From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase

Figure 7

Schematic representation of in situ Proximity Ligation Assay (PLA). (1) Protein interaction: the complex is formed between target proteins (light blue and pink). (2) Proximity probe binding: antibodies (black) with individual proximity probes (glay) bind to each of the target protein complexes. (3) Hybridization of connector oligo-DNAs: target protein complex serves to template the hybridization of connector oligo-DNAs (red). (4) Ligation to form complete DNA circle: adjacent connector oligos that are perfectly complementary to the target template are connected by DNA ligase. (5) Rolling Circle Amplification (RCA) and detection: after ligation, the RCA is initiated by the Phi29 DNA polymerase, by turning the target molecule primed by one of the proximity probes. The RCA product is detected through the hybridization of fluorescently labeled probes (green). Arrowheads indicate 3′ ends, and roundheads means 3′ ends modified with 2′ O-methyl RNA.