RESUMEN
We here present a method that combines genetic code expansion with CRISPR/Cas9 genome engineering to label endogenously expressed proteins with high spatiotemporal resolution. The method exploits the use of an orthogonal tRNA/tRNA synthetase pair in conjugation with noncanonical amino acids to create stop codon read through events. To demonstrate the functionality of the method, we pulse labeled endogenous ß-actin and tumor protein p53 with a minimally invasive HA tag at their C-termini. Targeting the protein label with a proximity ligation assay plus real time imaging facilitates seamless quantification of the protein synthesis rate and spatial localization at the single cell level. The presented approach does not interfere with any physiological control of cellular expression, nor did we observe any perturbation of endogenous protein functions.
Asunto(s)
Actinas/metabolismo , Sistemas CRISPR-Cas/genética , Codón de Terminación , Lisina-ARNt Ligasa/metabolismo , Ingeniería de Proteínas/métodos , Proteína p53 Supresora de Tumor/metabolismo , Actinas/genética , Ingeniería Genética/métodos , Células HEK293 , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Proteínas Luminiscentes/genética , Lisina/análogos & derivados , Lisina/genética , Lisina-ARNt Ligasa/genética , Methanosarcina barkeri/enzimología , Microscopía Fluorescente , Fragmentos de Péptidos/genética , ARN de Transferencia/genética , Proteína p53 Supresora de Tumor/genética , Proteína Fluorescente RojaRESUMEN
Here, we present an in silico, analytical procedure for designing and testing orthogonal DNA templates for multiplexing of the proximity ligation assay (PLA). PLA is a technology for the detection of protein interactions, post-translational modifications, and protein concentrations. To enable multiplexing of the PLA, the target information of antibodies was encoded within the DNA template of a PLA, where each template comprised four single-stranded DNA molecules. Our DNA design procedure followed the principles of minimizing the free energy of DNA cross-hybridization. To validate the functionality, orthogonality, and efficiency of the constructed template libraries, we developed a high-throughput solid-phase rolling-circle amplification assay and solid-phase PLA on a microfluidic platform. Upon integration on a microfluidic chip, 640 miniaturized pull-down assays for oligonucleotides or antibodies could be performed in parallel together with steps of DNA ligation, isothermal amplification, and detection under controlled microenvironments. From a large computed PLA template library, we randomly selected 10 template sets and tested all DNA combinations for cross-reactivity in the presence and absence of antibodies. By using the microfluidic chip application, we determined rapidly the false-positive rate of the design procedure, which was less than 1%. The combined theoretical and experimental procedure is applicable for high-throughput PLA studies on a microfluidic chip.
