Reagents and Methods for Assaying Protein Synthesis and Amino Acid Transport

We provide novel reagents - stable ternary complexes, and labeled tRNA and eF-Tu molecules - that together can be used to assay translation of mRNA into proteins in real time, or can be used in artificial vesicles impregnated with amino-acid transporters (AAT), to assay the activity of those transporters. The labels can be coordinated such that FRET, or single molecule FRET (smFRET), or quenching experiments can be conducted.

Using the reagents and methods, one can obtain a separate FRET reaction each time an amino acid is joined to the growing protein chain, and thus do protein sequencing.

The assay for AAT function is best conducted with fluorophore-quencher pairs. A vesicle is created, with an AAT in the membrane, containing tRNA for the amino acid and ternary complex including flurophore (or quencher) labeled EF-Tu and quencher (or fluorophore) labeled tRNA. In the ternary complex, the fluorophore is quenched, providing dark ternary complexes. Upon delivery of the charged tRNA to the ribosome, the ternary complexes dissociate and a burst of fluorescence is released. AATs are implicated in metabolic, psychiatric, and neurological diseases, and AAT modulators may also be useful to improve the distribution of drugs with poor bioavailability.

This invention also provides kits, methods for preparing stable ternary complexes, methods of protein sequencing, methods of detecting amino acid transport and methods of imaging translation events in single living cells.

Potential Applications

• Reagents for protein translation assays
• Reagents for amino-acid transporter activity assay
• Next-generation protein sequencing
• Drug screening assays to identify compounds that modulate amino acid transporter activity or protein translation