A layer-by-layer mass spectrometry intensity method for de novo direct sequencing of RNA therapeutics and impurities

Background:

The field of RNA-based therapeutics has experienced rapid growth due to the success of mRNA vaccines and the expanding use of small RNA molecules such as siRNA, miRNA, and CRISPR guide RNAs in medicine. These molecules offer precise mechanisms for gene regulation and editing, making them highly attractive for treating a wide range of diseases. However, the complexity of RNA synthesis and the need for chemical modifications to enhance stability and efficacy introduce significant challenges in ensuring product purity and safety. Regulatory agencies require rigorous characterization of these therapeutics, including the detection of minor variants and impurities, to mitigate safety risks and ensure consistent clinical outcomes. As the diversity and complexity of RNA drugs increase, there is a growing demand for technologies that can comprehensively sequence and profile all RNA species present in a sample, including low-abundance impurities and chemically modified variants. Current analytical approaches, such as next-generation sequencing (NGS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), have notable limitations when applied to RNA therapeutics. NGS methods typically rely on converting RNA to cDNA, a process that can obscure or miss important chemical modifications and may not accurately represent the full spectrum of RNA species, particularly impurities. LC-MS/MS, while effective for confirming target sequences, generally requires highly purified RNA samples and struggles to detect coexisting impurities or to provide de novo sequence information, especially for longer RNA molecules. These constraints hinder the ability to fully characterize the composition of RNA drug products, complicating quality control and regulatory compliance. As a result, there is a significant unmet need for analytical tools that can deliver unbiased, comprehensive, and direct sequencing of mixed and modified RNA samples without the limitations of current methodologies.

Technology Overview:

The 3D NGMS-Seq platform is an advanced mass spectrometry-based sequencing solution designed for the de novo analysis of mixed RNA samples, including those containing modified variants and impurities. By integrating mass spectrometry intensity data into an established framework, the platform computationally separates different RNA species. A sophisticated nested algorithm then further organizes hydrolyzed RNA fragments into distinct layers based on various characteristics, enabling the reconstruction of full-length RNA sequences from short fragments. This reconstruction leverages mass differences between adjacent fragments for accurate base-calling, a process enhanced by RNA acid hydrolysis kinetics and robust statistical modeling. The method has demonstrated high accuracy in sequencing synthetic siRNA, miRNA, and CRISPR/Cas9 sgRNAs, including the detection of low-abundance impurities, making it highly suitable for applications in RNA drug development and quality control. What sets this technology apart is its ability to provide comprehensive, unbiased, and de novo sequencing of complex RNA mixtures, overcoming the significant limitations of existing methods. Traditional LC-MS/MS techniques require purified RNA and often fail to detect coexisting impurities, while next-generation sequencing approaches typically rely on cDNA synthesis and miss crucial RNA modifications or impurity profiles. Furthermore, conventional mass spectrometry struggles with longer RNA molecules and lacks the capability for de novo sequence determination. The 3D NGMS-Seq platform uniquely addresses these challenges of existing analysis by enabling direct, high-resolution analysis of all RNA species present in a sample, regardless of length or modification status. This holistic approach ensures that even minor or modified impurities are detected and sequenced, supporting stringent regulatory requirements and enhancing the safety and efficacy of RNA-based therapeutics and vaccines.

Advantages:

•    Enables accurate de novo sequencing of mixed RNA samples, including modified variants and impurities, with claimed 100% accuracy.
•    Detects low-abundance impurities and chemical modifications critical for RNA therapeutic function and safety.
•    Overcomes limitations of traditional LC-MS/MS and NGS methods by providing comprehensive, unbiased RNA sequence and impurity profiling without requiring purified samples or cDNA synthesis.
•    Supports quality control, regulatory validation, and development of diverse RNA-based therapeutics such as siRNA, miRNA, mRNA vaccines, and CRISPR/Cas9 sgRNAs.
•    Facilitates improved safety and efficacy in RNA drug development by enabling holistic analysis of RNA species and impurities.

Applications:

•    RNA therapeutic quality control
•    Impurity profiling in RNA drugs
•    Regulatory validation of RNA medicines
•    De novo sequencing of modified RNAs
•    CRISPR guide RNA verification

Intellectual Property Summary:

Patent application filed

Stage of Development:

TRL 4

Licensing Status:

This technology is available for licensing.