The use of DNA Aptamers for the detection of Endotoxin.

摘要

The Gram negative bacterial cell wall, Lipopolysaccharide (LPS), is known as Endotoxin or Pyrogen, is of considerable biomedical interest since in mammals it may elicit symptoms of innate immunity such as fever and in extreme cases, death. The Horseshoe Crab blood coagulation system has been well studied, and their clotting cascade, which represents their immune system, responds to Endotoxin and 13-Glucan, both of which elicit innate immunity. The partially purified clotting cascade from the Horseshoe Crab has been used in assay and other diagnostic reagents. It is required by FDA for the release of all injectables and biological implants, as well as, any medical device that communicates with the extracorporeal environment. Several alternative test methods are available that do not rely on Horseshoe Crab blood, such as a recombinant proteins (Endotoxin Neutralization Protein), and antibodies. However, it was found that these proteins cannot provide the specificity and sensitivity that is required by the FDA. Aptamers are oligonucleotides (DNA or RNA) that can bind to a wide range of target molecules with high affinity and specificity, and some of them have been used as Diagnostic Reagents and Drug discovery tools. Aptamers against hydrophobic or amphiphilic molecule have not been examined well. For providing high specificity and sensitivity to Endotoxin, synthetic analogue was developed. The in vitro SELEX (systematic evolution of ligands by exponential enrichment) with this developed analogue and Pyrosequencing were applied for selecting Aptamer which binds to amphiphilic molecule; LPS. Five Aptamers were successfully selected and characterized. Using Surface Plasmon Resonance, Fluorescent Polarization and Molecular Beacons, we were able to demonstrate that our aptamer selection was active against LPS in the range of nM. We also demonstrated that the effect of Magnesium and Potassium paralleled the experience of others when using hydrophilic targets to Aptamers, however, in our case, using the hydrophobic target LPS, the effect of Sodium was found to be the opposite. These data are interpreted as suggesting that the binding modes and strengths of hydrophobic molecules to the moderately hydrophilic surfaces of Aptamers are significantly different.