DNA-Encoded Protein Display via Transcription Stalling
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Solution Overview
Problem
Current protein and peptide arrays are limited in throughput and stability due to laborious production processes and instability of proteins when immobilized on solid surfaces, allowing for only a few thousand members to be screened effectively, with many proteins being unstable and aggregating quickly.
Innovation Solution
A method for displaying ribosomal translation products on DNA templates using a promoter linked to an open reading frame and a molecular roadblock that stalls RNA polymerase and ribosomes, allowing for the association of proteins or peptides with their encoding DNA, enabling massively parallel high-throughput screening by immobilizing DNA templates on solid supports or in flow cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If proteins are immobilized on solid surfaces for array production, then protein display and screening are enabled, but protein stability deteriorates and proteins aggregate quickly
Solution Approach 1:
The patent uses DNA as an intermediary carrier between the solid support and the protein. The DNA template is immobilized on the solid support, and the protein is synthesized in association with the DNA through in vitro translation. This intermediary approach allows the protein to be displayed without direct immobilization on the solid surface, maintaining protein stability while enabling high-throughput screening.
Solution Approach 2:
The patent replaces the mechanical/physical immobilization method (direct attachment of proteins to solid surfaces) with a biochemical system (in vitro translation coupled with DNA templates). This substitution eliminates the harmful effect of protein aggregation on solid surfaces while maintaining the ability to display and screen proteins at high throughput.
2Quantity of substance
If conventional protein synthesis and array printing methods are used, then protein arrays can be produced, but the process becomes laborious and throughput is limited to several thousand members
Solution Approach 1:
The patent merges multiple functions into a single integrated system: DNA template immobilization, in vitro translation, and protein display occur simultaneously on the same solid support. This consolidation eliminates the need for separate steps of protein synthesis and array printing, enabling massively parallel production of millions to billions of protein members through high-throughput DNA sequencing methods.
Solution Approach 2:
The patent uses DNA as a copyable information carrier that can be massively replicated and sequenced. By encoding protein information in DNA sequences that can be read by high-throughput sequencing methods, the system enables parallel production and identification of millions of proteins without laborious individual synthesis and characterization steps.
3Reliability
If DNA templates are used to display ribosomal translation products, then protein stability and association with encoding DNA are maintained, but the system complexity increases
Solution Approach 1:
The patent extracts the essential function of protein display from the complex cellular machinery and implements it in a simplified in vitro system. By using purified DNA templates, isolated ribosomes, and defined translation factors, the system achieves stable protein-DNA association without requiring complex in vivo cellular environments, thus reducing overall system complexity while maintaining reliability.
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 method enables the generation of arrays displaying millions to billions of functional proteins or peptides, allowing for high-throughput sequencing and functional characterization, maintaining protein stability and facilitating high-throughput processing and screening.
Implementation Method 1
a molecular roadblock that blocks progress of an RNA polymerase during transcription of the DNA template
Implementation Method 2
a ribosome that binds to a ribosomal binding site on the mRNA, wherein the ribosome carries out protein translation
Implementation Method 3
such that the DNA template and transcribed mRNA remain associated
Data Source
AI summary
A novel method for displaying proteins and peptides is disclosed in which individual proteins or peptides remain associated with the DNA encoding them. Proteins or peptides can be generated by in vitro translation of DNA templates, either free in solution or arrayed on a solid support, such that the proteins or peptides remain immobilized on their DNA templates. In particular, high throughput sequencing can be combined with high throughput functional characterization of encoded proteins and peptides, wherein the identity of each protein or peptide is determined by DNA sequencing, and functional studies are carried out directly on each protein or peptide while immobilized on the DNA template encoding it. The methods of the invention should find numerous applications, for example, in high throughput genetic or pharmacological screening, epitope mapping, and protein engineering and directed evolution.