Top-Down Mass Spectrometry of Synthetic Single Guide Ribonucleic Acids Enabled by Facile Sample Clean-Up.

Abstract

In recent years, CRISPR-Cas9 genome editing has become an important technology in biomedical research and has demonstrated tremendous therapeutic potential. With Cas9 endonuclease, the use of single guide ribonucleic acids (sgRNAs) allows for sequence-specific cutting on target double-stranded deoxyribonucleic acids. Therefore, the design and quality of sgRNAs can greatly affect the efficiency and specificity of genome editing. Mass spectrometry (MS) has been a powerful tool to detect molecular features and sequence a variety of biomolecules; however, as the sizes of oligonucleotides get larger, it becomes more challenging to desalt samples and achieve high-quality intact spectra with effective fragmentation. Here, we develop a simple but effective online column-based clean-up method (reversed-phase column in a size exclusion mode) that removes formulation salts and metal adducts from larger oligonucleotides upon entering the mass spectrometer in a consistent manner. Using the top-down approach without any nuclease digestion, we characterized and sequenced 100-nucleotide-long sgRNAs by higher-energy collision dissociation (HCD), collision-induced dissociation (CID), ultraviolet photodissociation (UVPD), and activated electron photodetachment (a-EPD). In a single 10 min liquid chromatography-tandem MS (LC-MS/MS) run, CID yielded the best sequence coverage, of 67%. When adding complementary UVPD and a-EPD runs, we achieved 80% overall sequence coverage and 100% cleavages for the variable sequence, the first 20 nucleotides from the 5' end. This LC-MS/MS platform provides a facile top-down workflow to analyze and sequence larger chemically modified oligonucleotides with no sample treatment.