Detection Methods for Small Molecules and Advantages of Aptamers

Small molecules are involved in innumerable biochemical processes in life and can operate as substrate, catalyst or inhibitor in biochemical reactions. Because of these properties, small molecules are valuable as drugs in medicine or in biotechnological applications. In addition, in all cases the feasibility of quickly and reliably detecting them is essential.

Different and versatile techniques have been developed for the detection of small molecules. Classic detection methods are based on optical spectroscopic techniques. In these, electromagnetic radiation with a particular wavelength and intensity is applied to an object by which it is adsorbed, dispersed or emitted. Techniques like X-ray, ultraviolet-visible (UV/VIS), infrared (IR), electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy are used for small-molecule detection as well as for the structural analysis of molecules (Lottspeich and Zorbos 1998).

Other methods involve radioactive and non-radioactive-labelling. In recent years fluorescence, chemifluorescence and chemiluminescence have emerged as alternative technologies to traditional radioisotope-based systems. Convenience, speed and safety are strong arguments for non-radioactive labelling techniques, but use of radioisotopes may still offer significant advantages, i.e. because the insertion of a radioisotope does not change the structure of a molecule. In both application forms, the radioisotopes or the chromophores are directly embedded in the analyte or in the ligand, which exerts high affinity and high specificity for the analyte. The scintillation proximity assay (SPA) and fluorescence resonance energy transfer (FRET)-based assay exemplify the application of both techniques in high-throughput drug screening strategies (Clegg 1995; Woodbury and Venton 1999).

A further method is the surface plasmon resonance (SPR)-based interaction analysis technique. SPR is independent of any labelling. However, low molecular weight compounds can only be detected to a certain limit because they do not create strong enough signals (Szabo et al. 1995; Woodbury and Venton 1999).

Other techniques include chromatographic and electrophoretic methods, capillary electrophoresis (CE) and mass and microscope spectroscopy (Altria and Elder 2004; Lottspeich and Zorbos 1998; Woodbury and Venton 1999).

Most recently, the microarray technology has become a crucial tool for large-scale and high-throughput analysis (Glokler and Angenendt 2003; Walter et al. 2002; Zhu and Snyder 2003), its greatest advantage being the considerable reduction of sample consummation and the possibility to screen in parallel.

Using antibodies, rapid and simple immunoassays have been widely used for the detection of macromolecules like proteins, the most common format being the enzyme-linked immunosorbent assay (ELISA), performed as a sandwich assay. However, as the analyte becomes smaller, it is sterically impossible to be bound by two antibodies simultaneously. Also, small haptens often escape the immune system and specific antibodies are difficult to obtain via immunisation strategies. Technologies like phage display and ribosome display have been developed to overcome these difficulties, but there still is a constant search for a proper scaffold to recognise small molecules in a specific manner (Cicortas Gunnarsson et al. 2004; Vogt and Skerra 2004).

A competitive assay as described by us (see below and E. Ehrentreich-Forster, D. Orgel, A. Krause-Griep et al., submitted for publication) using aptamers can, however, detect a small molecule or a low molecular weight compound with high sensitivity and specificity. In recent years many aptamers as well as "aptazymes" (a combination of aptamer and ribozyme) have been developed as an alternative for small molecule detection (Burgstaller et al. 2002; Famulok 1999). Among those, aptamers against small molecules have been identified ranging from metabolic cofactors like Flavine adenine dinucleotide (FAD) (Clark and Remcho 2003; Roychowdhury-Saha et al. 2002), biotin (Nix et al. 2000; Wilson et al. 1998), vitamin B12 (Lorsch and Szostak 1994; Sussman et al. 1999) and elicitors like 3/-5/-cyclic adenosine monophosphate (cAMP) (Nonin-Lecomte et al. 2001; Koizumi and Breaker 2000) to toxins and drugs like antibiotics (Gold et al. 1995; Famulok 1999; Wilson and Szostak 1999).

We experienced that aptamers developed against a low molecular weight compound alone will hardly recognise the conjugated compound on a protein surface. We assume that the conjugated compound is probably buried or not fully accessible in its three-dimensional structure for aptamer binding, due to protein side chains or its being masked by the protein's immanent charges. To render aptamers capable of recognising a low molecular weight compound in a variety of milieus, it is necessary to develop the desired aptamers through varying steps and surroundings according to the anticipated chemical conditions and platforms.

An example of an aptamer developed against a low molecular weight compound is an aptamer that has been generated by us in co-operation with the Fraunhofer Institute for Biomedical Engineering (IBMT). We developed aptamers against the toxic agent trinitrotoluene (TNT) (E. Ehrentreich-Forster, D. Orgel, A. Krause-Griep et al., submitted for publication; Rimmele 2003; Rim-

mele and Ehrentreich-Förster 2004). This work opens a new field of aptamer applications for environmental analytics and chemical-process controlling using a biosensor approach. TNT and especially its degradation products are very toxic and can be recognised with antibodies only with low affinity (Kd value estimated in the millimolar range). Aptamers developed against TNT have a much higher affinity and specificity than antibodies, and they could be administered in a portable biosensor system (Fig. 1). A reason for the significantly higher affinity of aptamers to TNT in the sensor could be the highly flexible three-dimensional structure of nucleic acids (Hermann and Patel 2000; Rimmele 2003). The presented system also clearly demonstrates a further advantage of aptamers compared to proteinaceous antibody molecules. Aptamers are compatible with organic solvents that are needed for the solubilisation and detection of organic molecules like TNT (Baldrich et al. 2004; O'Sullivan 2001; Rimmele 2003). Our aptamers against TNT could be developed in buffers containing considerable amounts of methanol.

In the established biosensor assay, aptamers with high affinity have a high specificity for TNT, since the structurally similar explosive N-methyl-N-2,4,6-tetranitroaniline (Tetryl) displays no affinity to the TNT aptamers (data not shown).

The principle of measurement of the fibre-optic field biosensor is an indirect competitive assay. A fluorescence signal is detected with a photomultiplier tube (PMT). A scheme of the experimental set-up of the biosensor is shown in Fig. 2. The analyte TNT is covalently bound to the previously activated surface (glass fibre) of the measuring cell. The aptamers coupled with fluorescence beads and

Fig. 1 Overlay plot of real-time binding curves of aptamer versus antibody in the fibre-optic field sensor. The toxin (trinitrotoluene) was immobilised on a sensory glass fibre. Fluorescence-labelled aptamer or fluorescence-labelled antibody is pumped into the measuring cell and binds to the toxin on the fibre. Binding is shown as fluorescence intensity. The different curve progression mirrors a significant difference in toxin affinity of aptamer versus antibody. Binding measurements were performed by Eva Ehrentreich-Förster at the IBMT

Fig. 1 Overlay plot of real-time binding curves of aptamer versus antibody in the fibre-optic field sensor. The toxin (trinitrotoluene) was immobilised on a sensory glass fibre. Fluorescence-labelled aptamer or fluorescence-labelled antibody is pumped into the measuring cell and binds to the toxin on the fibre. Binding is shown as fluorescence intensity. The different curve progression mirrors a significant difference in toxin affinity of aptamer versus antibody. Binding measurements were performed by Eva Ehrentreich-Förster at the IBMT

Fig. 2 Instrumental set-up of the fibre-optic field biosensor. The sample is pumped through the measuring cell with an embedded sensing glass fibre. The excitation light passes through an interference filter and is guided by a fibre bundle to the measuring cell. The sensory glass fibre leads the fluorescence through a filter to a photomultiplier tube (PMT). The PMT signal is collected, converted and connected to a computer for data sampling

Fig. 2 Instrumental set-up of the fibre-optic field biosensor. The sample is pumped through the measuring cell with an embedded sensing glass fibre. The excitation light passes through an interference filter and is guided by a fibre bundle to the measuring cell. The sensory glass fibre leads the fluorescence through a filter to a photomultiplier tube (PMT). The PMT signal is collected, converted and connected to a computer for data sampling the analysed probe are injected together into the measuring cell. In the absence of TNT in the analysed probe the aptamers bind to the TNT molecules coupled on the glass fibre. The result is a strong fluorescence signal on the PMT. In the presence of TNT in the analysed probe, a number of aptamer molecules binds to the free TNT molecules of the probe. This leaves fewer aptamer molecules, coupled with fluorescence beads, that can bind to the TNT coupled on the glass fibre. The result is a reduced fluorescence signal on the PMT (Fig. 3).

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