1) Why is Achiko using DNA aptamer technology for a Covid-19 screening and diagnostic?
DNA aptamers bind to their targets with high specificity and high affinity. They are inexpensive to produce and scalable compared to antibody/antigen production. Aptamers are also very stable and can be easily stored without refrigeration.
2) What made Achiko choose the S1 virus protein over another virus protein (such as the N protein) to detect Covid-19?
The AptameX DNA-aptamer has high specificity and affinity for the SARS-CoV-2 spike protein (S1). The S1 protein was chosen as the target because it is a surface protein. Thus, the spike protein is more accessible, represents detection of actual viral particles (read more in FAQ #3) and provides better sensitivity and specificity. On the other hand, if the N protein were the target, since it is a core protein within the virus, viral particles need to be completely denatured for it to be detected. Furthermore, the N protein tends to have more non-specific interactions that may lead to false positives.
3) What is the difference in sample quality between a nasopharyngeal swab and saliva?
Samples taken from the nasopharyngeal area may include infected cells while infectious virions (whole viruses released from infected cells) are more likely to be detected in saliva.
The SARS-CoV-2 virus infects cells by binding to the ACE-2 receptor (the cellular doorway where the SARS-CoV-2 virus enters). Cells in the nasopharyngeal/oral area including the throat express high levels of ACE-2 receptor making them susceptible to SARS-CoV-2 viral infection.
Once the virus binds to the cell, it eventually enters the cell releasing its genome (RNA) into the cytoplasm where it uses the host enzymes to produce proteins encoded in the RNA genome. These newly expressed viral proteins include proteins that lead to replication or production of more copies of the RNA genome as well as proteins needed to produce new viral particles. In essence, the virus takes control of the entire cell and converts it into a factory for making new virus particles (virions). The infected cell therefore contains a large excess of RNA and viral proteins that have not yet been assembled into new viral particles. Once a virion is assembled, it is released from within the infected cell and from the cell surface, it can be dispersed by mucus to infect neighbouring cells.
When sampling from the nasopharyngeal area or throat it is most likely that infected cells are captured on the swabs. RT-PCR or rapid antigen tests does not distinguish whether the viral RNA or proteins come from the surface of or inside the cells. Thus, the swab sample contains viral RNA or protein that may or may not be derived from a virion. Surface RNA or proteins would be equivalent to sampling the virus. However, sampling the unassembled protein or RNA from inside the cell would represent an inaccurate contribution to viral load.
As virions are released from infected cells, a significant number will end up in saliva, ready to be transmitted to another host. The mouth is the probable source of viral transmission through the generation of aerosols. Thus, the RNA or protein tested in saliva samples is more likely to be from whole virions and should more accurately correlate with viral load.
4) Why are DNA aptamers better than RNA aptamers?
DNA aptamers are better than RNA aptamers because the former is more stable and resistant to ribonucleases which are abundant in cellular environments. In addition, DNA aptamers are cheaper to synthesise.
5) How can aptamers be designed and produced?
Aptamers are selected in a process called SELEX (Sequential Evolution of Ligands by Exponential Enrichment, see below for an illustration).
This involves a large oligonucleotide library (a pool of synthetic single-stranded nucleic acid chains where each chain contains approximately 20 nucleic acids) in an iterative process where non-binders (oligonucleotides that do not have affinity for the target) are eliminated from the process and binders are subjected to multiple rounds where selectivity is enhanced.
The selected aptamer is mass produced using methods for DNA synthesis.
6) What is the main disadvantage of aptamers?
Aptamers are highly specific molecules and subtle changes in its target or the environment from which it was selected may lead to loss of binding interaction. Thus, any mutation in the target (e.g. spike protein variant) that may change the conformation or interaction site in the target where the aptamer binds may lead to loss of detection.
To be absolutely certain that AptameX can detect a Covid-positive patient sample, Achiko has planned testing on an on-going basis, on the ability of AptameX to detect SARS-CoV-2 with the various mutations relevant to current and emerging strains.
7) What makes AptameX different from antigen tests in terms of sensitivity and specificity?
AptameX has higher sensitivity than other antigen tests. It targets the spike protein on the surface of SARS-CoV-2, enabling direct detection. Most antigen tests target the N protein, which is a core protein within the virus, whereby more viral particles would have to be present and completely denatured to enable detection.
AptameX is also inherently accurate as the DNA aptamer binds selectively to the SARS-CoV-2 spike protein.
AptameX should have higher specificity because it targets the spike protein. In comparison, the N protein tends to bind non-specifically to other molecules which can lead to false positives.
8) What is the AptameX sensitivity rate versus PCR for Covid-19?
Greater than 97% at CT values under 25, and between 25 to 33.
9) What is the AptameX specificity rate versus PCR for Covid-19?
Greater than 97% at CT values under 25, and between 25 to 33.
10) What other type of diagnostics would Achiko develop in the future?
Achiko will be developing other DNA aptamer-based diagnostics including a post-vaccination Covid-antibody serological test and a dengue diagnostic.
