Dynamic chemical labelling to profile circulating microRNAs in body fluids

Resumen

In the last few years, numerous studies are speedily expanding in search of various biochemical markers found in circulation. Strictly speaking, biomarkers are molecules that detect or confirm the presence of a pathophysiologic condition aiding to establish a diagnosis, refining the prognosis and modifying the treatment. The ideal biomarker exhibits high sensitivity and specificity and should be minimally-invasive. Micro-Ribonucleic Acids (miRNAs) have been proposed as a new class of biomarkers for multiple human diseases. miRNAs are small non-coding RNAs of 19–24 nucleotides in length that play a major role in fine-tuning the expression of protein-coding genes within the human organism as well as they perform a crucial role in the development and maintenance of numerous pathological processes. A substantial number of miRNAs are present outside the cells, circulating in blood and other body fluids. Recently, there has been a significant interest within the research community to discover and validate circulating miRNAs as clinical biomarkers. However, conventional techniques are not particularly suitable to interrogate small RNA species, such as miRNAs. Outside of these conventional tools, a unique PCR-free method for the direct detection of nucleic acids (NAs) based on dynamic chemistry, the so-called dynamic chemical labelling (DCL), has been recently proposed. The approach combines the specific labelling of an immobilized abasic peptide nucleic acid (PNA) capture probe with a biotinylated reactive nucleobase, through the templating action of target NA molecules, allowing NA reading with single base resolution. The DCL method harnesses Watson–Crick base pairing to template a dynamic reductive amination reaction on a strand of an abasic PNA, reaction which is thermodynamically controlled. Complementary NA strands also act as catalysts to accelerate the rate of reductive amination. Therefore, when there are not complementary NA strands, reactions do not happen within the assay timeframe. The aim of this project was to develop, optimise and implement the DCL method into different commercial reading platforms, some of them IVD (in-vitro diagnostic) certified, to reach an assay which allow interrogating patient’s biological fluids for obtaining clinical decisions. Different targets were studied: a) miR-451a, an erythroid cell-specific miRNA associated with human erythroid maturation. b) miR-122, a well-known specific miRNA of liver cells which is an early and more sensitive indicator of drug-induced liver injury (DILI) than other currently used biomarkers such as ALT or AST. c) Simultaneous detection of proteins and miRNAs in a single sample, which we have coined seqCOMBO assay. The DCL method was integrated into two different platforms: a) FLUOstar OMEGA, a conventional multi-mode fluorescent micro-plate reader for a bead-based in order to develop a novel cost-effective manner for profiling miRNAs. b) Luminex MAGPIX system, a bead-based fluorescent assay with multiplexing capabilities. Combining the DCL method with different platforms, direct quantification of miRNAs from biological fluids can be achieved. When used with clinical samples, the developed assay presented an AUC value of 0.94 in distinguishing pathological vs. non-pathological conditions, with no false-positive detected. In this Doctoral Thesis and for the first time, a multiplex assay of molecules from different natures (miRNAs and proteins) has been presented, leaving open a new door for the development of new diagnostic opportunities both in the R&D and IVD markets.