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Technological Advancements Support RNA and Oligonucleotide Sequence Confirmation

Advancements in gene therapy and the approval of mRNA vaccines have dramatically increased the demand for well-characterised RNA and oligonucleotides in the biopharmaceutical industry. To support this emerging need, researchers have developed new analytical tools to analyse nucleic acid sequences by mass spectrometry (MS). Software advancements can now take data from high isotopic fidelity Quadrupole time-of-flight (QTOF) instruments and enable the fully automatic confirmation of sequences based on intact mass and MS/MS data, as well as the quantitation and identification of synthesis by-products. Corresponding innovations in analytical instrumentation also have provided the robustness, sensitivity, and high dynamic range necessary for RNA and oligonucleotide sequence confirmation.

Introduction

The growing importance of oligonucleotides in research, diagnostics, and gene therapy – including tRNA, siRNA, and other modified oligos up to 120 bases – has increased interest in developing tools to verify their sequence, as well as identify and quantify related impurities. Liquid chromatography coupled with ultraviolet detection (LC-UV) and LC-MS/MS are the preferred methods to characterise highly modified oligonucleotide sequences. QTOF mass spectrometers are widely used for this purpose, as they can determine monoisotopic masses of intact oligonucleotides and their fragment ions with high mass accuracy. Improved data quality from high isotopic fidelity QTOF instruments has generated a growing need for enhanced capabilities from analytical software to automate confirmation of sequences based on intact mass and MS/MS data, as well as the quantitation and identification of synthesis byproduct. This technology holds the potential to simplify data interpretation, e.g. by accommodating user-defined sequence definitions to easily include the wide range of modified nucleotides typically used by the biopharmaceutical industry.

Challenges

RNA and oligonucleotide sequence accuracy is a critical attribute impacting molecular safety profiles to avoid off-target effects or toxicity, as well as compound efficacy to ensure correct activity and expression. While useful for oligonucleotides and RNA molecules made of canonical nucleotides, next-generation sequencing methods do not offer a comprehensive characterisation of highly modified RNA-based therapeutics.

Additionally, oligonucleotide by-products may include failed nucleotide additions, nucleotide variants, and cytidine-touridine conversions with either no or +1 Da molecular weight differences to the target product sequence, respectively. As these can be difficult to characterise solely based on their intact Advancements in gene therapy and the approval of mRNA vaccines have dramatically increased the demand for well-characterised RNA and oligonucleotides in the biopharmaceutical industry. To support this emerging need, researchers have developed new analytical tools to analyse nucleic acid sequences by mass spectrometry (MS). Software advancements can now take data from high isotopic fidelity Quadrupole time-of-flight (QTOF) instruments and enable the fully automatic confirmation of sequences based on intact mass and MS/MS data, as well as the quantitation and identification of synthesis by-products. Corresponding innovations in analytical instrumentation also have provided the robustness, sensitivity, and high dynamic range necessary for RNA and oligonucleotide sequence confirmation. mass, information at the nucleotide level is required to improve oligonucleotide sequence confirmation.