Nuclease Activity as a Biomarker in Cancer Detection

Sammanfattning: Nucleases are a group of enzymes that cleave the phosphodiester bonds in nucleic acids. As such, nucleases act as biological scissors that exhibit a plethora of fundamental roles, in prokaryotes and eukaryotes, dependent or non-dependent on their catalytic capability. Thus, differential status of nucleases between healthy and disease conditions might not be surprising, and can be deployed in disease detection. Specifically, there is growing body of research demonstrating the potential of nucleases as diagnostic biomarkers in several types of cancer. Biomarkers for early diagnosis are an immense need in the diagnostic landscape of cancer. In this sense, nucleases are promising biomolecules, and they possess a unique feature of catalytic activity that could be deployed for diagnosis and future therapeutic strategies.    In this thesis we aim to demonstrate the use of nucleases as biomarkers associated to cancer, and the capability of oligonucleotide substrates for targeting a specific nuclease.  The thesis work begins with comprehensive review of nucleases as promising biomarkers in cancer diagnosis (paper I). Then, we provide a methodological study in paper II, in which we propose a flexible approach for detection of disease associated nuclease activity using oligonucleotides as substrates. The probes utilized here are flanked with fluorophore at the 5’-end and a quencher at the 3’-end. Upon cleavage by nucleases, the fluorescent signal is increased in a proportional fashion to nuclease activity. This platform is suitable to implement in detection of any disease in which nuclease activity is altered.   We have applied this method in paper III, by using 75 probes as substrates to screen breast cancer cells, along with controls, for nuclease activity. We have identified a probe (DNA PolyAT) that discriminates between BT-474 breast cancer cells and healthy cells based on nuclease activity profile associated with cell membrane. Next, we screened tissue samples from breast tumors for nuclease activity, and we have identified a set of probes with the capability to discriminate breast tumor and healthy tissues in 89% of the cases (paper IV). To achieve a step forward towards non-invasive diagnosis, we have developed an activatable magnetic resonance imaging (MRI)-probe (paper V). The MRI-probe is oligonucleotide-based that works like a contrast agent, and it is activated only in presence of a specific nuclease. MRI-probes provide advantages over fluorescent probes, such as high spatial resolution and unlimited tissue penetration. In conclusion, our findings suggest the utility of nuclease activity as a biomarker in cancer detection. Moreover, we demonstrate the applicability of nuclease activity-based approaches in imaging modalities, such as MRI. Our future aim is to translate our findings into non-invasive detection of breast cancer by utilizing breast cancer activatable MRI-probes.