Cell-Free Translation Readthrough Assay System

A high-throughput cell-free assay system screens translation readthrough drugs using labeled reporter complexes and fluorescence-based measurements.
Tech Title: Technology:
Technology: The system includes a substrate with multiple regions, each containing a cell-free ribosome-dependent protein synthesis reporter complex. The reporter complex includes a ribosome, a modified Stop-IRES mRNA, and labeled peptidyl-tRNA or labeled-tRNA. In some assay formats, the modified RNA includes a portion of mRNA containing a premature stop codon mutation. The assay can be configured as a high-throughput format and can classify compounds based on measured signal changes.
Problem Title: Problem:
Problem: Premature termination codons can produce truncated proteins and are associated with disorders driven by nonsense mutations. Translation readthrough drugs may act through different mechanisms, making their effects difficult to interpret. This complexity has complicated efforts to determine how specific candidates work. It has also limited rational identification of additional translation readthrough drugs.
Solution Title: Solution:
Solution: The technology uses highly purified eukaryotic cell-free protein synthesis reporter complexes to screen translation readthrough drugs. Reporter complexes include a ribosome, a modified RNA with an IRES and termination codon, and labeled peptidyl-tRNA or labeled-tRNA. Candidate drugs are combined with the reporter complex and a release factor complex. Fluorescence anisotropy or changes in fluorescence or absorption are then measured.
Advantages Title: Advantages:
Advantages:

Uses highly purified eukaryotic cell-free protein synthesis systems.
Supports high-throughput screening.
Measures fluorescence anisotropy or changes in fluorescence or absorption.
Distinguishes compounds acting directly on protein synthesis machinery from compounds that act indirectly.
Can incorporate mRNA portions containing premature stop codon mutations.

Applications:

Drug Screening: Screens candidate compounds for translation readthrough activity using fluorescence-based measurements.
Mechanism Studies: Differentiates compounds that act directly on protein synthesis machinery from compounds that act indirectly.
Mutation-Specific Assays: Evaluates compounds using reporter constructs containing portions of mRNA with premature stop codon mutations.
PTC Disorder Research: Supports assays using mRNA portions associated with cystic fibrosis, Duchenne muscular dystrophy, β-thalassemia, or cancer.

Intellectual Property: