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In situ proximity ligation assay (PLA) is a method for detection of protein interactions, post-translational modifications (PTMs) and individual proteins that allows information about their localization in a cell or tissue to be extracted. The method is based on oligonucleotide-conjugated antibodies (proximity probes) that upon binding of two epitopes in close proximity give rise to an amplifiable DNA circle. Rolling circle amplification (RCA) is used to create a DNA bundle of over a thousand repeats to which fluorescently labeled detection oligonucleotides are hybridized. This thesis is focused on improving the existing in situ PLA method and on developing new approaches for detection of proteins, protein-protein interactions and PTMs in situ in cells and tissues.

In paper I, a new enzyme-independent method capable of in situ detection of protein-protein interactions was developed. The method combined the proximity requirement of in situ PLA and the amplification of hybridization chain reaction (HCR) creating a proximity-dependent initiation of hybridization chain reaction (proxHCR). Circumventing the need for enzymes resulted in a cost-efficient method that is less sensitive to storing conditions.

Paper II addresses the problem of irregularly formed RCA products that can appear to be split into several fluorescent objects. A compaction oligonucleotide system was designed to crosslink the DNA bundle with itself and thereby reduce the size and increase the brightness of each individual RCA product.

In paper III, the conventional in situ PLA was redesigned to increase the detection efficiency of protein interactions and PTMs in situ. The new set of proximity probes was designed to have circularization oligonucleotides incorporated that were unfolded through enzymatic digestion. The UnFold in situ PLA was able to generate more signals and had a higher sensitivity than the conventional in situ PLA.

In paper IV, an oligonucleotide system able to generate signals for individual proteins (A or B) and their interaction (A and B) in a molecular Boolean (MolBoolean) protein analysis was designed. The MolBoolean design was able to generate signals detecting both individual proteins and their interaction in situ.