DNA‐based molecular tools for monitoring cellular activity

Abstract

Methods to determine levels of protein‐protein interactions are essential in analysis of cellular signaling activity. High‐resolution analysis of proteins or protein complexes requires either advanced microscopy, or molecular methods that combine distance dependence with signal amplification. By using DNA molecules as tools, we have over the years developed several molecular methods utilizing a variety of enzymes to modify and amplify the DNA molecules used in the methods. One such method is Proximity Ligation Assay (PLA) where pairs of antibodies equipped with DNA oligonucleotides (so‐called proximity probes) are used to target proteins. Proximal binding of such probes template the creation of a circular DNA molecule, which can be amplified using rolling circle amplification (RCA). The single‐stranded RCA product from a single recognition event will contain several hundreds of repetitive motifs that can be visualized by hybridization of fluorophore‐conjugated oligonucleotides.Information on levels of both free and interacting proteins is important to determine if the interactions monitored are a result of a post‐translational protein modification, e.g. phosphorylations, that changes the conformation of the proteins and subsequently results in a different dissociation constant (KD), or if the amount of interactions reflects mere expression levels of the proteins. New more complex measurements are needed in order to model signaling pathway activity and information flow in single cells. We have hence further developed PLA to provided information on levels of both free and interacting proteins.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.