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Introduction

Development of new methods is essential to make great leaps in science, opening up new avenues for research, but the process behind method development is seldom described.

Areas covered

Over the last twenty years we have been developing several new methods, such as in situ PLA, proxHCR, and MolBoolean, using oligonucleotide-conjugated antibodies to visualize protein-protein interactions. Herein, we describe the rationale behind the oligonucleotide systems of these methods. The main objective of this paper is to provide researchers with a description on how we thought when we designed those methods. We also describe in detail how the methods work and how one should interpret results.

Expert opinion

Understanding how the methods work is important in selecting an appropriate method for your experiments. We also hope that this paper may be an inspiration for young researchers to enter the field of method development. Seeing a problem is a motivation to develop a solution.

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.

Hybridization chain-reaction (HCR) is technique to generate a linear polymerization of oligonucleotide hairpins, used in multiple molecular biology methods. The HCR reaction is dependent on that every hairpin is metastable in the absence of a triggering oligonucleotide and that every hairpin can continue the polymerization, which places a strong demand on oligonucleotide quality. In this paper we show how further purification can greatly increase polymerization potential. We found that a single extra PAGE-purification could greatly enhance hairpin polymerization both in solution and in situ. Purification using a ligation-based method further improved polymerization, yielding in situ immunoHCR stains at least 3.4-times stronger than non-purified control. This demonstrates the importance of not only good sequence design of the oligonucleotide hairpins, but also the demand for high quality oligonucleotides to accomplish a potent and specific HCR.

Determination of interactions between native proteins in cells is important for understanding function. Here the authors report MolBoolean as a method to detect interactions between endogenous proteins in subcellular compartments, using antibody-DNA conjugates for identification and signal amplification. Determining the levels of protein-protein interactions is essential for the analysis of signaling within the cell, characterization of mutation effects, protein function and activation in health and disease, among others. Herein, we describe MolBoolean - a method to detect interactions between endogenous proteins in various subcellular compartments, utilizing antibody-DNA conjugates for identification and signal amplification. In contrast to proximity ligation assays, MolBoolean simultaneously indicates the relative abundances of protein A and B not interacting with each other, as well as the pool of A and B proteins that are proximal enough to be considered an AB complex. MolBoolean is applicable both in fixed cells and tissue sections. The specific and quantifiable data that the method generates provide opportunities for both diagnostic use and medical research.

Fluorescence signal enhancement via isothermal nucleic acid amplification is an important approach for sensitive imaging of intra- or extracellular nucleic acid or protein biomarkers. Rolling circle amplification (RCA) is frequently applied for fluorescence in situ imaging but faces limitations concerning multiplexing, dynamic range, and the required multiple washing steps before imaging. Here, we show that Forster resonance energy transfer (FRET) between fluorescent dyes and between lanthanide (Ln) complexes and dyes that hybridize to beta-actin-specific RCA products in HaCaT cells can afford washing-free imaging of single beta-actin proteins. Proximity-dependent FRET could be monitored directly after or during (real-time monitoring) dye or Ln DNA probe incubation and could efficiently distinguish between photoluminescence from beta-actin-specific RCA and DNA probes freely diffusing in solution or nonspecifically attached to cells. Moreover, time-gated FRET imaging with the Ln-dye FRET pairs efficiently suppressed sample autofluorescence and improved the signal-to-background ratio. Our results present an important proof of concept of RCA-FRET imaging with a strong potential to advance in situ RCA toward easier sample preparation, higher-order multiplexing, autofluorescence-free detection, and increased dynamic range by real-time monitoring of in situ RCA.

The role of plasma membrane composition and dynamics in the activation process of receptor tyrosine kinases (RTKs) is still poorly understood. In this study we have investigated how signaling via the RTK, platelet-derived growth factor beta-receptor (PDGFR-beta) is affected by Dynasore or Dyngo-4a, which are commonly used dynamin inhibitors. PDGFR-beta preferentially internalizes via clathrin-coated pits and in this pathway, Dynamin II has a major role in the formation and release of vesicles from the plasma membrane by performing the membrane scission. We have found that dynamin inhibitors impedes the activation of PDGFR-beta by impairing ligand-induced dimerization of the receptor monomers, which leads to a subsequent lack of phosphorylation and activation both of receptors and downstream effectors, such as ERK1/2 and AKT. In contrast, dynamin inhibitors did not affect epidermal growth factor receptor (EGFR) dimerization and phosphorylation. Our findings suggest that there is a link between plasma membrane dynamics and PDGFR-beta activation, and that this link is not shared with the epidermal growth factor receptor.

Proximity based detection methods are invaluable tools in the field of molecular biology, increasing selectivity and allowing for analysis of protein interactions. ProxHCR utilizes pairs of antibodies labelled with oligonucleotides to probe for proximal binding and to initiate a hybridization chain reaction (HCR) to generate an amplified detection signal. As HCR is based upon hybridization of DNA hairpins, the performance is dependent on salt concentrations and temperature. Herein we have redesigned the proxHCR system to increase the performance and to reduce dependency on temperature and salt concentrations. The new oligonucleotides provide an increased signal when performed at physiological salt concentrations and in room temperature.

We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes -UnFold probes - where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic "unfolding" step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.

To accurately diagnose cancer and other aberrant cellular conditions, investigations of several parameters are required. Not only morphology, protein and gene expression but also more complex investigations, such as protein modifications, translocations and interactions can provide essential data. In order to investigate such cellular events, several molecular tools have been developed. Among them is proximity-dependent initiation of hybridization chain reaction (ProxHCR), an inexpensive tool developed to provide accurate analysis of proximity events within cells. While previous versions of ProxHCR has been capable of detecting protein-proximity, lacking signal strength has resulted in poor ability to properly quantify imaged results. Here we show how further purification of the HCR detection hairpins considerably increase signal strength. The increased signal strength allow for accurate quantification of proximity events in line with the established PLA method. Furthermore we show how ProxHCR can be used to track PDGFR-β phosphorylation and successive recruitment of GRB2 and PI3K over time. These results show how ProxHCR can be a valuable, enzyme free alternative to other proximity analysis tools.