Introduction of Oligonucleotides

Oligonucleotide-based therapeutics represent a rapidly advancing field in current drug development. According to their therapeutic mechanisms, oligonucleotide drugs can be categorized into antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA, anti-gene transcription factor inducers, and nucleic acid aptamers. Oligonucleotide sequences typically have lengths of around 12-30 nucleotides (nucleic acid aptamers may exceed 30 nucleotides), and their modes of action vary depending on the type. For example, ASOs have complementary base sequences to target RNA, allowing for specific binding. siRNA induces the degradation of target mRNA by cleaving it into specific-length and sequence fragments. Nucleic acid aptamers fold into stable three-dimensional structures, such as hairpins, pseudoknots, bulges, G-quadruplexes, etc., forming specific binding sites with the target molecule through these structures. The mechanism of action of nucleic acid aptamers is similar to antibodies, but they offer several advantages over traditional antibodies, including high stability, ease of synthesis and modification, low immunogenicity, and a broad range of target specificity.

Oligonucleotide Drug Bioanalytical Methods

In the pharmacokinetic studies of oligonucleotides, due to their susceptibility to rapid degradation by circulating plasma oligonucleotide enzymes in the body, it is necessary to employ quantitative analysis methods with high sensitivity, specificity, and stability for detection. Currently, several methods are used for analyzing oligonucleotide drugs, their complexes, and metabolites in biological samples (such as plasma and tissues).

Radiometric methods, such as liquid scintillation counting (LSC) and quantitative whole-body autoradiography (QWBA).

Liquid chromatography (LC), including high-performance liquid chromatography (HPLC), ion-exchange chromatography (IEC), and ion-pairing reverse liquid chromatography (IP-LC).

Capillary gel electrophoresis (CGE).

Mass spectrometry (MS), such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), capillary electrophoresis-mass spectrometry (CE-MS), or liquid chromatography-mass spectrometry (LC-MS).

Hybridization-based enzyme-linked immunosorbent assays (hybridization-based ELISA).

Quantitative polymerase chain reaction (qPCR).

Different analytical methods have varying requirements, advantages, and limitations. Therefore, it is essential to consider factors such as sample type (plasma, tissues, urine, etc.), concentration levels, and the analytical target (quantitative PK concentration analysis or qualitative identification of metabolites) to choose the optimal method for sample analysis.

Hybridization-Based ELISA for Nucleic Acids Analysis

In the study of the pharmacokinetics and toxicokinetics of oligonucleotide drugs, considering specificity, sensitivity, and sample processing requirements, the hybridization-based ELISA technique is widely employed in the analysis of biological samples. Hybridization-ELISA is a method that utilizes nucleic acid sequences labeled with enzymes (such as horseradish peroxidase, HRP) to specifically recognize target sequences. It mainly includes the sandwich hybridization assay, hybridization ligation assay, hybridization-based fluorescence assay, and competitive hybridization assay.

Sandwich Hybridization Assay

In the sandwich hybridization analysis, biotin-modified capture probes are immobilized on a streptavidin-coated plate. According to the principle of base complementarity, these probes bind to a partial sequence of the target. Simultaneously, digoxin-modified detection probes bind to another portion of the target. After the addition of anti-digoxin antibody-HRP, which binds to the complex, the quantitative analysis of the target analyte's concentration is performed based on the signal intensity generated by the enzymatic reaction between the enzyme and the substrate. This method is easy to operate and suitable for the detection of nucleic acid drugs with a length greater than 25 nt.

Hybridization-Ligation Assay

The hybridization-ligation assay relies on capture probes containing an additional nine nucleotides outside the sequence complementary to the target analyte and detection probes containing nine complementary nucleotides. Firstly, biotin-modified capture probes are immobilized on a streptavidin-coated plate, fixing them to a solid-phase carrier, and allowing them to bind to the target analyte. Subsequently, under the action of T4 DNA ligase, digoxin-modified detection probes bind to the target analyte through a phosphoester-hydroxyl reaction. Simultaneously, detection probes bind to capture probes. S1 nuclease is used to hydrolyze unbound single-stranded nucleic acids. Enzyme-labeled antibodies and substrates are then added to detect the reaction signal. This method exhibits higher specificity and is a better choice for some shorter nucleic acid drugs.

Hybridization-Based Fluorescence Assay

The hybridization-based fluorescence assay relies on the principle that the fluorescence dye (such as Hoechst or ethidium bromide) does not fluoresce or fluoresces weakly when interacting with single-stranded DNA (ssDNA) but enhances fluorescence when interacting with double-stranded DNA (dsDNA). Therefore, through the principle of base complementarity, the target ssDNA is hybridized with complementary ssDNA to form dsDNA, achieving quantitative analysis of the target analyte.

Competitive Hybridization Assay

The competitive hybridization assay utilizes a competition between the target nucleic acid and a primer with the same sequence immobilized on a microwell plate. Firstly, the nucleic acid primer complementary to the target nucleic acid is coated on the microplate. Then, the target analyte and a biotin-modified nucleic acid primer with the same sequence as the target are added for competitive binding. Finally, streptavidin-enzyme is added to bind with biotin, and the signal is generated after reacting with the substrate. In the competitive hybridization assay, the concentration of the target nucleic acid is inversely proportional to the response signal. In cases where the target nucleic acid chain is short in the sandwich hybridization method and the hybridization-ligation method, using shorter capture and detection nucleic acid primers can reduce the specificity of the molecular hybridization reaction. In such cases, the competitive hybridization assay can serve as an alternative to the sandwich hybridization method and the hybridization-ligation method.

Establishment and Challenges of the Hybrid-ELISA Method

For nucleic acid drugs with diverse chemical structures, the Hybridization ELISA method holds a crucial position in practical applications for analysis and assessment. This method not only exhibits high sensitivity but also requires minimal sample pretreatment. In the detection of nucleic acid drugs in plasma samples, there is no need for purification, enabling direct measurement. For nucleic acid drug detection in tissue samples, where the sample quantity is limited, the addition of proteinase K and non-ionic surfactant can disrupt the cell membrane lipid bilayer, allowing for the direct implementation of the Hybridization ELISA method. Therefore, the Hybridization ELISA method is widely applied due to its flexibility and ease of implementation.

Facing the complexity and variability of nucleic acid drugs, the hybridization ELISA method encounters various challenges, primarily in the following aspects.

Specificity: The Hybridization ELISA method, based on the principle of complementary base pairing between sequences, can differentiate between full-length oligonucleotide drugs and partial metabolites (with significantly different sequence lengths). However, it is challenging to completely distinguish modified oligonucleotide drugs and metabolites with high sequence similarity, such as N-1 and N-2. Additionally, the specificity of the method is reduced for target analytes with inherently short sequences.

Reliable reagent and supplier selection: The establishment of the Hybridization ELISA method relies heavily on the design of primer sequences targeting the specific analyte. Therefore, the quality of primer synthesis serves as the cornerstone for the successful development of the method. Choosing a reliable supplier is crucial, considering reagent delivery times and prices.

With the rapid development of oligonucleotide drugs, it is increasingly important to develop reliable analytical methods with high specificity, selectivity and sensitivity to support regulatory bioanalytical studies. Currently, the widely used LC-MS, hybridization ELISA and PCR have been optimized according to their respective methodological features, and developed to be more suitable for the detection requirements of different types of nucleic acid drugs. At the same time, various types of new methods and technologies have also emerged, such as droplet digital PCR (ddPCR) and branched DNA(bDNA) signal amplification, which have shown certain advantages and great application potential in the field of high-precision nucleic acid drug bioanalysis. In addition, the development of some automated linkage technologies, like SPE-LC-MS, is believed to further improve the efficiency of nucleic acid bioanalysis.

References

1. T, L, Bao.; et al. Antisense Oligonucleotides Targeting Angiogenic Factors as Potential Cancer Therapeutics. Molecular Therapy. 2019, 14: 142-157.

2. M, B, Thayer.; e?t al. Application of Locked Nucleic Acid Oligonucleotides for siRNA Preclinical Bioanalytics. Scientific Reports. 2019, 9: 3566.