for enzymatic ligation of the incoming oligonucleotide and the initial oligonucleotide to form an encoding oligonucleotide.