The COVID-19 pandemic is posing an unprecedented threat to public health and severely disrupting the global economy. Worldwide, millions of people are already infected, other hundreds of thousands lost their lives and livelihoods, and there is no immediate solution at sight. Existing drug repurposing has been promptly attempted as a quick measure, but with debatable and modest success. While several biopharmaceutical companies are racing to develop drugs and vaccines, it may still take months or even years before some direct therapeutic remedy comes out. Consequently, physical distancing and quarantining infected individuals remain to be the recommended strategy to arrest the spread of the contagion. Since the number of cases continues to grow, extensive availability of diagnostic testing and rapid identification is critical for the success of this approach.
Synthetic oligonucleotides have emerged as an important modality for both diagnostic and potential therapeutic purposes. The significant advantages of oligonucleotide-based strategies are that they can be quickly designed based on expected Watson-Crick hybridization and can be synthesized with well-developed solid-phase synthesis chemistries.
Commercially available COVID-19 tests presently can be classified into two major categories. The first category, also called molecular tests, relies on polymerase chain reaction (PCR)-based techniques to detect the viral ribonucleic acid (RNA). The second category depends on serological and immunological assays that detect antibodies generated in infected individuals.
In the PCR-based methods, synthetic oligonucleotides are directly being used to amplify and detect the viral RNA. There are several variations of the PCR-based techniques that are continually evolving to include automation, improve accuracy, and reduce assay duration. The majority of the molecular tests performed to date are based on real-time reverse transcription PCR (RT-PCR) technique that targets different genomic regions within the SARS-CoV-2 virus, which causes the COVID-19 disease. In the RT-PCR method, the DNA oligonucleotide primers and probes recognise and bind to the viral genomic RNA, if present in the patient sample. During the PCR amplification, the probe gets displaced and provides a detectable fluorescent signal for positive samples. Although the RT-PCR test is considered as the ‘gold standard’ for COVID-19 detection, it is laborious, requires expensive instrumentation, and takes days to get the result. These practical limitations are severely limiting mass screening, which is critical to combat the contagion.
Consequently, other versions of the PCR-based tests have been developed that rely on isothermal amplification technologies. Unlike the RT-PCR tests, where sophisticated thermal cyclers are required, isothermal tests are designed to work with simple incubation at a specific temperature. Examples of such tests are, reverse transcription loop-mediated isothermal amplification (RT-LAMP), transcription-mediated amplification (TMA), and rolling cycle amplification (RCA). Notably, all these methods employ synthetic oligonucleotides as essential reagents.
Other types of testing that can be used are the multiple versions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene-editing technology such as SHERLOCK, DETECTR, CREST, and FELUDA. In general, these methods use DNA-primers for reverse transcription and isothermal amplification of the viral RNA. CRISPR-based tests hold immense potential because they pledge to be inexpensive, rapid, and more specific than traditional tests.
In addition to the PCR and CRISPR-based methods, other molecular tests like next-generation sequencing (NGS) techniques have been developed for direct, high-throughput, and specific identification of infections in a variety of patient specimen types. Such technology first uses PCR-reactions and synthetic DNA primer sets to generate amplicons that are further sequenced to detect the presence of the virus infection.
Serological and immunological assays serve a complementary purpose to the molecular tests in managing the pandemic progression. Whereas the molecular tests detect the existing infections, serological assays could be used to identify individuals who were previously infected. The serological tests rely on detecting the presence of viral antigen or produced antibodies in the patient blood sample. Like the molecular tests, there are several variations of the serological tests. For example, in the enzyme-linked immunosorbent assay (ELISA), an immobilised viral antigen is being exposed to patient samples to detect the presence of bound antibodies. Unlike the molecular tests, synthetic oligonucleotides are not directly used in the serological tests. Nevertheless, many of the serological assays are based on recombinant proteins, and synthetic oligonucleotide primers remain as inevitable cloning reagents for the preparation/modification of the corresponding expression plasmids.
Antisense oligos and SiRNAs
While significant effort is underway, an effective antiviral strategy to treat SARS-CoV-2 infection does not exist to date. Along with the small (e.g., antiviral drug) and large molecule (e.g., vaccines, antibodies) ̶ based approaches, oligonucleotide therapeutics signify an emerging strategy in combating the contagion.
Synthetic oligonucleotides can inhibit gene expression by several avatars such as antisense oligos (ASOs) and silencing RNAs (siRNAs). They hybridize with the complementary disease-causing target gene sequence and inhibit the corresponding protein synthesis, either by blocking the ribosome access or by enzyme-assisted destroying the target mRNA. Such oligonucleotides have been used for antiviral applications with some success. For example, the US FDA approved the ASO fomivirsen (Vitravene) in 1998 for the treatment of cytomegalovirus (CMV) retinitis — an infection of the retina in HIV patients that can lead to blindness. Similarly, ASOs and siRNAs have been proposed that can target the critical component of the virus itself, e.g., the spike protein. In the past, morpholino ASOs have been shown to inhibit some other coronavirus classes. Currently, several research groups are working on the application of such therapeutic oligonucleotides for many viral diseases including COVID-19. Unlike small molecules, oligonucleotide drug design often follows established rules and are easier to develop. The success of these approaches will, therefore, lead to a sustainable therapeutic strategy for the current and future outbreaks.
Additionally, since synthetic oligonucleotides are indispensable reagents in recombinant molecular biology techniques, along with direct applications as siRNAs or ASOs, synthetic oligonucleotides are also employed as primers for potential DNA/mRNA-based vaccine development. Advances in gene synthesis using oligonucleotide technologies can help to generate viral antigen subunit vaccines rapidly, avoiding the use of dangerous viral pathogens.
In the light of all these facts that are emerging about the superiority of synthetic oligonucleotides, it is becoming clearer that they will remain critical reagents in viral disease diagnostics and therapeutics that are not limited to only COVID-19. Moreover, considering the myriad of ways the scientific community is trying to tackle the COVID-19 pandemic, applications of oligonucleotides are likely to spread in many newer directions.
(The author is Principal Investigator, Chemical Development – Oligonucleotides Process Research and Development, Syngene International Ltd, Bangalore.)