Skip to main content
NCBI home page
ACS AuthorChoice logoLink to ACS AuthorChoice
. 2024 Oct 11;96(42):16994–17003. doi: 10.1021/acs.analchem.4c04384

Development of a Flow Through-Based Limited Digestion Approach for High-Throughput and High-Sequence Coverage Mapping of Therapeutic mRNAs

Shuli Tang 1, Gao-Yuan Liu 1, Yuetian Yan 1,*, Shunhai Wang 1,*, Ning Li 1
PMCID: PMC11503513  PMID: 39391985

Abstract

graphic file with name ac4c04384_0006.jpg

Messenger RNAs (mRNAs) have rapidly emerged as a pivotal class of biotherapeutics with great promise in the prevention and treatment of various diseases. As with other biotherapeutics, the sequence accuracy and integrity of mRNAs are critical quality attributes (CQAs), influencing the translation efficiency and protein expression fidelity of mRNAs. Due to the generation of short and repetitive oligonucleotides, traditional sequence mapping methods, which utilize in-solution RNase T1 digestion followed by LC-MS/MS analysis, face challenges in achieving high sequence coverage. In this study, we developed a novel flow through (FT)-based strategy to achieve the limited RNase T1 digestion of therapeutic mRNAs, leading to improved mRNA sequence mapping by LC-MS/MS analysis. Compared with the traditional in-solution digestion methods, the FT-based digestion method could consistently generate an increased number of unique oligonucleotides with miscleavages, which significantly boosted the overall sequence coverage (over 93%) of therapeutic mRNAs of varying sizes. Moreover, the automated digestion workflow using the AssayMAP platform offers significant advantages in method reproducibility and throughput. The high throughput and high sequence coverage features of this method could facilitate its wide application in the development of mRNA-based therapeutics.

Since their discovery in the 1960s,1 messenger RNAs (mRNAs) have been a target of interest as therapeutic tools by researchers.2,3 Early investigation of mRNA-based therapeutics dates back to the late 1990s4 but was fraught with challenges related to the stability, cell delivery efficiency, and the induced immune responses of the mRNA molecules. To overcome these obstacles, decades of research efforts were made, including developing modified nucleosides to reduce immunogenicity5−8 and the creation of lipid nanoparticles (LNPs) for cellular delivery and degradative protection of the mRNA molecules.9,10 With accelerated technology breakthroughs that improved the stability and therapeutic efficacy of mRNAs,11 mRNA therapeutics became a viable option in the 2000s.12,13 This groundwork was crucial for the successful development of the mRNA-based vaccines in combating the coronavirus disease 2019 (COVID-19) pandemic.14,15 This historic achievement led to a surge in interest in developing mRNA-based therapeutics for the prevention and treatment of various diseases, including infectious diseases,16 genetic diseases,17 cancers,18 and cardiovascular diseases.19

The mRNA drug substance (DS) is commonly manufactured by the in vitro transcription (IVT) process before formulation and encapsulation with LNPs.20 As with other biotherapeutics, the integrity and accuracy of the mRNA sequence are critical quality attributes (CQAs)21 due to the impact on the protein translation efficiency and fidelity.22 Hence, analytical characterization of the mRNA primary structure is an important task during the development and manufacturing of mRNA therapeutics.23 Conventionally, oligonucleotide sequencing techniques,24−26 such as Sanger sequencing and next-generation sequencing (NGS), have been applied as the standard approaches for sequence analysis of mRNAs, due to their excellent reliability, cost effectiveness, and method throughput. Alternatively, mass spectrometry (MS)-based approaches are also known for their exceptional specificity, sensitivity, ability to multiplex, and quantitation performance and therefore are frequently explored for the characterization of new therapeutic modalities.27−30 Recently, ion-paring reversed-phase liquid chromatography (IP RP-LC)31−34 and hydrophilic interaction chromatography (HILIC)35 coupled with tandem MS (MS/MS) have seen increasing applications and showed great promises in sequence mapping of mRNAs. Similar to protein sequence mapping, LC-MS/MS-based mRNA sequence mapping relies on effective enzymatic digestion (commonly by endoribonucleases) of the mRNA molecules followed by LC-MS/MS identification of the resulting oligonucleotide fragments. In this workflow, generating fragments that can be uniquely mapped to the protein or mRNA sequences is crucial for improving the sequence coverage. However, unlike proteins that are composed of 20 different amino acid residues, mRNAs only contain four nucleotide building blocks, namely, adenosine monophosphate (AMP, A), guanosine monophosphate (GMP, G), uridine monophosphate (UMP, U), and cytidine monophosphate (CMP, C).36 This feature makes the generation of oligonucleotide fragments that are uniquely mappable to mRNAs much more challenging compared with that from the enzymatic digestion of proteins. For example, commonly used endoribonucleases for mRNA sequence mapping, such as RNase T137 (G-specific) and MC138 (U-specific), predominantly generate short oligonucleotides that are often identical or isomeric to each other. These fragments can often be mapped to multiple locations within the mRNA sequence. Thus, they cannot be used for unambiguous sequence confirmation. To overcome this limitation, endoribonucleases with stricter cleavage specificities that produce longer (more likely to be uniquely mappable) oligonucleotides could be used either in parallel with RNase T132 or in solo34 to improve the overall sequence coverage of mRNAs. For example, Hua et al. reported the addition of colicin E539 (cleaves between GU) and Escherichia coli MazF40 (cleaves at the 5′ end of ACA) digestions to RNase T1 digestion and achieved a 1- to 2-fold increase in combined sequence coverages of three mRNAs (from 30–53 to 73–87%).32 Recently, Corrêa et al. also showed that the use of human RNase 4,41,42 a semispecific enzyme that mainly cleaves between URs (R = A or G), resulted in improved sequence coverage of mRNA by LC-MS/MS.34

Alternatively, limited digestion, which intentionally reduces the endoribonuclease digestion efficiency and produces an increased number of oligonucleotides that are often longer and uniquely mappable due to the occurrence of miscleavages, is another viable approach to improving the sequence mapping of mRNAs. For example, Dickman et al. developed a partial digestion method using immobilized RNase T1 and improved the sequence coverages of multiple mRNAs to above 80%.33 By immobilizing RNase T1 on magnetic particles that can be easily removed from the solution, the enzymatic reaction time can be easily tuned to achieve the desired degree of digestion. Leveraging a similar concept, here, we developed a novel flow through (FT)-based limited RNase T1 digestion workflow that facilitated the production of longer and uniquely mappable oligonucleotides with miscleavages at the guanosine (G) site to achieve ultrahigh coverage for mRNA sequence mapping. In this workflow, mRNA samples were digested as they were aspirated through an RNase T1-immobilized cartridge on an Agilent AssayMAP Bravo automation platform under a controlled temperature and flow rate. The digested samples were collected as the postcartridge flow through fraction and subjected to subsequent IP RP-LC-MS/MS analysis. Using EGFP mRNA as a model system, it was demonstrated that this method could efficiently generate high abundances of miscleavage-containing oligonucleotides, primarily in the 2′,3′-cyclic phosphorylated form, and achieve over 96% sequence coverage for EGFP mRNA. Furthermore, the optimized method was also applied to three other mRNAs of varying lengths (582–4245 nt) and with uridine nucleobase modifications (i.e., 5-methoxyuridine), demonstrating its universal applicability across different mRNAs by consistently achieving high sequence coverages of over 93%. Notably, due to the flow through nature of the digestion and its execution on an automation platform, the degree of digestion using this method can be precisely controlled, leading to excellent consistency. Along with its high-throughput feasibility, this method can be routinely applied in sequence analysis for mRNA therapeutics.

Experimental Section

Materials

Deionized water was provided by a Milli-Q integral water purification system installed with a MilliPak Express 20 filter (MilliporeSigma, Burlington, MA). Acetonitrile (Optima LC/MS grade), RNase T1 (1000 U/μL), EZ-Link NHS-Biotin, dimethyl sulfoxide (DMSO, Anhydrous), EDTA (0.5 M, pH 8.0, RNase-free), ultraPure 1 M Tris-HCI buffer (pH 7.5), Gibco 1× phosphate buffered saline (PBS, pH 7.4), and Pall Lab Nanosep centrifugal devices with Omega Membrane-3K were purchased from Thermo Fisher Scientific (Waltham, MA). Urea, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, LC-MS LiChropur), and N,N-diisopropylethylamine (DIPEA, purified by redistillation, 99.5%) was purchased form Sigma-Aldrich (St. Louis, MO). All oligonucleotide samples including EGFP mRNA, EGFP-5moU mRNA, EPO-5moU mRNA, and Cas9–5moU mRNA were purchased from Trilink Biotechnologies (San Diego, CA). AssayMAP 5 μL streptavidin (SA-W) cartridges were obtained from Agilent Technologies (Santa Clara, CA).

Biotinylation of RNase T1

The biotinylated RNase T1 was prepared by mixing 25 μL of RNase T1 (1000 U/μL), 25 μL of NHS-biotin solution (10 mM in DMSO), and 400 μL of PBS (1×, pH 7.4), followed by incubation for 30 min at 25 °C with shaking at 650 rpm. The reaction was quenched by adding 50 μL of 1 M Tris-HCl (pH 7.5). The quenched reaction mixture was centrifuged at 14,000g in a Nanosep centrifugal device (3K) for 25 min to remove the excess NHS-biotin reagent. The recovered biotinylated RNase T1 was resuspended by adding 400 μL of PBS (1×, pH 7.4), and the final concentration was measured using a NanoDrop spectrophotometer at 280 nm absorbance with an extinction coefficient of 2.9. The full procedure for the preparation of biotinylated RNase T1 required 1 h.

Immobilization of Biotinylated RNase T1 on AssayMAP Cartridges

Immobilization of biotinylated RNase T1 on the streptavidin cartridges was performed in a multiplexed fashion (up to 96 cartridges) on the AssayMap Bravo platform by using the “immobilization” application (Figure S1). The priming/equilibration buffer, cartridge wash buffer, and syringe wash buffer were prepared by mixing 4 mL of 0.5 M EDTA, 100 mL of 1 M Tris-HCl (pH 7.5), and 896 mL of Milli Q water together. The biotinylated RNase T1 solution (1–30 U/μL) was prepared in 1× PBS (pH 7.4). The immobilization protocol follows the following steps sequentially: (1) streptavidin cartridges were primed and equilibrated each with 100 μL of priming/equilibration buffer at a flow rate of 300 μL/min and 10 μL/min; (2) 100 μL of biotinylated RNase T1 solution of varying concentrations was loaded at a flow rate of 5 μL/min; and (3) the cartridges were washed with 100 μL of cartridge wash buffer at a flow rate of 10 μL/min to remove excess/nonbinding biotinylated RNase T1. The total task time for the immobilization step was 45 min. The tested loading amount of biotinylated RNase T1 on the AssayMAP cartridges ranges from 100 to 3000 U.

Flow Through (FT)-Based Limited Digestion of mRNAs

FT-based limited digestion of mRNA was executed using the “on-cartridge reaction” application (Figure S2) on the AssayMAP Bravo platform, which enables the automated aspiration of a temperature-controlled mRNA solution through immobilized AssayMAP cartridges for limited digestion. The equilibration/chase buffer, cartridge wash buffer, and syringe wash buffer were prepared by mixing 4 mL of 0.5 M EDTA, 100 mL of 1 M Tris-HCl (pH 7.5), and 896 mL of Milli Q water together. The mRNA solution (0.2 μg/μL) was prepared in the equilibration/chase buffer and 100 μL was used for each digestion. The on-cartridge digestion followed the following steps sequentially: (1) The RNase T1-immobilized cartridges were re-equilibrated with 100 μL of equilibration buffer at a flow rate of 10 μL/min. (2) 100 μL of mRNA solution was aspirated onto the cartridge at a fixed temperature (10, 25, 35, and 45 °C) and flow rate (7, 10, and 20 μL/min). The digestion temperature (25, 35, and 45 °C) was controlled through a 96 red PCR plate thermal insert on the Peltier thermal station where the mRNA samples were seated on; thus, the actual digestion temperature is slightly lower than the preset station temperature. The digestion temperature of 10 °C was controlled by storing the mRNA samples and the PCR thermal insert plate in the 4 °C refrigerator prior to limited digestion, which returned to approximately 10 °C during the digestion process under room temperature. The aspiration flow rate was controlled by the total reaction time (5–15 min). (3) The reaction was stopped by loading 5 μL of chase buffer at a flow rate of 5 μL/min. The total task time for the FT-based limited digestion step was 20–30 min. The combined flow through fraction containing mRNA digestion products was collected and 5–10 μg of each sample was subjected to LC-MS/MS analysis.

In-Solution Complete Digestion of mRNAs

In-solution digestion of mRNAs was performed by mixing 20 μL of mRNA or mRNA mixtures (1 μg/μL) with 30 μL of 8 M urea, 6 μL of 1 M Tris-HCI (pH 7.5), and 0.4 μL of 0.5 M EDTA and placed at 80 °C for 10 min for sample denaturation. The denatured sample was then cooled to room temperature before being mixed with 10 μL of 1000 U/μL RNase T1, followed by incubation at 37 °C for 15 min. After digestion, 34 μL of PBS (1×, pH 7.4) solution was added to dilute the mRNA solution to 0.2 μg/μL prior to LC-MS/MS analysis.

IP RP-LC-MS/MS Analysis of mRNA Digests

The IP RP-LC separation of mRNA digests was performed on a Waters ACQUITY H-Class UPLC equipped with an ACQUITY UPLC oligonucleotide BEH C18 (130 Å, 1.7 μm, 2.1 mm × 150 mm) column with the column compartment set to 60 °C. The mobile phase A (MPA) was 1% HFIP and 0.1% DIPEA in H2O, and the mobile phase B (MPB) was 0.075% HFIP and 0.0375% DIPEA in 65% ACN and 35% H2O. The LC gradient operated at 0.35 mL/min was set as follows: 0 min, 3% B; 5 min, 3% B; 20 min, 15% B; 23 min, 25% B; 23.01 min, 3% B; 25 min, 3% B; 28 min, 95% B; 35 min, 95% B; 35.01 min, 3% B; and 40 min, 3% B. The post-LC flow was subjected to online UV detection at a wavelength of 260 nm followed by MS/MS analysis performed on a Thermo Q-Exactive plus mass spectrometer equipped with a heated electrospray ion source (HESI). The instrument HESI source parameters were set as follows: spray voltage: 3.0 kV, negative polarity, sheath gas: 40 (arbitrary units), aux gas: 10, capillary temperature: 275 °C, aux gas temperature: 350 °C, and S-lens RF level: 65. MS analysis was performed in the data-dependent acquisition mode. The full MS scan was performed with an instrument resolving power set to 35,000, scan range of 500–3000, maximum IT of 50 ms, and the AGC target of 3E6. For data-dependent MS2 analysis, the following scan parameters were used: NCE: 24, resolving power: 17,500, scan range: 200–2000, maximum IT: 100 ms, AGC target: 5E4, and dynamic exclusion: 5 s.

LC-MS/MS Data Processing

LC-MS/MS data processing was performed using Byonic software and Byologic “Digested Oligonucleotides” workflow from Protein Metrics (Cupertino, CA) to identify oligonucleotide fragments and calculate the sequence coverage of mRNA. The digestion and instrument parameters were set as follows: the cleavage site, G; cleavage side, C-terminal; digestion specificity, fully specific (limiting cleavage to only the 3′ side of guanosine residues in the RNA); maximum missed cleavages, 4; precursor mass tolerance, 20 ppm; fragment mass tolerance, 50 ppm; and fragmentation type, NUVPD. For the fixed and variable modification settings, hydroxylation of the 5′ terminal (−79.9663 Da) was set as fixed, and phosphorylation (+79.9663 Da) and cyclic phosphorylation (+61.9558 Da) of the 3′ terminal were set as variable common modifications. For analyzing mRNAs fully substituted with 5-methoxyuridine, 5-methoxyuridine (+30.0106 Da) was added as a fixed modification. The maximum number of common modifications was set to 1. The maximum precursor mass was set as 10,000 Da. The oligonucleotide output option was set to a score cutoff threshold of 275.

Results and Discussion

Flow Through (FT)-Based Limited RNase T1 Digestion Workflow

RNase T1 digestion of mRNAs, when executed under limited digestion conditions, can be used to produce more miscleavage-containing oligonucleotides that are more likely to be uniquely mappable to the mRNA sequence, thereby improving the overall coverage of mRNA sequence mapping. However, as RNase T1 cannot be readily inactivated by either high temperature or common denaturants, its effective removal at the desired digestion end point is key to the success and has been the biggest challenge in developing a reliable limited digestion method. Here, to enable precise control of enzyme–substrate contact time, limited digestion was executed using an FT-based approach by passing the mRNA samples through a solid matrix immobilized with RNase T1. This was achieved on an AssayMAP Bravo automation platform to take advantage of the high precision and multichannel capabilities of this liquid-handling system. In this workflow, RNase T1 was first biotinylated and immobilized onto the streptavidin cartridges using streptavidin–biotin chemistry (Figure 1a). Subsequently, the mRNA sample prepared in the digestion buffer was aspirated through the cartridge at a controlled flow rate and temperature (Figure 1b). Notably, as the volume of each cartridge is fixed (5 μL), the contact time between the enzyme and the substrate can be precisely controlled by modulating the flow rate of the mRNA sample solution through the cartridge. By optimizing the enzyme–substrate contact time, the digestion temperature, and the amount of the immobilized enzyme, the RNase T1 digestion efficiency can be fine-tuned to produce increased levels of miscleavage-containing oligonucleotides, thereby achieving higher sequence coverage of mRNAs (Figure 1c).

Figure 1.

Figure 1

Open in a new tab

Schematic illustration of FT-based limited RNase T1 digestion of mRNAs established on an AssayMAP Bravo platform: (a) RNase T1 was biotinylated and immobilized onto streptavidin cartridges; (b) temperature-controlled mRNA solution was aspirated through RNase T1-immobilized cartridges at a determined flow rate; (c) representation of FT-based limited digestion of mRNA showing the generation of an increased number of oligonucleotides that are often longer and more likely to be uniquely mappable compared to those from complete digestion (created with BioRender.com; Agreement Number QK273H38DS).

Three major factors that may impact the digestion efficiency, the amount of immobilized RNase T1 (or enzyme-to-substrate ratio when a fixed amount of mRNA is used), the reaction time (or flow rate), and the reaction temperature, were evaluated using EGFP mRNA (720 nt) as a model system. First, to optimize the amount of immobilized RNase T1 for each digestion, four sets of immobilized cartridges were prepared by loading 100, 500, 1500, and 3000 U of RNase T1, onto each cartridge during the immobilization step. Each cartridge was used to digest 100 μL of EGFP mRNA by aspirating the sample solutions through the cartridges at a flow rate of 10 μL/min. Throughout the digestion process, the mRNA samples were seated on a Peltier thermal station with the station temperature kept at 45 °C (the actual digestion temperature being slightly lower). The digestion products were then analyzed by LC-MS/MS to identify the generated oligonucleotides. Since the goal of applying limited digestion is to improve mRNA sequence coverage by producing more miscleavage-containing oligonucleotides that are more likely to be uniquely mappable, the number of identified oligonucleotide sequences, as well as the overall sequence coverage, were used as key indicators for method evaluation (Figure 2). Further, the total identified oligonucleotide sequences were categorized into two groups, including the fully cleaved (gray) and miscleaved products (red), to better understand the impact of experimental conditions on digestion efficiency. As shown in Figure 2a, when 500–1500 U of biotinylated RNase T1 was used, the digestion resulted in the most identified oligonucleotide sequences and the highest sequence coverage of EGFP mRNA. In contrast, when the amount of RNase T1 was decreased to 100 U or increased to 3000 U, both the number of identified oligonucleotide sequences and the achieved sequence coverage dropped. This is presumably attributed to insufficient or overdigestion at low or high enzyme-to-substrate ratios, respectively. Additionally, comparisons of the total ion chromatograms (TICs) from IP RP-LC-MS/MS analyses of the four different digests also revealed a scarcity of resulting oligonucleotides in the 100 U digestion sample, agreeing with the expected low digestion efficiency under this condition (Figure S3). Meanwhile, an increasing trend in the relative abundances of the early eluting oligonucleotides was observed in samples digested with increasing amounts of RNase T1. It is worth noting that IP RP-LC separates oligonucleotide fragments primarily in a size-based order, with shorter oligonucleotides eluting earlier than longer oligonucleotides, which often contain more miscleavages. Therefore, the increasing abundance of short oligonucleotides in the presence of a higher enzyme amount was consistent with the expectation that more complete digestion would occur under such conditions.

Figure 2.

Figure 2

Open in a new tab

Optimization of the FT-based limited RNase T1 digestion conditions, including (a) amount of immobilized RNase T1, (b) flow rate of the mRNA samples through the cartridge, and (c) digestion temperature, using EGFP mRNA (720 nt). (d) EGFP mRNA sequence coverage map under the optimized conditions with 35 °C digestion temperature, 1500 U of immobilized RNase T1, and flow through rate set at 10 μL/min. The LC-MS injection amount was 5 μg. A minimum Byonic score of 275 for each matched oligonucleotide from MS and the MS/MS database search was required for its positive identification.

Second, the on-cartridge reaction time was also optimized. Considering the nature of the FT-based digestion, the effective enzyme–substrate contact time (digestion time) is directly determined by the sample flow rate. Therefore, three different sample flow rates, including 7, 10, and 20 μL/min, were evaluated for the digestion of EGFP mRNA, while the amount of RNase T1 and digestion station temperature were fixed at 1500 U and 45 °C, respectively. As shown in Figure 2b, a concomitant increase in both the sequence coverage and the number of identified oligonucleotide sequences was observed with the increase of flow rate from 7 to 20 μL/min. This is consistent with the expectation that a lower sample flow rate (longer reaction time) could lead to overdigestion of the mRNA, generating predominantly short and non-unique oligonucleotides. On the other hand, a sample flow rate that is too high could lead to insufficient digestion, producing oligonucleotides that are too long to be effectively identified by MS/MS. Under the tested ranges, it was found that both flow rates at 10 or 20 μL/min could produce desired levels of miscleaved oligonucleotides and achieve high sequence coverage (>90%) of EGFP mRNA. Notably, considering that the bed volume of each cartridge was 5 μL, the equivalent enzyme–substrate contact time at the sample flow rate of 10 μL/min was only 30 s. This extremely short reaction time highlights the necessity of precise method execution using the automation platform, as the alternative manual approach is expected to face significant challenges in achieving the required accuracy and consistency.

Similarly, the impact of digestion temperature was investigated by evaluating the digestion outcome at four sample station temperatures (10, 25, 35, and 45 °C), using the previously optimized RNase T1 amount (1500 U) and sample flow rate (10 μL/min). As shown in Figure 2c, a digestion temperature of 10 °C resulted in the fewest digestion products and the poorest sequence coverage compared to the other three conditions, presumably due to the decreased catalytic activity of RNase T1 at this low temperature. On the other hand, digestion carried out at 35 °C performed slightly better than that at 25 and 45 °C, resulting in the most identified oligonucleotides and the highest achieved sequence coverage of EGFP mRNA.

In addition to EGFP mRNA, another mRNA of approximately 6× length (Cas9, >4000 nt) was also utilized in similar method optimization experiments to evaluate the impact of mRNA size on optimal digestion parameters (Figure S4). Interestingly, despite the significant differences in their lengths, the optimal digestion conditions identified for both EGFP and Cas9 mRNA were nearly identical. In particular, both mRNAs required an optimal digestion temperature of 35 °C and 1500 U of immobilized RNase T1 to achieve the highest sequence coverage. In the meantime, while digestion of EGFP mRNA at the sample flow rate of 20 μL/min produced slightly better sequence coverage than that at 10 μL/min (Figure 2b), both conditions yielded similar performance for the digestion of Cas9 mRNA (Figure S4b). To ensure general applicability and robust execution, the slightly lower sample flow rate of 10 μL/min, along with a digestion temperature of 35 °C and 1500 U of immobilized RNase T1, were selected as the platform conditions for FT-based limited digestion of mRNAs. Additionally, as indicated from the method optimization results, reasonable deviations from these selected conditions imposed only small changes to the overall digestion outcome, highlighting good method robustness in tolerating possible systematic errors. Under these conditions, FT-based limited digestion of EGFP mRNA resulted in a total of 194 unique oligonucleotides, including 54 fully cleaved products and 140 miscleaved products, achieving an overall sequence coverage of 96.3% with an LC-MS injection amount of 5 μg (Figure 2d). This achieved sequence coverage represents an unprecedented improvement from those previously reported using conventional complete digestion or limited digestion protocols,32,33 highlighting the great potential of this method in mRNA sequence mapping.

Characterization of Oligonucleotide Products from FT-Based Limited RNase T1 Digestion of EGFP mRNA

To gain a comprehensive understanding of the FT-based RNase T1 digestion and its improvement in sequence coverage, the digestion products of EGFP mRNA were thoroughly characterized and compared with those from a conventional solution-based digestion. An increased LC-MS injection amount (10 μg) was used for both digestion methods to facilitate the detection of low-abundance oligonucleotide fragments. The LC-MS/MS-identified oligonucleotide sequences were first screened and grouped by the number of miscleavages in the sequence (Figure 3a). Triplicate analyses revealed that a total of 261 ± 4 oligonucleotide sequences were identified from FT-based limited digestion, of which 54 ± 1 were fully digested (with 0 miscleavage) and 207 ± 3 contained 1–4 miscleavages. Notably, oligonucleotides with more than 4 miscleavages may also be present in the digested samples at low abundances. However, their contributions to further improving the overall sequence coverage were found to be negligible owing to the existence of other oligonucleotides with overlapping sequences; hence, they were not included. Together, these identified oligonucleotides accounted for an overall sequence coverage of 96.5 ± 1.1% for EGFP mRNA by FT-based limited digestion. In comparison, the solution-based digestion produced a similar number of fully digested oligonucleotides (53 ± 1) but significantly fewer miscleavage-containing products (62 ± 4), leading to a much lower sequence coverage (79.8 ± 2.2%).

Figure 3.

Figure 3

Open in a new tab

Comparison of RNase T1 digestion products of EGFP mRNA from FT-based limited digestion and in-solution complete digestion. (a) Comparison of the number of identified oligonucleotides from both digestions grouped by the number of miscleavages (0–4) in the sequence. The number of uniquely mappable sequences within each group is also shown. (b) Catalytic mechanism of RNA cleavage by RNase T1. Comparison of oligonucleotide products based on 3′ structural differences and the number of miscleavages from digestions carried out using (c) in-solution and (d) FT-based limited digestion workflow. “Others” represents oligonucleotides associated with the 3′–OH form by itself or together with 2′,3′-cP and 3′-P forms (Table S1). The LC-MS injection amount was 10 μg.

It is worthwhile to mention that not all of the identified oligonucleotide sequences could be uniquely mapped to the mRNA sequence; thus, they are only counted once toward sequence coverage. In addition, as only the oligonucleotides that are uniquely mappable can be used for unambiguous mRNA sequence confirmation, the identified oligonucleotides were further grouped by the number of their occurrences within the mRNA sequence (Figure 3a). It was not surprising that a significant portion (35.2% (19/54) by FT-based digestion and 32.1% (17/53) by in-solution digestion) of the fully digested oligonucleotides could be mapped to more than one location within the EGFP mRNA sequence due to their short and repetitive nature. In contrast, the majority of the miscleavage-containing oligonucleotides were uniquely mappable, accounting for 87.6% (181/207) by FT-based digestion and 80.6% (50/62) by solution-based digestion. Moreover, among the 145 miscleavage-containing oligonucleotides that were uniquely identified from FT-based limited digestion, 90.3% (131/145) were uniquely mappable to the EGFP mRNA sequence. These results highlighted the critical role of miscleavage-containing oligonucleotides in improving mRNA sequence mapping and supported the validity of employing limited digestion strategies to achieve this goal.

Like other endoribonucleases, RNase T1 digestion of mRNAs often produces a mixture of digestion products, which may include 2′,3′-cyclic phosphorylated, 3′-phosphorylated, and 2′,3′-hydroxylated forms (Figure 3b). The 2′,3′-cyclic phosphate (2′,3′-cP) is an intermediate species generated by the initial RNase T1 digestion, known as transphosphorylation. Subsequently, the 2′,3′-cP intermediates could undergo hydrolysis and form 3′-phosphate (3′-P) products. As transphosphorylation occurs much faster than the hydrolysis reaction,37 it is common to find both 2′,3′-cP and 3′-P products in the final digested sample. Despite being less common, 3′-hydroxylated (3′–OH) oligonucleotides could also be produced via further hydrolysis of the 3′-P species. This heterogeneity of digestion products, arising from different phosphorylation structures, could potentially convolute and reduce the sensitivity of LC-MS/MS analysis and, therefore, was also evaluated for both the FT-based limited digestion and solution-based digestion products. Analysis of the solution-based digestion products of EGFP mRNA (Figure 3c) showed that the majority of the identified oligonucleotides adopted a single 3′-P form (68/115), while smaller populations existed solely in the 2′,3′-cP form (24/115) or in both 2′,3′-cP and 3′-P forms (10/115). The remaining 13 identified oligonucleotides were found to exist in the less common 3′–OH form, either by itself or together with the 3′-P form (Table S1a). Interestingly, the 2′,3′-cP products were found to distribute disproportionally between fully digested and miscleavage-containing oligonucleotide populations, accounting for 13.2% (7/53) of the former and 43.5% (27/62) of the latter. This “enrichment” of digestion intermediates (i.e., 2′,3′-cP) in miscleavage-containing oligonucleotides is not surprising and likely pointed to local regions of the mRNA sequence that were less efficiently digested by RNase T1 (e.g., due to secondary structures). In contrast, FT-based limited digestion of EGFP mRNA generated predominantly homogeneous digestion products mainly composed of 2′,3′-cP species (Figure 3d). Specifically, 69.7% (182/261) of the identified oligonucleotides were found exclusively in the 2′,3′-cP form, 24.9% (65/261) existed in both 2′,3′-cP and 3′-P forms, and the remaining 5.0% (13/261) mostly adopted the 3′-P/3′–OH forms (Table S1b). Interestingly, among the 67 oligonucleotides that were associated with multiple 3′ structures, 49 were contributed by fully digested products and only 18 were contributed by miscleavage-containing products, each representing 90.7% (49/54) and 8.7% (18/207) of their corresponding populations, respectively. The generation of a homogeneous 3′ structure in the miscleavage-containing oligonucleotides is highly desirable, as it enhances the detectability of the oligonucleotides by consolidating the MS signal. This is particularly beneficial in the context of limited digestion, where the abundance of individual oligonucleotides is usually reduced due to the generation of multiple fragments with overlapping sequences. Notably, the favorable generation of 2′,3′-cP over 3′-P products by the FT-based limited digestion aligns well with the catalytic mechanism of RNase T1, which renders a rapid generation of 2′,3′-cP intermediate species but a slower subsequent hydrolysis reaction to form 3′-P products. Specifically, the priorly formed 2′,3′-cP intermediates likely failed to undergo further hydrolysis to form 3′-P products, due to the extremely short reaction time (30 s) allowed under the optimized FT-based digestion conditions. Together, these results highlighted the advantage of the FT-based limited digestion method in generating homogeneous 2′,3′-cP products and their crucial roles in improving the overall sequence coverage for mapping mRNAs.

General Applicability of FT-Based Limited Digestion for Different mRNAs

Synthetic mRNAs with modified uridine have shown great potential for therapeutic applications, as the modifications can improve mRNA stability and reduce immunogenicity.5−8 To test if the FT-based limited digestion discriminates between unmodified mRNAs and those with chemical modifications, a modified EGFP mRNA, in which the uridine was substituted with 5-methoxyuridine (EGFP-5moU), was analyzed and compared with an unmodified EGFP mRNA. Similarly, the digestion performance was compared by assessing the number of identified oligonucleotide sequences and the overall achieved mRNA sequence coverage. As shown in Figure S5a (and Figure 4b), both EGFP and EGFP-5moU mRNAs digested by the FT-based method produced comparable numbers of identified oligonucleotides, which were calculated at 261 ± 4 and 254 ± 6 from triplicate analyses, respectively. In particular, these identified oligonucleotides from both sample digests were predominantly composed of miscleavage-containing products (207 ± 3 and 197 ± 5, respectively), which aligned well with the expected outcome from the limited digestion approach. As a result, similar sequence coverages of EGFP-5moU mRNA (98.3 ± 0.3%) and unmodified EGFP mRNA (96.5 ± 1.1%) were achieved.

Figure 4.

Figure 4

Open in a new tab

(a) Sequence coverages of EGFP (720 nt), EGFP–5moU (720 nt), Epo–5moU (582 nt), and Cas9–5moU (4245 nt) using FT-based limited digestion and in-solution complete digestion methods. (b) Comparison of the two digestion methods in producing unique oligonucleotides, including both fully cleaved products (gray) and miscleaved products (red) from the four mRNAs. The LC-MS injection amount was 10 μg.

Furthermore, an exemplary miscleavage-containing oligonucleotide (UUCGAGGGCG) was selected to show nondiscriminatory enzymatic digestion and successful LC-MS/MS detection regardless of the 5moU modification (Figure S5b–e). Specifically, the extracted ion chromatograms (XICs) displayed nearly identical intensities of both the unmodified and 5moU-modified oligonucleotides from the corresponding digests (2.5–2.7E6, Figure S5b,c), suggesting a similar digestion efficiency for both samples. Additionally, it was also observed that the 5moU-modified oligonucleotide eluted slightly later than the unmodified one under IP RPLC separation, presumably due to the increased hydrophobicity from the 5′-methoxyl group. Finally, MS/MS analysis of the oligonucleotide was unaffected by the uridine modification and produced an identical fragmentation pattern to that of the unmodified sequence (Figure S5d,e). Together, these results demonstrated that the FT-based RNase T1 digestion method can be generically applied to chemically modified mRNAs.

In addition to its utility for chemically modified mRNAs, the general applicability of a sequence mapping method across different mRNA molecules (e.g., varying sizes and sequence features) is equally important. Considering that mRNAs are built by repetitions of four nucleotides and are largely “unstructured”, it is reasonable to hypothesize that the same limited digestion method could produce similar digestion outcomes for most mRNAs regardless of the size and identity. To test this hypothesis, two additional mRNAs, Epo–5moU (582 nt) and Cas9–5moU (4245 nt), each representing a shorter or longer sequence, were analyzed together with EGFP mRNA (720 nt), using the same FT-based limited RNase T1 digestion method. Specifically, in addition to the previously optimized RNase T1 amount, digestion temperature, and sample flow rate, the mRNA input (100 μL at 0.2 μg/μL) of each sample was also kept consistent. In addition, solution-based complete digestion of these mRNAs was also conducted to enable a comparison in sequence mapping performance. The resulting quantitative comparison of the identified oligonucleotides and the calculated sequence coverages of each mRNA from both digestion methods are summarized in Figure 4. As expected, the FT-based limited digestion method consistently delivered significantly higher sequence coverages for all three mRNAs (93.2–98.3%) compared to those from the solution-based complete digestion method (61.5–83.8%) (Figure 4a). Consistently, an apparent increase in the number of identified oligonucleotides, almost exclusively attributed to those with miscleavages, was observed by the FT-based digestion method compared to the solution-based digestion method (Figure 4b). Interestingly, the obtained sequence coverages of mRNAs by the solution-based digestion method appeared to be highly dependent on the mRNA length, which displayed a catastrophic drop to 61.5% for the Cas9 mRNA (4245 nt). This decrease in sequence coverage can be explained by the disproportionately large increase in mRNA lengths (a 6-fold increase from EGFP to Cas9 mRNA) compared to the relatively small increase in the total number of identified oligonucleotides generated by the solution-based digestion method (a 3.6-fold increase from 115 for EGFP to 411 for Cas9 mRNA), due to the highly repetitive nature of mRNA sequences. On the contrary, FT-based digestion effectively overcame this limitation by producing a larger number of miscleavage-containing oligonucleotide sequences. For example, while both digestion methods yielded a comparable number of fully digested oligonucleotides from Cas9 mRNA (n = 181), FT-based digestion produced significantly more miscleavage-containing oligonucleotides (n = 787 vs n = 230, Figure 4b), resulting in an impressive increase in the sequence coverage of Cas9 mRNA from 61.5 to 93.2%. Lastly, the consistently high sequence coverage achieved for mRNAs ranging from 582 to 4245 nt in length further highlights the excellent versatility of this developed FT-based digestion method, making it suitable for characterizing a variety of therapeutic mRNAs.

Consistency of FT-Based Limited RNase T1 Digestion

To evaluate if the developed FT-based limited RNase T1 digestion method can robustly generate miscleavage-containing oligonucleotides with reproducible patterns, three replicate digestions of EGFP mRNA were conducted, and the final products were analyzed by LC-MS/MS. The results showed that similar TIC profiles were obtained across the three replicates (Figure 5a). Close examination of the peak features and retention times of individual peaks also demonstrated high consistency among the triplicate analyses, suggesting that highly consistent digestion products were produced.

Figure 5.

Figure 5

Open in a new tab

Repeatability of the FT-based limited digestion workflow using the AssayMAP automation platform. (a) TICs from triplicate analyses of RNase T1-digested EGFP mRNA using the FT-based limited digestion method (replicate 1: gray; replicate 2: blue; and replicate 3: red) and (b) Venn diagram depicting the overlap of identified oligonucleotides in the triplicate analyses. Each circle represents a distinct replicate, illustrating the numbers of unique and shared oligonucleotides identified among the replicates. The LC-MS injection amount was 10 μg.

Furthermore, the identified oligonucleotide sequences from each analysis were compared and grouped by their occurrence among different replicates. As shown in Figure 5b, the identified oligonucleotides exhibited high reproducibility across the triplicate analyses, with a total of 192 oligonucleotides consistently identified in all replicates, accounting for more than 73% of the entire population. Although a small number of oligonucleotides were uniquely identified in each replicate, they were often present at low abundances, which led to inconsistent identification by data-dependent MS2 analysis (e.g., due to exclusion from MS2 or poor MS2 quality). Nevertheless, because of the presence of other redundant oligonucleotides with overlapping sequences, the overall sequence coverage was not impacted. Indeed, similar sequence coverages of EGFP mRNA were also achieved from the triplicate analyses, with an average of 96.5% and a narrow deviation (±1.2%). Together, these results indicated that the miscleavages were not generated randomly under the FT-based limited RNase T1 digestion conditions. Instead, the enzyme preferentially cleaves more accessible regions within the mRNA sequence and cleaves only less accessible regions (e.g., those with secondary structures) in a limited fashion. It is hence concluded that the pattern of oligonucleotides can be consistently produced by this FT-based limited digestion approach, therefore leading to reliable mRNA sequence mapping. Additionally, with the multiplexing capability from the AssayMAP platform, this workflow can be implemented in a high-throughput manner, allowing for the digestion of up to 96 samples simultaneously.

Conclusions

Endoribonuclease digestion coupled with LC-MS/MS analysis is an attractive approach for mRNA sequence mapping. Traditional in-solution digestion methods using RNase T1 often generate short and repetitive oligonucleotide fragments that cannot be unambiguously mapped to the mRNA sequence and, therefore, are limited in achieving sufficient sequence coverages. Alternative methods have been reported to improve MS-based sequence mapping of mRNAs by either using endoribonucleases with stricter cleavage specificities or crafting limited digestion conditions to intentionally generate longer oligonucleotides that are more likely to be uniquely mappable to the sequence. Leveraging the concept of limited digestion, here, we developed an FT-based limited RNase T1 digestion method in combination with LC-MS/MS to enable high-sequence coverage mapping of therapeutic mRNAs. The workflow was built on an AssayMAP Bravo automation platform that enables precise control of the digestion conditions. Using EGFP mRNA as a model system, the FT-based digestion approach was optimized to generate high abundances of miscleavage-containing oligonucleotides with unique sequence information, achieving an unprecedentedly high sequence coverage (>96% for EGFP mRNA). In particular, it was demonstrated that unlike the conventional in-solution digestion method, this FT-based limited digestion method produced primarily homogeneous 2′,3′-cyclic phosphorylated oligonucleotide products, which was essential for improving MS detection sensitivity. Moreover, it was also demonstrated that this FT-based limited digestion method can be universally applied to chemically modified mRNAs and mRNAs of different lengths (up to 4245 nt tested), consistently achieving high sequence coverage (>93%). Additionally, as the workflow was executed on an automation platform, it demonstrated excellent consistency and provided opportunities for high-throughput analysis. Together, these features should make this FT-based limited digestion method highly desirable during the development of therapeutic mRNAs, allowing for its broad applications in industrial laboratories.

Acknowledgments

This study was sponsored by Regeneron Pharmaceuticals Inc. The authors would like to thank Ilker Sen and Maria Basanta-Sanchez from Protein Metrics Inc. for data processing software support.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.4c04384.

  • AssayMAP-based immobilization application setting overview, AssayMAP-based on-cartridge reaction application setting overview, optimization of the flow through (FT)-based RNase T1 digestion conditions by using different amounts of immobilized biotinylated RNase T1 (TICs), optimization of the flow through (FT)-based RNase T1 digestion conditions using Cas9–5moU mRNA (4245 nt), RNase T1 digestion product of EGFP mRNA from in-solution complete digestion and FT-based limited digestion, and FT-based RNase T1 digestion of EGFP and EGFP-5moU mRNAs (PDF)

Author Contributions

† S.T. and G.-Y.L. are cofirst authors and contributed equally.

The authors declare the following competing financial interest(s): S.T., G.L., Y.Y., S.W., and N.L. are fulltime employees and shareholders of Regeneron Pharmaceuticals Inc.

Supplementary Material

ac4c04384_si_001.pdf (880.9KB, pdf)

References

  1. Brenner S.; Jacob F.; Meselson M. An Unstable Intermediate Carrying Information from Genes to Ribosomes for Protein Synthesis. Nature 1961, 190 (4776), 576–581. 10.1038/190576a0. [DOI] [PubMed] [Google Scholar]
  2. Dong Y.; Anderson D. G. Opportunities and Challenges in mRNA Therapeutics. Acc. Chem. Res. 2022, 55 (1), 1 10.1021/acs.accounts.1c00739. [DOI] [PubMed] [Google Scholar]
  3. Rohner E.; Yang R.; Foo K. S.; Goedel A.; Chien K. R. Unlocking the promise of mRNA therapeutics. Nat. Biotechnol. 2022, 40 (11), 1586–1600. 10.1038/s41587-022-01491-z. [DOI] [PubMed] [Google Scholar]
  4. Wolff J. A.; Malone R. W.; Williams P.; Chong W.; Acsadi G.; Jani A.; Felgner P. L. Direct Gene Transfer into Mouse Muscle in Vivo. Science 1990, 247 (4949), 1465–1468. 10.1126/science.1690918. [DOI] [PubMed] [Google Scholar]
  5. Badieyan Z. S.; Evans T. Concise Review: Application of Chemically Modified mRNA in Cell Fate Conversion and Tissue Engineering. Stem Cells Transl. Med. 2019, 8 (8), 833–843. 10.1002/sctm.18-0259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Pardi N.; Hogan M. J.; Weissman D. Recent advances in mRNA vaccine technology. Curr. Opin. Immunol. 2020, 65, 14–20. 10.1016/j.coi.2020.01.008. [DOI] [PubMed] [Google Scholar]
  7. Gao M.; Zhang Q.; Feng X.-H.; Liu J. Synthetic modified messenger RNA for therapeutic applications. Acta Biomater. 2021, 131, 1–15. 10.1016/j.actbio.2021.06.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kim S. C.; Sekhon S. S.; Shin W.-R.; Ahn G.; Cho B.-K.; Ahn J.-Y.; Kim Y.-H. Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency. Mol. Cell. Toxicol. 2022, 18 (1), 1–8. 10.1007/s13273-021-00171-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hou X.; Zaks T.; Langer R.; Dong Y. Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 2021, 6 (12), 1078–1094. 10.1038/s41578-021-00358-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Xiao Y.; Tang Z.; Huang X.; Chen W.; Zhou J.; Liu H.; Liu C.; Kong N.; Tao W. Emerging mRNA technologies: delivery strategies and biomedical applications. Chem. Soc. Rev. 2022, 51 (10), 3828–3845. 10.1039/D1CS00617G. [DOI] [PubMed] [Google Scholar]
  11. Qin S.; Tang X.; Chen Y.; Chen K.; Fan N.; Xiao W.; Zheng Q.; Li G.; Teng Y.; Wu M.; Song X. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduction Targeted Ther. 2022, 7 (1), 166. 10.1038/s41392-022-01007-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heiser A.; Coleman D.; Dannull J.; Yancey D.; Maurice M. A.; Lallas C. D.; Dahm P.; Niedzwiecki D.; Gilboa E.; Vieweg J. Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J. Clin. Invest. 2002, 109 (3), 409–417. 10.1172/JCI0214364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Weide B.; Carralot J.-P.; Reese A.; Scheel B.; Eigentler T. K.; Hoerr I.; Rammensee H.-G.; Garbe C.; Pascolo S. Results of the First Phase I/II Clinical Vaccination Trial With Direct Injection of mRNA. J. Immunother. 2008, 31 (2), 180–188. 10.1097/CJI.0b013e31815ce501. [DOI] [PubMed] [Google Scholar]
  14. Vogel A. B.; Kanevsky I.; Che Y.; Swanson K. A.; Muik A.; Vormehr M.; Kranz L. M.; Walzer K. C.; Hein S.; Güler A.; et al. BNT162b vaccines protect rhesus macaques from SARS-CoV-2. Nature 2021, 592 (7853), 283–289. 10.1038/s41586-021-03275-y. [DOI] [PubMed] [Google Scholar]
  15. Baden L. R.; El Sahly H. M.; Essink B.; Kotloff K.; Frey S.; Novak R.; Diemert D.; Spector A. S.; Rouphael N.; Creech C. B.; et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N. Engl. J. Med. 2021, 384 (5), 403–416. 10.1056/NEJMoa2035389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Chaudhary N.; Weissman D.; Whitehead K. A. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat. Rev. Drug Discovery 2021, 20 (11), 817–838. 10.1038/s41573-021-00283-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Martini P. G. V.; Guey L. T. A New Era for Rare Genetic Diseases: Messenger RNA Therapy. Hum. Gene Ther. 2019, 30 (10), 1180–1189. 10.1089/hum.2019.090. [DOI] [PubMed] [Google Scholar]
  18. Liu C.; Shi Q.; Huang X.; Koo S.; Kong N.; Tao W. mRNA-based cancer therapeutics. Nat. Rev. Cancer 2023, 23 (8), 526–543. 10.1038/s41568-023-00586-2. [DOI] [PubMed] [Google Scholar]
  19. Huang C.-K.; Kafert-Kasting S.; Thum T. Preclinical and Clinical Development of Noncoding RNA Therapeutics for Cardiovascular Disease. Circ. Res. 2020, 126 (5), 663–678. 10.1161/CIRCRESAHA.119.315856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Webb C.; Ip S.; Bathula N. V.; Popova P.; Soriano S. K. V.; Ly H. H.; Eryilmaz B.; Nguyen Huu V. A.; Broadhead R.; Rabel M.; et al. Current Status and Future Perspectives on MRNA Drug Manufacturing. Mol. Pharmaceutics 2022, 19 (4), 1047–1058. 10.1021/acs.molpharmaceut.2c00010. [DOI] [PubMed] [Google Scholar]
  21. Daniel S.; Kis Z.; Kontoravdi C.; Shah N. Quality by Design for enabling RNA platform production processes. Trends Biotechnol. 2022, 40 (10), 1213–1228. 10.1016/j.tibtech.2022.03.012. [DOI] [PubMed] [Google Scholar]
  22. Buccitelli C.; Selbach M. mRNAs, proteins and the emerging principles of gene expression control. Nat. Rev. Genet. 2020, 21 (10), 630–644. 10.1038/s41576-020-0258-4. [DOI] [PubMed] [Google Scholar]
  23. Gunter H. M.; Idrisoglu S.; Singh S.; Han D. J.; Ariens E.; Peters J. R.; Wong T.; Cheetham S. W.; Xu J.; Rai S. K.; et al. mRNA vaccine quality analysis using RNA sequencing. Nat. Commun. 2023, 14 (1), 5663 10.1038/s41467-023-41354-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ozsolak F.; Milos P. M. RNA sequencing: advances, challenges and opportunities. Nat. Rev. Genet. 2011, 12 (2), 87–98. 10.1038/nrg2934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stark R.; Grzelak M.; Hadfield J. RNA sequencing: the teenage years. Nat. Rev. Genet. 2019, 20 (11), 631–656. 10.1038/s41576-019-0150-2. [DOI] [PubMed] [Google Scholar]
  26. Shendure J.; Balasubramanian S.; Church G. M.; Gilbert W.; Rogers J.; Schloss J. A.; Waterston R. H. DNA sequencing at 40: past, present and future. Nature 2017, 550 (7676), 345–353. 10.1038/nature24286. [DOI] [PubMed] [Google Scholar]
  27. Beccaria M.; Cabooter D. Current developments in LC-MS for pharmaceutical analysis. Analyst 2020, 145 (4), 1129–1157. 10.1039/C9AN02145K. [DOI] [PubMed] [Google Scholar]
  28. Kenderdine T.; Fabris D. The multifaceted roles of mass spectrometric analysis in nucleic acids drug discovery and development. Mass Spectrom. Rev. 2023, 42 (4), 1332–1357. 10.1002/mas.21766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meissner F.; Geddes-McAlister J.; Mann M.; Bantscheff M. The emerging role of mass spectrometry-based proteomics in drug discovery. Nat. Rev. Drug Discovery 2022, 21 (9), 637–654. 10.1038/s41573-022-00409-3. [DOI] [PubMed] [Google Scholar]
  30. Tamara S.; den Boer M. A.; Heck A. J. R. High-Resolution Native Mass Spectrometry. Chem. Rev. 2022, 122 (8), 7269–7326. 10.1021/acs.chemrev.1c00212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Gau B. C.; Dawdy A. W.; Wang H. L.; Bare B.; Castaneda C. H.; Friese O. V.; Thompson M. S.; Lerch T. F.; Cirelli D. J.; Rouse J. C. Oligonucleotide mapping via mass spectrometry to enable comprehensive primary structure characterization of an mRNA vaccine against SARS-CoV-2. Sci. Rep. 2023, 13 (1), 9038 10.1038/s41598-023-36193-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Jiang T.; Yu N.; Kim J.; Murgo J.-R.; Kissai M.; Ravichandran K.; Miracco E. J.; Presnyak V.; Hua S. Oligonucleotide Sequence Mapping of Large Therapeutic mRNAs via Parallel Ribonuclease Digestions and LC-MS/MS. Anal. Chem. 2019, 91 (13), 8500–8506. 10.1021/acs.analchem.9b01664. [DOI] [PubMed] [Google Scholar]
  33. Vanhinsbergh C. J.; Criscuolo A.; Sutton J. N.; Murphy K.; Williamson A. J. K.; Cook K.; Dickman M. J. Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry. Anal. Chem. 2022, 94 (20), 7339–7349. 10.1021/acs.analchem.2c00765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wolf E. J.; Grünberg S.; Dai N.; Chen T.-H.; Roy B.; Yigit E.; Corrêa I. R. Jr. Human RNase 4 improves mRNA sequence characterization by LC–MS/MS. Nucleic Acids Res. 2022, 50 (18), e106 10.1093/nar/gkac632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Goyon A.; Scott B.; Yehl P.; Zhang K. Online Nucleotide Mapping of mRNAs. Anal. Chem. 2024, 96 (21), 8674–8681. 10.1021/acs.analchem.4c00873. [DOI] [PubMed] [Google Scholar]
  36. Šponer J.; Bussi G.; Krepl M.; Banáš P.; Bottaro S.; Cunha R. A.; Gil-Ley A.; Pinamonti G.; Poblete S.; Jurečka P.; et al. RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview. Chem. Rev. 2018, 118 (8), 4177–4338. 10.1021/acs.chemrev.7b00427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Loverix S.; Winqvist A.; Strömberg R.; Steyaert J. Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond. Chem. Biol. 2000, 7 (8), 651–658. 10.1016/S1074-5521(00)00005-3. [DOI] [PubMed] [Google Scholar]
  38. Thakur P.; Atway J.; Limbach P. A.; Addepalli B. RNA Cleavage Properties of Nucleobase-Specific RNase MC1 and Cusativin Are Determined by the Dinucleotide-Binding Interactions in the Enzyme-Active Site. Int. J. Mol. Sci. 2022, 23 (13), 7021. 10.3390/ijms23137021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ogawa T.; Inoue S.; Yajima S.; Hidaka M.; Masaki H. Sequence-specific recognition of colicin E5, a tRNA-targeting ribonuclease. Nucleic Acids Res. 2006, 34 (21), 6065–6073. 10.1093/nar/gkl629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Muñoz-Gómez A. J.; Santos-Sierra S.; Berzal-Herranz A.; Lemonnier M.; Díaz-Orejas R. Insights into the specificity of RNA cleavage by the Escherichia coli MazF toxin. FEBS Lett. 2004, 567 (2–3), 316–320. 10.1016/j.febslet.2004.05.005. [DOI] [PubMed] [Google Scholar]
  41. Shapiro R.; Fett J. W.; Strydom D. J.; Vallee B. L. Isolation and characterization of a human colon carcinoma-secreted enzyme with pancreatic ribonuclease-like activity. Biochemistry 1986, 25 (23), 7255–7264. 10.1021/bi00371a002. [DOI] [PubMed] [Google Scholar]
  42. Terzyan S. S.; Peracaula R.; de Llorens R.; Tsushima Y.; Yamada H.; Seno M.; Gomis-Rüth F. X.; Coll M. The three-dimensional structure of human RNase 4, unliganded and complexed with d(up), reveals the basis for its uridine selectivity11Edited by R. Huber. J. Mol. Biol. 1999, 285 (1), 205–214. 10.1006/jmbi.1998.2288. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ac4c04384_si_001.pdf (880.9KB, pdf)

Articles from Analytical Chemistry are provided here courtesy of American Chemical Society

RESOURCES