Oligonucleotide therapeutics are an exciting class of drugs due to their potential to treat diseases previously considered undruggable. Currently, there are over 20 approved oligonucleotide therapeutics. Structurally, they consist of single-stranded antisense oligonucleotides (ASOs), small interfering RNA (siRNA), and aptamers.
This blog focuses on the approved ASOs and siRNAs, which are used to treat a wide variety of genetic disorders, including:
Each drug binds a specific mRNA, resulting in downregulation of a target protein or production of a more productive isoform. Their use can dramatically increase the quality of life for patients, providing optimism that future development of this drug class may lead to additional breakthroughs in disease treatment.
Oligonucleotides are comprised of individual nucleotides linked together by one of the following types of linkages:
As shown in Figure 1, each nucleotide type includes a base of A, C, 5-methyl-C, G, T, or U corresponding to adenine, cytosine, 5-methyl cytosine, guanine, thymine, or uracil, respectively. These therapeutics may include modifications such as fluorine (F), O-methyl (O-Me), or O-methoxyethyl (O-MOE) to the 2’ position of the ribose sugar, increasing both stability and binding affinity. Additionally, the replacement of a single oxygen atom with sulfur in the PS backbone provides additional nuclease resistance.
As with any drug, quality attributes such as identity, safety, purity, quality, and potency must be studied extensively during the development of oligonucleotides. Methodologies must be established to assess and monitor each of these attributes during development, production and ultimately, release. The definition of the oligonucleotide in terms of its structural identity is fundamental. In this context, sequence is hugely important since it can significantly impact binding efficiency, stability, and even delivery.
Mass spectrometry is considered one of the most reliable techniques for sequencing oligonucleotides. Traditional methods such as PCR are somewhat limited by the structural diversity and modifications of many oligonucleotide therapeutics. The most commonly used mass spectrometry techniques for oligonucleotide sequencing include:
Both techniques use “soft” ionization to generate intact pseudomolecular ions.
One MALDI-MS approach requires pre-digestion of the oligonucleotide by enzymes, while another utilizes in-source fragmentation. However, both techniques may be significantly constrained by the structure of the oligonucleotide, often resulting in limited sequence information.
Another MALDI-MS approach takes advantage of the synthetic route used for most oligonucleotides, where nucleotides are added one at a time. Since no synthetic procedure is 100% complete, incomplete, or failure, sequences are possibly observed. These sequences align with stepwise fragments from the initial to final nucleotide, where the MALDI mass spectrum will appear as a “ladder” of products with mass differences, corresponding to the masses of each sequentially added nucleotide.
Sequencing by ESI involves generating intact pseudomolecular ions in the gas phase, then subjecting them to Collisionally Activated Dissociation (CAD). By optimizing the mass spectrometer parameters, fragmentation can be focused along the oligonucleotide backbone between adjacent nucleotides, as shown for PO/PS oligos (Figure 2A) and PMOs (Figure 2B).
For PMOs, which have phosphorodiamidate linkages, Electron Capture Dissociation (ECD) can be beneficial since this technique may provide increased fragmentation along the PMO backbone as opposed to increased loss of bases when using CAD.1
Overall, ESI-MS(MS) is the preferred technique for oligonucleotide sequencing because:
Figure 3 illustrates how mass spectrometry confirms oligonucleotide sequence for a small oligonucleotide. Its single-letter sequence (5’ – T C T G A – 3') is displayed in the top row, followed by its full-length structure, which has a mass of 1831 Da, shown in row 2.
Below this, experimentally observed fragments are displayed, including a1 to a4 and w4 to w1 in rows 3 to 6. The respective a fragments, extending from the 5´ end, begin with the T nucleotide, followed by successive C, T, and G. Meanwhile, the w fragments (starting at the 3’-end, bottom row right) consist of A followed by G, T, and C.
As indicated in the figure, each subsequent fragment includes the additional mass of the sequential nucleotide. By combining all a and w fragments along with the full-length structure, the “sequence” of the oligonucleotide is confirmed in both directions.
This availability of numerous fragments is both the beauty and complexity of mass spectrometry data.
Design effective structural characterization studies for oligonucleotide therapeutics.
Example data from the analysis of unmodified Inotersen and Inotersen.
In closing, sequencing confirmation of oligonucleotide therapeutics is one of the most important processes during oligonucleotide drug development, production, and routine batch release.
With its incredible ability to produce and identify specific fragments consistently, mass spectrometry stands out as the ideal sequencing technique for this class of drugs. As oligonucleotide structures continue to increase in complexity, mass spectrometry remains ideally suited to analyse virtually any type of structure.
References
1 Karasawa, K.; Duchoslav, E.; Burton, L.; Kawakami, J.; Baba, T. Anal. Chem. 2023, 95 (44), 16352-16358
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