Therapeutic oligonucleotide drugs are a rapidly developing field in drug research today. They can be divided into anti-sense oligonucleotides (ASO), ribozymes/deoxyribozymes (DN Azyme), small interfering RNAs (siRNA), microRNAs, anti-gene transcription factor inducers, nucleic acid aptamers, and more. The length of oligonucleotide sequences is usually around 12-30 nucleotides (nucleic acid aptamers may exceed 30 nucleotides), and their mechanisms of action vary depending on the type. For example, ASOs have a base sequence complementary to the target RNA and can specifically bind to it; siRNAs are small RNA fragments with specific lengths and sequences produced by cleavage of the target RNA, which can specifically induce the degradation of the target mRNA; nucleic acid aptamers fold into stable high-order structures through interaction, such as hairpins, pseudoknots, bulges, G-quadruplexes, etc., forming specific binding sites with target molecules. The mechanism of action of nucleic acid aptamers is similar to antibodies. Still, they have many advantages over traditional antibodies, such as high stability, easy synthesis and modification, low immunogenicity, and a wide range of targets.
Bioanalytical Methods for Oligonucleotide Drugs
In the pharmacokinetic study of oligonucleotides, because they are easily degraded by circulating plasma oligonucleotide enzymes in the body, it is necessary to use highly sensitive, highly specific, and relatively stable quantitative analysis methods for detection. The current methods for analyzing oligonucleotide drugs and their conjugates and metabolites in biological samples (such as plasma or tissues) include:
- Radiometric methods, such as liquid scintillation counting (LSC) and quantitative whole-body autoradiography (QWBA)
- Liquid chromatography (LC), such as high-performance liquid chromatography (HPLC), ion-exchange chromatography (IEC), ion-pairing reverse phase 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 different requirements and analytical advantages and disadvantages. Therefore, it is necessary to comprehensively consider the type of test sample (plasma, tissues, urine, etc.), concentration level, and detection target (i.e., quantitative PK concentration analysis or qualitative metabolite identification) to select the best method for sample analysis.
In the study of pharmacokinetics and toxicokinetics of oligonucleotide drugs, considering specificity, sensitivity and sample processing requirements, nucleic acid hybridization technology based on ELISA is widely used in the analysis of biological samples.
Hybridization-ELISA uses enzyme-labeled (such as horseradish peroxidase, HRP) nucleic acid sequence fragments to recognize target sequences specifically. It mainly includes sandwich hybridization assay, hybridization ligation assay, hybridization-based fluorescence assay, and competitive hybridization assay.
Sandwich Hybridization Assay
In sandwich hybridization analysis, the biotin-modified capture probe is fixed on the streptavidin-coated plate. It binds to a part of the test substance according to the principle of base complementary pairing. The digoxin-modified detection probe binds to another part of the test substance. Then, after adding anti-digoxin antibody-HRP, it binds to the complex. Finally, the concentration of the target analyte is quantitatively analyzed according to the signal intensity produced by the enzyme’s reaction with the substrate. This method is simple to operate and suitable for detecting nucleic acid drugs of more than 25 nucleotides in length.
Hybridization-Ligation Assay
The hybridization-ligation method relies on a capture probe containing an additional link to nine nucleotides of sequence complementary to the target analyte and a complementary detection probe containing nine nucleotides. First, the biotin-modified capture probe binds to the streptavidin-coated plate, anchoring it to the solid phase carrier and binding it to the target analyte. Then, under T4 DNA ligase, the digoxin-modified detection probe binds to the target analyte through a phosphate group-hydroxyl reaction. At the same time, the detection probe binds to the capture probe. Also, S1 nuclease is used to hydrolyze single-stranded nucleic acids that have not been successfully bound. Subsequently, enzyme-labeled antibodies and substrates are added, and the reaction signal is detected. This method has a higher specificity and is a better choice for some shorter nucleic acid drugs.
Hybridization-Based Fluorescence Assay
The hybridization fluorescence assay is based on the principle that fluorescence dyes (Hoechst or ethidium bromide) do not fluoresce or fluoresce weakly when interacting with single-stranded DNA (ssDNA) but fluoresce strongly when interacting with double-stranded DNA (dsDNA). Therefore, through the principle of base complementary pairing, the target ssDNA is hybridized with complementary ssDNA to form dsDNA, thereby achieving quantitative analysis of the test substance.
Competitive Hybridization Assay
The competitive hybridization assay involves the test nucleic acid and a probe with the same sequence competing to bind to a complementary nucleic acid probe fixed on a microplate. First, a nucleic acid probe complementary to the test nucleic acid is coated on the microplate. Then, the target analyte and a nucleic acid probe with the same sequence and biotin modification are added to compete for binding. Finally, streptavidin-enzyme is added to bind with biotin, and a signal is produced after reacting with the substrate. In the competitive hybridization assay, the concentration of the test nucleic acid is inversely proportional to the response signal. In the sandwich hybridization assay and hybridization-ligation assay, when the chain of the test nucleic acid is short, the capture nucleic acid probe and the detection nucleic acid probe used are also relatively short, leading to a reduction in the specificity of the molecular hybridization reaction. At this time, the competitive hybridization assay can serve as an alternative to the sandwich hybridization assay and the hybridization-ligation assay.
Establishment and Challenges of the Hybridization ELISA Method
For nucleic acid drug molecules with diverse chemical structures, the Hybridization ELISA method plays an important role in practical applications of analysis and evaluation. The Hybridization ELISA method has high sensitivity and requires almost no sample pretreatment. For the detection of nucleic acid drugs in plasma samples, there is no need for purification, and direct measurement is possible; for the detection of nucleic acid drugs in tissue samples, as the amount of tissue sample is small, proteinase k and non-ionic surfactants can be added to disrupt the lipid bilayer of the cell membrane and then directly perform the Hybridization ELISA method. Therefore, the Hybridization ELISA method is widely used due to its flexibility and ease of implementation.
In the face of the complexity and variability of nucleic acid drugs, the Hybridization ELISA method also faces various challenges, mainly in the following aspects:
- Specificity. The Hybridization ELISA method is based on the principle of base complementary pairing between sequences, which can distinguish full-length oligonucleotide drugs and partial metabolites (with a large difference in sequence length). However, it is more difficult to distinguish completely between modified oligonucleotide drugs and metabolites such as N-1 and N-2, which have high sequence similarity. In addition, the specificity of the method will be reduced for target analytes with shorter sequences.
- Reliable reagent and supplier selection. The establishment of the Hybridization ELISA method largely depends on the probe sequence design for the target analyte. Therefore, the quality of probe synthesis, as the cornerstone of the method establishment, is key to its success. Considering the delivery time and price of reagents, it is crucial to choose a reliable supplier.
The analysis strategy can also attempt PCR or LCMS platforms for nucleic acid drugs with complex or secondary structures.
A Final Word
With the rapid development of oligonucleotide drugs, it is increasingly important to develop reliable analytical methods with high specificity, selectivity, and sensitivity to support regulated bioanalytical research. Currently, widely used platforms like LC-MS, Hybridization ELISA, and PCR are constantly optimizing, developing, and establishing methods more applicable to the detection requirements of different types of nucleic acid drugs. At the same time, various new methods and technologies have emerged, such as droplet digital PCR (ddPCR) and branched DNA (bDNA), which show certain advantages and huge application potential of high-precision nucleic acid drug bioanalysis. In addition, the development of some automated connection technologies, like SPE-LC-MS, is believed to improve the efficiency of nucleic acid bioanalysis further.
