Overview of ASO Quantification and Bioanalysis Using qPCR

Polymerase Chain Reaction (PCR) is a fundamental biological technique that can produce millions of copies of a target DNA sequence, enabling the analysis and engineering of these basic building blocks of life. Since its conception by the maverick Cary Mullis, potentially during his LSD travels, it has become a cornerstone of molecular biology and biotechnology, gaining widespread recognition during the COVID-19 pandemic.

In tackling difficult-to-treat diseases, Antisense Oligonucleotide Therapies (ASOs) are short, synthetic, single-stranded oligodeoxynucleotides that can bind to a patient’s mRNA to reduce, restore, or modify protein expression through various mechanisms. While the pharmacokinetic (PK) properties of small molecules are well understood, making these larger ASO molecules more drug-like is one of the chief challenges faced by ASO developers. Techniques for PK studies, where a drug and its metabolites are tracked and quantified, also must be adapted for ASOs and often cannot reach the same power and resolution as for small molecules.

ASOs, being RNA-based, should be targetable using reverse-transcriptase quantitative PCR (RT-qPCR), which allows for the quantification of RNA sequences. Naturally, drug developers looked to this technique for quantification as part of PK bioanalysis to overcome the limited sensitivity of LC-MS. Unfortunately, the synthetic characteristics introduced to make these RNA molecules more suitable as drugs also make them challenging for PCR.

In this blog post, we will provide a summary overview of the current state of the art in bioanalytical approaches for ASOs using qPCR.

Challenges in qPCR

qPCR for measurement of ASOs during bioanalysis must overcome two major challenges:

1. Short length of ASOs:
   Designing PCR primers for the available 19–25 nucleotides (nt) on an ASO is counter-productive. Typically, the optimal length of a single PCR primer lies between 18 and 24 base pairs (bp), which is comparable to the length of an ASO itself. Shorter primers are less specific during the annealing phase, leading to more non-specific binding and amplification. This necessitates an elongation step to create cDNA that can accommodate effective primer binding.
2. PCR enzyme compatibility with ASO chemical modifications:
   ASO structures are chemically modified, via the backbone or other moieties, to improve their drug properties such as stability and tissue specificity. Unfortunately, these chemical modifications alter the sterics of enzyme binding, reducing the recognition and activity of transcriptase required to create cDNA for PCR. This causes a high degree of variability in experimental runs.

Development of Splint-Ligation qPCR

Splint ligation, originally developed for microRNA quantification, was first reported in the literature for ASO targets in 2021. Its use in drug development is presumably more established. This technique works around the aforementioned issues by translating the short, chemically modified ASO into a more suitable input for qPCR.

Splint Ligation Process

Rather than directly detecting the ASO, the ASO molecule hybridises and bridges two DNA template sequences. These template sequences can then be ligated together by DNA ligase to create a new single template, which is then used to create cDNA for amplification. Chemical ligation can also be used during this step but is generally a less sensitive approach.

Drawbacks of this technique include the complexity of developing a splint-ligation assay (both templates and primers must be optimised) and the skill required to run it well. Splint ligation is also less effective for advanced ASOs with 2'-O-methoxyethyl (2'-MOE) modifications, suffering from reduced sensitivity.

Conclusion

While developers value the sensitivity and lower switchover costs of using a qPCR assay for different ASOs, there is still a long way to go in creating easy-to-use, reagent-based assays that developers can feel confident in.

This is where Nanovery’s Nucleic Acid Nanorobotics (NANs), a completely novel, non-enzymatic approach, can help. Specially built for ASO quantification, NANs are reagent-based but enzyme-free and comparably sensitive to qPCR for a wide range of modifications and patterns.

Some advantages of using NANs are that they work directly in samples, removing extraction steps, and are incredibly simple to design and operate.

Get in touch with us to find out more.

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References

[1] Boos JA et al. "Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides." Nucleic Acids Research Aug; 41(15): e145, (2013)

[2] Marcial-Quino J et al. "Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia." Genes (Basel) Dec; 7(12): 131, (2016)

[3] Hills AC et al. "Peptide Conjugates of a 2′-O-Methoxyethyl Phosphorothioate Splice-Switching Oligonucleotide Show Increased Entrapment in Endosomes." ACS Omega 8, 43, 40463–40481 (2023)

[4] Gagnon KT et al. "Guidelines for Experiments Using Antisense Oligonucleotides and Double-Stranded RNAs." Nucleic Acid Therapeutics 29, 3 75.1, 21-30 (2023)