Michael Chan's CIAP Aptamer Project (2019)

RNA Aptamer and Calf Intestinal Alkaline Phosphatase Utilized to Diagnose Sepsis More Efficiently in Blood Samples Using ELONA

Sepsis is a systemic inflammatory response to microbial infection with a 25-30% mortality rate that can potentially result into septic shock, which has a higher rate of mortality of 50-85% (Minasyan, 2019). Early detection and therapeutic measures, such as antibiotics and intravenous fluid, can reduce mortality by treating the infection and ultimately reduce organ damage (Hotchkiss et al, 2016). The cause of such a high mortality is due to the difficulty in diagnosing the condition, the long amount of time required for blood cultures to provide results, and the severe amount of damage that sepsis can inflict on the body. A possible aptamer solution for the quicker diagnosis of sepsis is with an antigen enzyme-linked oligonucleotide assay (ELONA) that uses another aptamer that binds to specific antigens to detect for antigens in the sample. This is made possible because the presence of bacteria is used as an indicator of sepsis via blood cultures (Gul, Arslantas, Cinel, & Kumar, 2017). Calf Intestinal Alkaline Phosphatase (CIAP) can function as an indicator, or reporter molecule, which is an enzyme that acts as a homodimeric enzyme with detection applications, such as emitting color in the presence of other molecules (Stovall, 2019). By finding an aptamer that can bind to CIAP and a separate aptamer for an antigen specific only to Staphylococcus aureus, it will be possible to detect sepsis early and prevent further deterioration into septic shock and potentially death.

For this experiment, the ELONA is used to analyze blood samples for bacterial antigens, but the general concept can be replicated using another aptamer to detect other biomarkers as well, since CIAP’s capabilities as a reporter molecule is the primary focus. The disease or condition that the ELONA is detecting for can also change given modifications such as replacing antibody or aptamer that binds to the biomarker. For example, the difficulty of finding a single unique antigen specific only to sepsis-causing strains of S. aureus diminishes the specificity of an ELONA, which is its highly sensitive detection abilities, low cost, and shorter incubation time compared to the gold standard of blood cultures (Lamy, Dargère, Arendrup, Parienti, & Tattevin, 2016). Further research needs to be conducted in the future to improve the application of the CIAP aptamer.

Aptamers are oligonucleotides that bind to a certain target. Aptamers are preferable to antibodies because they are cheaper to produce, have a longer shelf life, are more thermally stable, and non-immunogenic, allowing for more efficient drug delivery and accurate diagnostic results (Lakhin, 2013). Instead of using antibodies to bind to their respective antigens, aptamers will be used to create a cheap, yet accurate diagnostic tool. The aptamer against CIAP will serve a diagnostic application by binding the reporter molecule, CIAP, to the antigen of interest that will then bind to an aptamer linked to a solid support such as a 96 well plate (Figure 1). By analyzing the colorimetric response of CIAP in a blood sample, it will be possible to detect the presence of bacteria to allow for the prediction and subsequent prevention of systemic inflammations that could lead to fatal conditions such as septic shock. For example, the Nutiu et al. lab produced results using aptamers for CIAP that found a structure-switching signaling aptamer against CIAP that allows real-time reporting of enzymatic activity by noting the changes in levels of fluorescence (Nutiu, 2004).

To select for high binding affinity aptamers, the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was utilized (Figure 2). Oligonucleotides will be bound with the target protein over many rounds and washed with the selection buffer to remove unbounded species to select for an aptamer with the highest affinity to the protein. Each iterative round, the nucleic acids will undergo reverse transcription to make ssDNA to be amplified, large scale PCR to create large amounts of dsDNA, transcription to synthesize RNA from dsDNA, and polyacrylamide gel electrophoresis to purify the RNA that will be used in the following round to select for the aptamer with the highest binding affinity.

The goal of the first round of selection is to narrow the pool to find an aptamer that will aid in the detection of sepsis in blood using CIAP for later rounds of selection. In the first round of selection, there were difficulties with ccPCR bands not showing up. Through a series of efficacy checks to check for reagent contamination and guidance from mentors, it was concluded that the binding and selection step was too stringent causing no bands to appear in W3 and E1. To avoid this problem in the future, the ratio of target to pool will be adjusted and the binding and selection will be carefully conducted in the future. The next step will be to complete Round 2 of selection to find a diagnostic aptamer for the early detection of sepsis in hospital patients.

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