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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.  

Cellular functions are governed by intricate chains of interactions between proteins. In order to properly understand cellular biology one must look into, not only protein function, but also its interacting network. Furthermore, due to the large heterogeneity between cells within cultures and tissue samples it is important to retain spatial information to enable investigations on a single cell level. In order to achieve this, in situ methods play a large importance in further elucidation of these interacting networks.

In order to investigate interactions between macromolecules such as proteins and nucleic acids, many outstanding methods have been developed. Some focusing on larger scale analysis, some on live cell imaging and some on detecting novel interactions. Our own group has focused on in situ methods utilizing DNA conjugated antibodies. DNA itself is a great macromolecule to work with, it can be produced synthetically, DNA hybridization is highly predictable and there is a large repertoire of DNA-modifying enzymes. This has been used in the development of methods such as proximity ligation assay (PLA) and Proximity-dependent initiation of hybridization chain reaction (ProxHCR).

Both methods utilize antibodies conjugated with DNA in order to detect proximity events between two proteins. PLA utilizes ligation to confirm proximity, while ProxHCR utilizes a chain of strand displacements to do the same. Both methods work well, but no method is beyond further optimization.

For PLA, a general concern lies in the formation of incorrectly interacting probes, resulting in incorrect ligations that yield linear fragments, incapable of producing visible signal. As a result PLA can produce a substantial amount of false negatives. To address this, we produced a similar method, Unfold, to streamline the probe interactions and ligations to improve efficiency.

For ProxHCR the original method required overly stringent reactions conditions to allow for efficient strand displacements and thus strong signal. Furthermore, signal strength was further compromised by oligonucleotide quality. To improve these issues, the ProxHCR method was completely redesigned and oligonucleotide quality along with signal strength was improved by further purification.

Both optimizations resulted in more efficient and versatile methods suitable for routine lab work and potential diagnostic use.