Evoking picomolar binding in RNA by a single phosphorodithioate linkage.

RNA aptamers are synthetic oligonucleotide-based affinity molecules that utilize unique three-dimensional structures for their affinity and specificity to a target such as a protein. They hold the promise of numerous advantages over biologically produced antibodies; however, the binding affinity and specificity of RNA aptamers are often insufficient for successful implementation in diagnostic assays or as therapeutic agents. Strong binding affinity is important to improve the downstream applications. We report here the use of the phosphorodithioate (PS2) substitution on a single nucleotide of RNA aptamers to dramatically improve target binding affinity by ∼1000-fold (from nanomolar to picomolar). An X-ray co-crystal structure of the α-thrombin:PS2-aptamer complex reveals a localized induced-fit rearrangement of the PS2-containing nucleotide which leads to enhanced target interaction. High-level quantum mechanical calculations for model systems that mimic the PS2 moiety and phenylalanine demonstrate that an edge-on interaction between sulfur and the aromatic ring is quite favorable, and also confirm that the sulfur analogs are much more polarizable than the corresponding phosphates. This favorable interaction involving the sulfur atom is likely even more significant in the full aptamer-protein complexes than in the model systems.

Surface Plasmon Resonance imaging: a method to measure the affinity of the antibodies in allergy diagnosis.

AIM: The biological diagnosis of type I hypersensitivity reactions is based on the quantification of specific IgEs. However, the IgE titer is not always strongly related to the clinical symptoms or predictive of the evolution of the disease. The specificity and affinity of antibodies of other isotypes may contribute to the allergic status of the patients. The aim of the present work was to develop a method that simultaneously detects the complex antibody response to various allergens and measures the avidity of the antibodies directed to each allergen. METHODS: A chip based on a covalent binding of 3 major milk allergens on a gold-activated surface was developed. The binding of specific antibodies to α-lactalbumin, ß-lactoglobulin or caseins was monitored using Surface Plasmon Resonance imaging (SPRi). The sensitivity and specificity of the method were compared to those obtained by ELISA, the reference method. RESULTS: The specificity of the antibodies characterized by SPRi was identical to the one obtained by ELISA. The intensity of the signal was proportional to the quantity of antibodies bound to each allergen. The sensitivity of the SPRi detection was about 8-10 times lower than for ELISA but the SPRi is faster and the analysis of association/dissociation kinetics allowed the determination of the avidity of the antibody response. CONCLUSION: The present study shows that SPRi allows a multiplex monitoring of the complex antibody response to the major allergens of an allergenic source. This label-free method constitutes a new tool that may be added to IgE detection to improve allergy diagnosis.