Cyclic Peptide Library Screening with Segmented Co-Compartmentalisation
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Solution Overview
Problem
Existing methods for generating and screening cyclic peptide libraries face challenges in compatibility of biochemical reactions within droplets, droplet fusion difficulties, and limitations in library size and functional assays, particularly in non-specialist settings, which hinder high-throughput screening and identification of peptides with desired pharmacological activities.
Innovation Solution
A method for co-compartmentalising cyclic polypeptides with their encoding polynucleotides, involving expression and cyclisation within compartments like droplets or beads, allowing for passive or enzymatic cyclisation, and subsequent screening for desired activities using fluorescence-activated sorting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If droplet fusion is used to combine IVTT containing droplets with droplets enclosing amplified DNA, then co-compartmentalisation is achieved, but the process becomes complex and difficult to implement in continuous workflow
Solution Approach 1:
The system separates DNA amplification and protein expression into distinct compartments (droplets or beads) rather than requiring droplet fusion. Each compartment independently contains either amplified DNA or IVTT machinery, eliminating the need for complex fusion processes while maintaining co-compartmentalisation benefits.
Solution Approach 2:
The invention introduces an intermediary approach where compartments are designed with complementary surfaces or recognition elements that enable spontaneous association without requiring active fusion mechanisms. This mediator-based approach simplifies the combining process while achieving the desired co-compartmentalisation.
2Adaptability or versatility
If different biochemical reactions are performed in the same droplet, then multi-step processes are enabled, but compatibility issues arise between reactions
Solution Approach 1:
Different biochemical reactions (DNA amplification and protein expression) are segregated into separate compartments rather than being performed in the same droplet. This physical separation eliminates compatibility issues between reactions while maintaining the ability to process multiple steps through sequential compartment operations.
Solution Approach 2:
DNA amplification is performed in advance in separate compartments before protein expression. This preliminary action allows optimization of each reaction condition independently, ensuring reliable performance of each step without interference from other reactions.
3Manufacturing precision
If single templates are isolated for protein expression, then monoclonal units are generated, but insufficient protein is produced to reach detection threshold
Solution Approach 1:
DNA templates are pre-amplified in separate compartments to generate sufficient quantities before protein expression. This preliminary amplification ensures that when IVTT occurs in monoclonal units, enough protein is produced to reach detection thresholds while maintaining the precision of single-template isolation.
Solution Approach 2:
The system separates template amplification from protein expression into distinct compartments. This segmentation allows independent optimization of each process, enabling sufficient protein production for detection while maintaining monoclonal unit integrity through separate compartment processing.
4Reliability
If cyclic peptides are generated with improved pharmacokinetic properties, then drug candidate quality increases, but screening and identification efficiency decreases
Solution Approach 1:
The system incorporates fluorescent tagging or colorimetric indicators in compartments containing cyclic peptides. This allows rapid optical detection and sorting of active peptides through fluorescence-activated sorting, maintaining high screening efficiency while evaluating peptides with improved pharmacokinetic properties.
Solution Approach 2:
Traditional mechanical sorting methods are replaced with optical detection and fluorescence-activated sorting. This substitution enables high-throughput identification of cyclic peptides based on their activity, maintaining productivity while assessing pharmacokinetic properties through in vitro compartmentalization assays.
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 high-throughput screening and identification of cyclic peptides compatible with pharmaceutical assays, facilitating the generation and tagging of cyclic peptide libraries for functional assays, with improved pharmacokinetic properties and ease of identification.
Implementation Method 1
Aqueous droplets link genotype to phenotype since the compartmentalization they provide mimics that of nature's cells. In each man-made compartment, a single gene is transcribed and translated by cell-free means to give multiple copies of the protein it encodes.
Implementation Method 2
subsequent screening for desired activities using fluorescence-activated sorting
Data Source
AI summary
A method for co-compartmentalising a cyclic polypeptide with a polynucleotide encoding the cyclic polypeptide, comprising the steps of a) forming a compartment containing a polynucleotide encoding the cyclic polypeptide, b) expressing a polypeptide from the polynucleotide, and c) cyclising the polypeptide. Co-compartmentalised cyclic polypeptides and encoding polynucleotides. Libraries of co-compartmentalised cyclic polypeptide and encoding polynucleotide. Methods for screening libraries of co-compartmentalised cyclic polypeptide and encoding polynucleotide. Incorporation of non-canonical nucleic acids into such libraries.