Closing in on life: proximity dependent methods for life sciences

Method development is a corner stone in science, providing the tools required to address gaps in knowledge. For life sciences, methods for genomics and proteomics have facilitated the endeavors to map the human genome and proteome. These achievements have and will continue to profoundly influence our understanding of life and diseases, providing the blue print on cells and how the genetic information is translated into biological processes in a cell. For a multicellular organism, as ourselves, information at a single cell level is fundamental for the understanding of how individual cells are organized into organs, and how this is disrupted in malignancies. Hence, tools that preserve information on tissue architecture while obtaining information on activity status of each individual cell will be needed to determine intercellular communication and response to the microenvironment [1]. To analyze functional states of cellular signaling networks protein-protein interactions and post-translational modifications of proteins will have to be assayed, and to achieve this on endogenous proteins, affinity reagents, e.g. antibodies, will have to be used. The next task is to determine if the antibodies are in close proximity, i.e. pairwise binding to epitopes on interacting proteins, or a post-translational modification in combination with an epitope on the protein. By labeling antibodies or proteins with different fluorophores, Förster resonance energy transfer can be applied, monitoring changes in emission spectra from donor and acceptor fluorophores, or changes in lifetime [2]. Another approach to generate a signal, as a consequence of dual binding of antibodies, is to use DNA as reporter molecules. An advantage with DNA is the abundance of enzymes that can be used to modify and copy it, such as restriction enzymes, ligases and polymerases, and that hybridization between different oligonucleotides can be well predicted, which facilitates the design of the oligonucleotides used in an assay [3]. The in situ proximity ligation assay (in situ PLA) [4] utilizes DNA-conjugated antibodies (proximity probes) as hybridization templates for a pair of circularization oligonucleotides. Addition of a DNA-ligase will create a circular DNA molecule, as a surrogate marker for the pairwise binding of the proximity probes. This circle can then be amplified by phi29 DNA-polymerase, using the oligonucleotide on the proximity probe as a primer. The rolling circle amplification (RCA) product will be included as an extension of the proximity probe and can be visualized by hybridization of fluorophore-labeled oligonucleotides, complementary to the RCA product. As each RCA product consists …