High-Throughput Amenable MALDI-MS Detection of RNA and DNA with On-Surface Analyte Enrichment Using Fluorous Partitioning
Year: 2020
Type: article
Abstract: High-throughput matrix-assisted laser desorption/ionization mass spectrometry (HT-MALDI-MS) has garnered considerable attention within the drug discovery industry as an information-rich alternative to assays using light-based detection methods. To date, these efforts have been primarily focused on assays using protein or peptide substrates. Methods for RNA or DNA analysis by HT-MALDI-MS have not been extensively reported due to the challenges associated with MALDI-MS of oligonucleotides, including the propensity to form multiple salt adducts, low ionization potential, and ease of fragmentation. The objective of this work was to develop a platform suitable for HT-MS analysis of RNA and DNA substrates that overcomes these hurdles by combining on-surface sample preparation with soft ionization. This has been accomplished through the selective immobilization of fluorous-tagged oligonucleotides on a fluorous-modified MS target plate, followed by on-surface enrichment, matrix addition, and direct laser desorption/ionization, a process dubbed fluorous HT-MS (F-HT-MS). The work has resulted in methods by which RNA and DNA substrates can be detected at nanomolar concentrations from a typical assay buffer system using procedures that are amenable to full automation. The protocols were applied to an miRNA biogenesis assay, demonstrating its potential for RNA processes and thereby filling a prominent gap in RNA drug discovery: the paucity of in vitro functional assays.
Source: SLAS DISCOVERY
Institution: University of Pittsburgh
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $3,000
Awards: DNA Computation in Cells, G6000670052, Digestive Health and Diseases (DHD) Training Grant, RNA and DNA analysis and detection by fluorous high-throughput MS, G7962024199, Probing short and long term consequences of Small and Large Bowel Microbiota Transplants on Host Physiology: Implications for the development of future live biotherapeutics
OpenAlex ID: W3090636803