Signal amplification methods for the imaging of protein synthesis and neurotransmitter transport

Abstract

The present invention describes the synthesis of biological samples that can be used for the purpose of enhancing the signal-to-noise ratios achievable during the imaging of protein synthesis, amino acid transport and neurotransmitter transport, particularly in applications where single-molecule resolution is demanded. The present invention provides quencher-labeled elongation factor (EF-Tu) and fluorophore-labeled tRNA. When these molecules are present in a ternary complex with GTP, the fluorescently-labeled tRNA is quantitatively quenched. Once the tRNA is incorporated into an actively translating ribosome, however, a burst of fluorescence is released and can be detected by a variety of techniques, including smFRET imaging. The invention further provides novel EF-Tu constructs for achieving quencher labeling at high levels while retaining native or near native activity in the translation reactions, as well as methods for preparing stable ternary complexes, methods of protein sequencing, methods of detecting amino acid transport using a proteoliposome assay system and the proteoliposomes systems and methods of imaging translation events in single living cells. The present invention should have an immediate impact on next-generation sequencing technologies and the detection of neurotransmitter transporter activities in both in vitro and in vivo settings, a critical component of drug activity/screening assays targeting this important class of molecules.