In a recent study published in Nature Communications, researchers proposed a simplified approach for analyzing messenger ribonucleic acid (mRNA) vaccines using long-read sequencing.

Background

Messenger RNA vaccines demonstrated safety and efficacy during the COVID-19 pandemic, but extensive quality and purity testing is required to verify their efficacy and safety. Manufacturing advances have enabled billions of doses to be manufactured with acceptable quality and safety.

Various approaches are now utilized to assess mRNA vaccines; however, the efficacy of novel therapies depends on speedy and safe manufacture. Rigorous analytics are required at each stage of the production process to detect impurities and assure the safety of mRNA vaccines.

About the study

In the present study, researchers investigated the VAX-seq method for quality analysis of messenger RNA vaccines.

The researchers developed VAX-seq, a simplified procedure for analyzing mRNA vaccines and therapeutics using long-read sequencing. This procedure compares VAX-seq to industry standards, including chromatography, capillary and agarose electrophoresis, and immunoblotting. The researchers employed a variety of methodologies, including Illumina plasmid DNA sequencing, ONT cDNA-PCR sequencing, and Oxford Nanopore direct ribonucleic acid sequencing.

Key messenger RNA quality features assessed by VAX-seq were sequence similarity, integrity, 3′-poly(A) nucleotide tail dimension, and RNA and DNA contamination. To assist VAX-seq, a software toolbox was created that provides thorough and automated reporting on mRNA quality. An enhanced green fluorescent protein (eGFP) messenger RNA was created and generated as a reference to demonstrate the application and validity of the methodology,

The plasmid template was amplified in Escherichia coli, isolated, purified, and linearized as the initial stage in the preparation process. The linearized pDNA template was then employed as a template for synthetic mRNA transcription in vitro. To examine the isolated mRNA, the program was combined with complementary deoxyribonucleic acid (cDNA) sequencing. VAX-seq attached a reverse transcriptase primer to the 3′ terminus of the poly(A) nucleotide tail, allowing the length of the tail to be measured.

The researchers used the tailfindr program to normalize deletion mistakes and the read-specific nucleotide translocation rate. As part of the VAX-seq process, the complementary DNA library preparation introduced two flanking-type adaptors to the messenger RNA’s 5′ and 3′ ends.

To identify complete-length molecules of mRNA from truncated messenger RNA, sequencing reads contained both flanking adaptors. Off-target RNA contaminants were identified using VAX-seq, which was used to assess fragmented and off-target RNA contaminants in cDNA libraries.

Results

The analysis revealed that VAX-seq, a technique for sequencing mRNA vaccines, can detect sequence, length, integrity, and purity. It also enabled the examination of linearized plasmid DNA templates and the detection of impurities from plasmid amplification. VAX-seq easily established the length and similarity of mRNA vaccine sequences. The eGFP mRNA size profile revealed a major peak (77%) that was within 5.0% of the predicted length [1,153 nucleotide (nt)-long], as well as a varied spectrum of smaller, fragmented mRNAs. Short-read sequencing provided insufficient and inconsistent coverage, while heterogeneous alignment coverage was highly repeatable across replicates.

Most sequences were aligned with the on-target messenger RNA product, and only a few reads revealed Escherichia coli contamination. The remaining seven percent of ribonucleic acid species were off-target RNA molecules, with 0.3% presumably originating from initiation sites of cryptic transcription. Direct ribonucleic acid sequencing libraries produce lower yields than comparable complementary DNA sequencing genetic libraries and cannot be multiplexed at the moment.

The researchers did, however, identify biases particular to direct ribonucleic acid sequencing, such as inferior-quality poly(A) nucleotide tail deletion. Direct ribonucleic acid sequencing found changed nucleosides in messenger RNA vaccines, demonstrating that including modified nucleosides in mRNA vaccines might lessen the innate immunological response while improving stability and translation.

Modified nucleosides had minimal effect on messenger RNA quality features and complementary DNA sequencing errors between messenger RNAs, including native N1-methylpseudouridine and uridine, but direct ribonucleic acid sequencing had a larger error rate.

Complementary DNA and direct ribonucleic acid sequencing revealed that modified messenger RNA vaccines had more truncated-type transcripts, with 41% complete-length and 54% truncated messenger RNA molecules, especially those less than 500 nt in length. Direct ribonucleic acid sequencing discovered nucleosides of N1-methylpseudouridine with a distinctive base-calling mistake that miscategorized N1-methylpseudouridine into cytosines, skewing the messenger RNA length profile.

Conclusions

Overall, the study findings showed that VAX-seq was a procedure based on sequencing long reads that assessed essential mRNA quality characteristics such as integrity, contamination, and sequence identity. This technique can potentially become important to developing and producing mRNA medicines, offering a thorough and integrated evaluation at various manufacturing stages. VAX-seq employed full-length complementary DNA sequencing using Nanopore chemistry, which allowed for accurate assessment of the poly(A) molecular tail length as well as various off-target readings.

The approach offered a sensitive and quantitative assessment of mRNA characteristics, making it a more efficient alternative to conventional analytical techniques. VAX-seq enabled real-time identification of antisense RNA and messenger RNA integrity, allowing for swift testing lasting a few hours post-manufacture.

It might also identify complicated off-target ribonucleic acid contaminants created during transcription in vitro, as well as the degradation or sharing of messenger RNA vaccines during manufacturing, storage, and transportation. VAX-seq needed only a small quantity of messenger RNA as input and may be integrated to allow for large-scale and low-cost validation of vaccination batches.