Kinetic binding analysis of aptamers targeting HIV-1 proteins by a combination of a microbalance array and mass spectrometry (MAMS).

An enhanced chip-based detection platform was developed by integrating a surface acoustic wave biosensor of the Love-wave type with protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). The system was applied to characterize the interaction of aptamers with their cognate HIV-1 proteins. The aptamers, which target two proteins of HIV-1, were identified using an automated in vitro selection platform. For aptamers S66A-C6 and S68B-C5, which target the V3 loop of the HIV-1 envelope protein gp120, KD values of 406 and 791 nM, respectively, were measured. Aptamer S69A-C15 was shown to bind HIV-1 reverse transcriptase (HIV-1 RT) with a KD value of 637 nM when immobilized on the biosensor surface. HIV-1 RT was identified with high significance using MALDI-ToF MS even in crude protein mixtures. The V3-loop of gp120 could be directly identified when using chip-bound purified protein samples. From crude protein mixtures, it could be identified indirectly with high significance via its fusion-partner glutathione-S-transferase (GST). Our data show that the combination of the selectivity of aptamers with a sensitive detection by MS enables the reliable and quantitative analysis of kinetic binding events of protein solutions in real time.

Selection process generating peptide aptamers and analysis of their binding to the TiO2 surface of a surface acoustic wave sensor.

The set-up presented in this article is intended for the selection of peptides which serve as specific binders to suitable materials. Additionally, the interaction of such binders with material surfaces can be characterized. Using this approach, a subset of peptides which adhere to the mineral TiO(2) was generated by means of a cell surface display library. The peptides are constrained by a thioredoxin scaffold. Selection of proteins was carried out on a silicium wafer sputtered with TiO(2) in anatase conformation. To verify binders and to analyze the binding kinetics of the diluted suspension of the purified proteins, the chip-based S-sens K5 surface acoustic wave sensor system was used. The surface of the sensor chips was also TiO(2), resembling the material of the Si wafer selection target retaining the peptides. Several peptides were identified. The respective binding behavior differed. The data derived from real-time interaction analysis were evaluated to select for strong and specific binders. For one of these peptides, the binding kinetics was analyzed. On- and off-rate binding constants were extracted from the fitted curves. With the resulting association rate constant k(on) and the dissociation constant k(off), the affinity of the peptide for the TiO(2) surface was calculated, represented by the equilibrium dissociation constant K(D)=81 nM.

Discrimination of single mutations in cancer-related gene fragments with a surface acoustic wave sensor.

Here, we report on using a surface acoustic wave sensor for the highly sensitive and accurate detection of individual point mutations in cancer-related gene DNA fragments from single injections. Our sensor measures both the mass and viscosity signals and, thus, allows discriminating between mass effects resulting from hybridization of short DNA strands and viscosity effects due to increasing amounts of DNA deposited on the sensor. Single nucleotide exchanges or deletions are distinguished reliably and with exceeding simplicity from the wild-type sequences, on the basis of differences in their dissociation or association rates starting at low nanomolar concentrations. Mutant oligonucleotides were identified immediately from viewing the recorded signal and without further processing of the data. Multiple repeated binding cycles were possible over days without affecting sensitivity. To achieve signal amplification, our new bioassay can also apply multiple hybridization steps based on sandwich hybridizations. Kinetic evaluations gave insight into the physicochemical properties of the fragments used that explain the differences in their binding processes.