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Global analysis of DNA-protein interactions by sensitive proximity-dependent DNA ligation assays and tag arrays

Final Activity Report Summary - HIGH-THROUGHPUT PROX (Global analysis of DNA-protein interactions by sensitive proximity-dependent DNA ligation assays and tag arrays)

The main objective of this project was the development of a test system for the analysis of interactions between deoxyribonucleic acid (DNA) and proteins. These interactions are of great scientific interest as they regulate the way in which genetic information is used.

In this regard, we designed and implemented a novel approach based on the principle of proximity ligation, in which the target-specific binding of affinity probes induced proximity between oligonucleotides attached to these probes and thereby supported their enzyme-mediated catenation, i.e. DNA ligation. Here, one of the affinity probes was an antibody with strong affinity for the DNA-binding protein to be analysed while the other probe was made up of a DNA sequence with unknown affinity for that protein. In this setup, the degree of affinity of the studied DNA-binding protein to a particular DNA sequence was proportional to the number of complexes in which the two affinity probes were in non-random proximity. DNA ligation was used to convert this invisible spatial information into an amplifiable molecule that could be detected and quantified by a method termed quantitative real-time polymerase chain reaction (PCR).

A major advantage of our approach was its ease of use and the fact that it required no more than standard laboratory equipment. We confirmed the assay performance by analysing DNA-binding of several well-studied transcription factors and published our findings in the prestigious scientific journal ‘Proceedings of the National Academy of Sciences of the United States of America (PNAS)’. We thereafter encountered problems in the process of multiplexing the assay, i.e. when we tried to extend it towards the parallel analysis of many DNA-protein interactions. At this point we decided to adjust the objectives of the project and focus more on other aspects of the proximity ligation technology.

Initially, the fellow worked on optimising a protocol that bound analytes to a solid support so that substances interfering with the assay could be removed by washes. Additionally, he worked on procedures to improve PLA performance in complex samples such as cell lysate, nuclear lysate, blood serum and plasma. These were important issues in the context of biomarker analysis, as the available samples usually consisted of blood or blood derivatives. A biomarker was a biological molecule, often but not necessarily a protein, which was found in blood, other body fluids or tissues and was a sign of a normal or abnormal process, or of a condition or disease. A biomarker might also be used to examine how well the body responded to a treatment for a disease or condition. In this regard, the fellow was involved in a project established at Stanford University about profiling of putative plasma biomarkers relevant to pancreatic and ovarian cancer. This cooperation yielded a co-authored publication together with distinguished scientists such as Rob Tibshirani and Ronald W. Davis.

The fellow was also involved in a project dealing with the diagnosis of nine world organisation for animal health (OIE) list A diseases and was currently in the process of finalising a publication on which he was the first author by the time of the project completion. The contribution of the fellow was to develop proximity ligation assays for the detection of viruses that were causative of some of these diseases. Sensitive real-time PCR assays were available for this purpose; nevertheless they required time consuming DNA preparation or ribonucleic acid (RNA) preparation and reverse transcription. Additionally, they could be sensitive for minor mutations of the virus genome. An antibody-based method avoided these limitations of PCR technology, but standard enzyme-linked immunosorbent assay (ELISA) usually suffered from limited analytical sensitivity. PLA detection of viruses combined the advantages of both approaches, i.e. high sensitivity through exponential PCR amplification, relative insensitivity towards minor mutations as well as speed and ease of use because of dispensable sample preparation.

He firstly focussed on vesicular diseases of pigs and other domestic animals. Swine vesicular disease (SVD) was a contagious disease of pigs, caused by an enterovirus, called the swine vesicular disease virus (SVDV) and characterised by vesicles on the coronary band, heels of the feet and occasionally on the lips, tongue, snout and teats. It was not an economically important disease, but it should be controlled so that diagnostic confusion with foot and mouth disease (FMD) could be avoided. Vesicular stomatitis (VS), caused by the vesicular stomatitis virus (VSV) was a vesicular disease of horses, cattle and pigs that was indistinguishable from FMD or SVD. In addition, sheep, goats and many other wild species could be infected, while humans were also susceptible.

He secondly focussed on poultry diseases. Avian influenza, or ‘bird flu’, was a contagious disease of animals caused by viruses that normally infected only birds and, less commonly, pigs. Avian influenza viruses were highly species-specific, but had, on rare occasions, crossed the species barrier to infect humans as they were closely related to the viruses causing human infections. In domestic poultry, infection with avian influenza viruses caused two main forms of disease. The so-called ‘low pathogenic’ form commonly caused only mild symptoms such as a drop in egg production and could easily go undetected. The highly pathogenic form was far more dramatic, spread very rapidly through poultry flocks, caused disease affecting multiple internal organs, and had a mortality that could approach 100 %, often within 48 hours. The ability to rapidly recognise avian influenza virus (AIV) was of paramount importance to ensure that appropriate measures could be taken quickly to contain the spread of the virus.

Infectious laryngotracheitis virus (ILTV), an alpha herpesvirus, was an important pathogen of poultry in the North America, Europe and Australia, responsible for egg production losses and death. It caused an acute disease of chickens which was characterised by respiratory depression, gasping and expectoration of bloody exudate. Viral replication was limited to cells of the respiratory tract wherein infection of the trachea gave rise to tissue erosion and haemorrhage. Newcastle disease was a highly contagious disease that affected the digestive, respiratory and nervous systems of domestic poultry, cage and aviary birds and wild birds. It was caused by a virus, namely the Newcastle disease virus (NDV) of the family paramyxoviridae. Some strains of the disease caused only minor symptoms while others were fatal. The severity of the symptoms depended on the strain of the virus and the age and health of the bird.

Overall, sensitive PLA assays were developed for the detection of three economically and epidemiologically important poultry diseases. A logical next step would be to combine these assays into a multiplex panel for the parallel detection and discrimination of these diseases. For reasons of time, this step lay beyond the scope of the project but was pursued by collaborators from the ‘Swedish National Veterinary Institute’ (SVA, Prof. Sandor Belak et al.).