Implementation and technology transfer of naops for in vitro detection of scpecifc nuclease activity in liquid biopsy samplessalmonella and leukemia

Resumen

Traditionally, the detection of nucleases in cancer has been performed by quantifying their mRNAs usingPCR (1) or by immunohistochemistry (2, 3). Likewise, the presence of nucleases, like any other cancerbiomarker, has been usually determined by PCR or antibodies, but their functionality to degrade specificoligonucleotides as substrates has not been exploited in depth yet. For this reason, the main goal of thisPhD research was to develop a detection method based on Nuclease-Activated Oligonucleotide Probes(NAOPs) with the ability to detect in a sensitive and specific way the nuclease activity derived from thewhite blood cells (WBC) of patients diagnosed, mainly, with Chronic Lymphoid Leukemia (CLL), as analternative strategy for the in vitro detection of Minimal Residual Disease (MRD). In particular, chemicalmodifications such as 2'-O-methyl and 2'-fluor were used in the NAOPs tested to increase their stabilityagainst endogenous nucleases (4) and favoring their specificity by optimizing reaction conditionregarding chemicals (cations and chelators) and time. In this way, based on the knowledge and previousexperience of the biothecnology company SOMAprobes S.L. in the development of NAOPs to detect S.aureus (5) and breast cancer cells (6), in addition to a previos report of nucleaes alteration in leucocytes(7) a library of approximately 100 NAOPs was design with natural and chemically modifiedoligonucleotide sequences to be experimentally evaluated in biological samples (i.e. pure bacterial culturesupernatants, food safety testing field samples and human liquid biopsies) by in vitro assays and in silicovirtual docking analysis, in order to obtain NAOPs able to detect Salmonella spp. and leukemia underoptimized by Fluorescence Resonance Energy Transference (FRET) and Lateral Flow Immuno-Chromatography (LFIC).References:1.Tsuchiya N, et al. Cancer research. 2007 Oct 1;67(19):9568-76. PubMed PMID: 179090682.Singh P, et al. Molecular cancer research. MCR. 2008 Nov;6(11):1710-7. Pubmed Central PMCID:2948671.3.Kim M-H, et al. The Journal of Clinical Investigation. 123(8):3211-30. PubMed PMID: PMC3726152.4.Opalinska JB, et al. Nature reviews Drug discovery. 2002 Jul;1(7):503-14. PubMed PMID: 12120257.5. Hernandez FJ, et al. Nature medicine. 2014 Mar;20(3):301-6. PubMed PMID: 24487433. PubmedCentral PMCID: 3949172.6. Hernandez LI, et al. Chemical Communications, 2016 Oct 25; 52(83):12346-12349. PMID: 27722495.7. Shiokawa D, et al. Cell Death and Differentiation (2007) 14, 992¿1000.