Oligo Mapping of sgRNA Digests: Leveraging Xevo MRT Mass Spectrometer Performance and Streamlining Data Analysis

Recent advances in liquid chromatography-mass spectrometry (LC-MS) and bioinformatics technologies have enhanced our detailed understanding of therapeutic RNA molecules. This application note presents a compliance-ready workflow for digested single-guide RNA (sgRNA) oligo mapping. The workflow incorporates sample preparation using the RapiZyme™ MC1 ribonuclease, UPLC™-MS^E data acquisition on the Xevo™ MRT Mass Spectrometer and automated data analysis on the waters_connect™ MAP Sequence App (ver 1.0). The Xevo MRT Mass Spectrometer combines cutting-edge multi-reflecting time-of-flight technology with hybrid quadrupole time-of-flight (QTOF) capabilities, delivering sub-ppm mass accuracy that ensures confident identification of digested RNA products. In this study, complete (100%) unique (non-ambiguous) sequence coverage of an sgRNA was achieved based on the unique accurate masses of the sgRNA digestion products. The CONFIRM Sequence App was also employed to assign isomeric digestion products by validating their sequence from elevated energy fragmentation data gathered in the same run used for digestion product mapping.

Benefits

• Confident characterization of sgRNA digestion products with sub-ppm mass accuracy of oligonucleotide precursors, outstanding MS resolution (~100,000), and sensitivity at high acquisition speed
• A compliance-ready informatics workflow featuring the waters_connect MAP Sequence App (ver 1.0) streamlines oligonucleotide mapping of sgRNA digests
• RapiZyme MC1, a new ribonuclease - offers unique cleavage specificity and the opportunity to generate overlapping digestion products, enabling complete (100%) sequence coverage for sgRNAs

Single guide RNAs (sgRNAs) were first described in 2012¹ when two RNA molecules essential for CRISPR-Cas9-mediated DNA cleavage – the trans-activating CRISPR RNA (tracrRNA), which serves as a scaffold for the Cas9 nuclease, and the CRISPR RNA (crRNA), responsible for DNA target recognition - were fused into a single RNA construct. This fusion created the sgRNA, a critical component of the CRISPR-Cas9 gene editing system.^1–2 The sgRNA molecule directs the Cas9 nuclease to introduce precise double-stranded breaks in DNA, enabling targeted genetic modifications. 

Since its discovery, which was honored with the Nobel Prize in Chemistry, CRISPR technology has transformed gene editing applications. Beyond basic research, it has been adapted for rapid diagnostic tools, such as COVID-19 tests developed during the 2020 global pandemic³, and holds promise for therapeutic interventions in genetic diseases, cancer, and infectious diseases. As sgRNAs are typically synthesized via solid-phase oligonucleotide synthesis, their analytical characterization requires confirmation of both the intact molecular weight⁴ and the sequence verification to ensure functional accuracy.⁵

Traditional LC-MS workflows for sgRNA digest oligo mapping have been labor-intensive and time-consuming, relying heavily on manual data analysis and interpretation. Recently⁶, a UPLC-MS and informatics workflow designed for automated sgRNA sequence mapping was presented following enzymatic digestion with a panel of ribonucleases, including RNase T1, hRNAse 4 and two RNase T2 enzymes: RapiZyme MC1 and Cusativin. This application note extends the effectiveness of this workflow (Figure 1) when implemented on a Xevo MRT Mass Spectrometer (Figure 1), while focusing specifically on MC1 digestion of the Waters™ sgRNA LC-MS standard, where complete unique digested product coverage was obtained.

Reagents and Sample Preparation

N,N-diisopropylethylamine (DIPEA, 99.5% purity, catalog number 387649-100ML) was purchased from Millipore Sigma (St Louis, MO) and 1,1,1,3,3,3-hexafluoro-2-propanol (IonHance HFIP, p/n:186010781) was obtained from Waters (Milford, MA). Methanol (LC-MS grade, catalog number 34966-1L) and acetonitrile (LC-MS grade, catalog number 34967-6XL) were obtained from Honeywell (Charlotte, NC). HPLC grade Type I deionized (DI) water was purified using a Milli-Q system (Millipore, Bedford, MA). Mobile phases were prepared fresh daily. Ultrapure nuclease-free water (catalog number J71786.AE) for sgRNA digestions was purchased from Thermo Fisher Scientific (Waltham, MA).

The Waters sgRNA LC-MS standard (p/n:186011357) is a 100-mer single-guide RNA (sgRNA), encoding specifically for the mouse GATA2 transcription factor. Its sequence is: 5’ - C*U*U* CAA CCA UCU CGA CUC GCG UUU UAG AGC UAG AAA UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC U*U*U* U-3’. It contains a 2’-OMe modification on its first three 5’ nucleotides (C*U*U*, denoted as the WYY sequence shown in Figure 5), as well as on its last three 3’ nucleotides (U*U*U*, sequence YYY in Figure 5). The asterisk indicates that all these six nucleotides are phosphorothioated. A 50 µM stock solution of the Waters sgRNA LC-MS standard was prepared using nuclease-free water.

RapiZyme MC1 (Waters p/n:186011190, 10000 units/tube) is a novel RNA digestion enzyme recently introduced by Waters Corporation.^6-7 10.6 µL of 50 µM Waters sgRNA standard (p/n:186011357) was denatured at 90 ^oC for 2 minutes in a buffer containing 200 mM ammonium acetate (catalog no AM9071-500ML, Thermo Fisher, Waltham, MA) pH 8.0. The sample was cooled on ice and microcentrifuged to collect the sample droplets. After adding 100 units of digestion enzyme (2 µL of RapiZyme MC1) and 7.4 µL of nuclease-free water to obtain a final volume of ~ 20 µL, the sgRNA was digested at 30 ºC for 30 minutes in an Eppendorf thermomixer. The digest was then analyzed immediately by LC-MS using 5 µL injections.

All datasets were acquired with waters_connect (ver 4.1.0.17) and subsequently processed using the MAP Sequence App (ver 1.0), assisted by the mRNA Cleaver and Coverage Viewer MicroApps.

An informatics workflow (Figure 1) featuring the waters_connect MAP Sequence App (version 1.0) was used to facilitate automated data processing of UPLC-MS^E datasets acquired following the enzymatic digestion of an sgRNA standard with RapiZyme MC1.^6–7 The informatics processing workflow consisted of three steps:

1. In-silico digestion: The mRNA Cleaver MicroApp was used to generate predicted, in-silico digested oligonucleotide digestion products, based on the target RNA sequence.

2. Data processing and mass matching: The waters_connect MAP Sequence App processed the UPLC-MS^E data, matching the predicted neutral monoisotopic masses of digested oligonucleotides to the experimentally acquired MS1 data.

3. Sequence coverage visualization: The resulting sequence coverage for the RNA digest was summarized and visualized using the Coverage Viewer MicroApp.

All data acquisition and processing were conducted within the waters_connect Informatics Platform to ensure regulatory compliance.

Digested Oligo Mapping of sgRNA

An UPLC-MS^E oligo map dataset was acquired for the Waters sgRNA LC-MS oligo standard digested with RapiZyme MC1. The UV and TIC chromatograms (Figure 2) indicate that the MC1 digested products were detected with comparable elution patterns by both UV and MS detection. The UPLC-MS^E data was acquired on the Xevo MRT Mass Spectrometer, which uses the multi-reflecting time-of-flight technology (MRT) to achieve exceptional MS resolution, speed, sensitivity and sub-ppm mass accuracy.

The waters_connect MAP Sequence App (ver 1.0) was used for automated processing of the UPLC-MS^E dataset and a portion of the results table is displayed in Figure 3. The green circles displayed next to each digestion product indicate unique assignments based on MS1 (precursor mass) data. Digestion products were assigned with high confidence based on matching the experimentally measured monoisotopic oligonucleotide precursors against the in-silico predicted monoisotopic masses. The mass accuracy observed for all digestion products displayed is in the sub-ppm range (< 1 ppm).

Five different negative charge states (-1 through -5) were detected for the ions corresponding to the highlighted MC1 digestion product (U56:U63, shown in blue), as indicated by the panel labeled “Details” (Figure 3, Right Side). The detailed isotopic distribution of the [M-5H]^5- charge state of the U56:U63 (8-mer) MC1 digestion product (Figure 4) displays the MS resolution achieved by the Xevo MRT Mass Spectrometer instrument (~100,000), which fully resolves the multiple isotopes of this (-5) negatively charged precursor.

The digested oligo mapping results produced by the Coverage Viewer MicroApp are shown in Figure 5. The Rapizyme MC1 enzyme achieved full (100%) coverage for the Waters sgRNA standard though unique digestion specificity (five distinct dinucleotide cleavage sites), and through its ability to generate controlled missed cleavages. These longer digestion products, generated through 2–4 missed cleavages, are more likely to have unique masses. RapiZyme MC1 cleaves at the 5’-end of uridine residues, with three major cleavage sites (A_U / C_U / U_U) and two minor cleavage sites (C_A / C_G)^6-11. MC1 produces mainly digested products ending with a 3’ cyclic phosphate (cP). As a result of these enzymatic features, MC1 exbibits higher coverage, compared to traditional RNase T1 digestion.^6–11

Elucidating Ambiguous Sequence Assignments with CONFIRM Sequence

In cases where oligonucleotide sequence assignments are ambiguous, the CONFIRM Sequence waters_connect App¹² can be employed to analyze the elevated energy MS^E data and assign fragment ions to predict digest product sequences. To illustrate this approach, two 7-mer digested products generated by RapiZyme MC1 treatment of the Waters sgRNA standard are highlighted. These products, U10:C16 (sequence UCU CGA C) and U12:C18 (sequence: UCG ACU C), are structural isomers with identical nucleotide compositions and predicted MS1 mass values.

The MAP Sequence App automatically assigned both sequences (Figure 6, top) by evaluating multiple precursor ions across charge states 1 to 4 and flagged them as “alternative product assignments”. The extracted ion chromatogram (EIC) for the triply charged precursor (m/z = 732.75), also presented in Figure 6, reveals a chromatographic peak doublet with retention times of 14.36 and 14.60 minutes, likely corresponding to these sequence isomers. The MAP Sequence processing result labeled these assignments as having “1 alternative peak”, reporting the most abundant peak at 14.61 minutes as the primary observed retention time.

Subsequently, the CONFIRM Sequence App was utilized to automatically assign the fragment ions observed in the elevated energy ESI-MS^E spectral channel corresponding to the doublet. Based on this analysis, the peak at 14.36 minutes was clearly assigned to the U12:C18 product, while the peak at 14.60 minutes corresponded to the U10:C16 isomer (Figure 7). In both cases, the elevated energy MS^E fragmentation achieved 100% MS2 sequence coverage, providing complete linear sequence verification for each digestion product.

• An informatics workflow, incorporating the waters_connect MAP Sequence App (ver 1.0) was successfully demonstrated for UPLC-MS^E digested oligo mapping analysis of sgRNA digests using the Xevo MRT QTOF Mass Spectrometer.

• Confident peak assessments for analysis of sgRNA digests were achieved with the Xevo MRT QTOF Mass Spectrometer, which delivered excellent sensitivity, high resolution MS (100,000), and mass accuracy of less than 1 ppm.

• Complete sgRNA sequence coverage (100%) was achieved by employing RapiZyme MC1, a novel endonuclease introduced by Waters, which provided unique cleavage specificity and predictable missed cleavages to generate longer, unique, overlapping digestion products.
• Oligonucleotide isomeric sequences resulting from enzymatic digestion were effectively differentiated using the waters_connect CONFIRM Sequence App, which matched elevated energy fragment ions patterns to their respective sequences, allowing unambiguous isomer identification.

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