When analyzing large RNA such as mRNA, the first denaturation step can be performed in the presence of 8 M urea and heat (90 ☌) (14) to unfold the RNA and improve the access of the nuclease to single strands. (9,14,16) The two nucleases generate 3′-linear phosphate fragments. RNases T1 and A are commonly used for the digestion of large RNA molecules due to their high nucleobase specificity and availability from several suppliers. The final HILIC-UV profiles for the three RNases were obtained with this setup. The Fusion instrument was coupled to Thermo Scientific UltiMate 3000 RS pump, sampler, column compartment, and DAD modules. The optimization of the HILIC method was done using an Agilent Technologies (Waldbronn, Germany) 1290 instrument equipped with a quaternary pump, sampler, column thermostat, and a diode array detector (DAD) module containing a 2.5 μL flow cell volume. A UV detector was equipped with a 2.5 μL flow cell volume and operated at 260 nm. A Binary Pump H and Split Sampler HT Vanquish modules were assembled with UltiMate 3000 rapid separation (RS) thermostatic column compartment and a multiple-wavelength detector module. The development of the digestion procedures and analysis of sgRNA sequences and impurities was performed using Thermo Scientific (Madison, WI, USA) Vanquish and UltiMate 3000 modules. This demonstrated the robustness and flexibility of the process to adapt to different instrumentation. Three LC instruments were used to develop this process. (14) In these studies, the oligonucleotide fragments generated via RNase digestions were separated by ion-pairing reversed-phase liquid chromatography (RPLC). (13) In 2019, greater than 70% sequence coverage was achieved for therapeutic mRNA near 3000 nucleotides long via RNase T1, Colicin E5, and MazF digestions. (12) Next to RNA sequencing, the advances were also applied for the quantification of RNA nucleoside modifications using the bottom-up approach. Major breakthroughs include the (i) sequencing of tRNA via parallel RNase T1, A, and U2 digestions (9) (ii) use of a MS-compatible hydrophilic interaction liquid chromatography (HILIC) method in the absence of ion-pairing agents and (iii) expression of novel nucleobase-specific RNases, that is, RNase U2, (10) MC1, (11) and cusativin. Limbach’s group has pioneered the characterization of modified ribonucleic acids via the use of enzymatic digestions and MS detection. (8) The size reduction of large RNA oligonucleotides for further MS/MS sequence confirmation can be achieved via ribonuclease (RNase) digestions. (7) Nearly full sequencing (99% sequence coverage) of a 76-mer transfer RNA (tRNA) has been achieved by Fourier transform ion cyclotron resonance top-down MS combining collisionally activated dissociation and electron detachment dissociation fragmentation. Small oligonucleotides up to 30 nucleotides in length can be completely sequenced by top down MS using collision-induced dissociation (CID) fragmentation. To our knowledge, it is the first time that (i) RNA digests are separated and identified by HILIC-HRMS and (ii) chemically modified sgRNAs are directly sequenced via a bottom-up approach. A biocompatible ethylene-bridged hybrid amide column was evaluated for the separation of RNase digestion products. Thorough optimization of sgRNA digestions was performed by varying the nuclease-to-sgRNA ratio, buffer conditions, and reaction times. Full sgRNA sequencing was achieved using unique fragments generated by RNase T1, A, and U2 parallel digestions. When using RNase T1 digestion alone, a maximal sequence coverage of 81% was obtained excluding the nonunique fragments. In this paper, we present the full sequencing of sgRNA via parallel ribonuclease (RNase) T1, A, and U2 digestions and the simultaneous separation and identification of the digestion products by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS). Accurate sequencing of sgRNA is critical to patient safety and is the expectation by regulatory agencies. CRISPR/Cas9 is a powerful genome editing approach in which a Cas9 enzyme and a single guide RNA (sgRNA) form a ribonucleoprotein complex effectively targeting site-specific cleavages of DNA.
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