Poster abstracts
Poster number 52 submitted by Rachel Robertson
A mass spectrometry-based diagnostic method for detection of clinically relevant RNA biomarkers
Rachel M. Robertson (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Anju Sheregar (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Abraham Badu-Tawiah (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Venkat Gopalan (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210)
Abstract:
Extracellular RNAs in the blood have diagnostic and prognostic value in various cancers. Importantly, distinctive circulating RNA signatures could exemplify gene expression changes unique to specific cancers. Despite this promise of non-invasive cancer diagnosis based on detection of RNA released by tumor cells, abundance as well as the necessity for multiplexed detection of a panel of RNA biomarkers pose difficulties for reliable diagnostics. Here, we seek to address this challenge by developing an affordable, mass spectrometry-based multiplexed RNA detection method that can be used for point-of-care diagnostics. For proof-of-concept, we used as our first target RNA, n-Tr20-ext, a mouse tRNA-Arg-UCU isodecoder that was engineered with 5' and 3' extensions. A biotin-streptavidin based pull-down system was used to capture n-Tr20-ext by its 3' extension. In parallel, we developed a detection conjugate by attaching an in-house-synthesized ionic probe to a poly-amidoamine dendrimer, which in turn was attached to a DNA oligonucleotide that is complementary to the 5' extension of n-Tr20-ext. Multiple copies of the ionic probe, which upon alkali hydrolysis release a diagnostic fragment with m/z 87, ensure sensitive detection through amplification of the signal and not the analyte. The specific binding of the detection bioconjugate to the 5' extension of n-Tr20-ext was validated by comparing the results obtained using two variants with or without the 5' extension. Next, we demonstrated selectivity of the assay by showing that the readout for n-Tr20-ext was unaffected by the presence of a second RNA that lacked any complementarity to the detection oligonucleotide (bioconjugate). Lastly, we established the platform’s multiplexing capabilities by using two n-Tr20-ext variants with different 5' extensions, each interrogated with an ionic probe that yields a characteristic m/z (either 59 or 87). The use of different mass-resolved probes showcases the assay’s potential for assessing a panel of RNA biomarkers in a single assay.
Keywords: Extracellular RNA, Mass spectrometry, Cancer diagnosis