Ligands for streptavidin

Weber, P.C, Pantoliano, M.W., Simons, D.M., and Salemme, F.R. (1994) Structure-based design of synthetic azobenzene ligands for streptavidin. Journal of the American Chemical Society, 116,2717-2724. 

Both the AFM rupture experiments as well as our simulation studies focussed on the streptavidin-biotin complex as a model system for specific ligand binding. Streptavidin is a particularly well-studied protein and binds its ligand biotin with high affinity and specificity [51]. Whereas previous experiments (see references in Ref. [49]) and simulation studies [52] referred only to bound/unbound states and the associated kinetics, the recent AFM... 

Figure 5.11 illustrates the basic performance of the on-line MS assay. For comparison, a homogenous fluorescence assay has been set up in parallel. For this purpose, the carrier flow was split after the second microcoU reactor, with 90% of the total flow being directed to a fluorescence detector (Fig. 5.11a) and 10% to the MS (Fig. 5.11b). The affinity interaction between streptavidin and biotin was chosen to study the characteristics of an on-line MS biochemical assay. Fluorescein-biotin was used as reporter ligand for both fluorescence and MS in the SIM mode (m/z 390) detection. In the fluorescence mode, the homogeneous biochemical assay is based on the quenching of the fluorescein-biotin fluorescence upon binding to streptavidin.

FarreraJ-A, Hidalgo-Femandez P, HanninkJM, et al. Divalent ligand for intramolecular complex formation to streptavidin. Org Biomol Chem. 2005 3 2393-2395. 

In an extensive SFA study of protein receptor-ligand interactions, Leckband and co-workers [114] showed the importance of electrostatic, dispersion, steric, and hydrophobic forces in mediating the strong streptavidin-biotin interaction. Israelachvili and co-workers [66, 115] have measured the Hamaker constant for the dispersion interaction between phospholipid bilayers and find A = 7.5 1.5 X 10 erg in water. 

Streptavidin-single-stranded DNA covalent conjugates were described as the building blocks for assembling nanostructured scaffolds [31], The amount and type of biotinylated ligands were used to modulate the affinity of duplex formation between solid-phase-bound nucleic acid templates and DNA-streptavidin conjugates. This system has been proposed for the design of fine-tuned sequence detection systems. 

Also recently, Liao and collaborators [89] proposed a homogeneous noncompetitive assay of a protein in biological samples based on FRET by using its tryptophan residues as intrinsic donors and its specific fluorescent ligand as the FRET acceptor, which was defined as an analytical FRET probe. To evaluate this method, a naphthylamine derivative, namely /V-biotinyl-/V -(l -naphthylj-ethylene-diamine (BNEDA) 33 was used as an analytical FRET probe for the homogeneous noncompetitive assay of streptavidin. 

The biochemical MS assay performance was studied for various biotin derivatives, such as biotin [m/z 245), N-biotinyl-6-aminocaproic acid hydrazide (m/z 372), biotin-hydrazide (m/z 259), N-biotinyl-L-lysine (m/z 373) and biotin-N-succinimi-dylester m/z 342). These five different bioactive compounds were consecutively injected into the biochemical MS assay. Figure 5.12 shows triplicate injections in the biochemical MS-based system of the different active compounds. Each compound binds to streptavidin, hence the MS responses of peaks of the reporter ligand (fluorescein-biotin, m/z 390) are similar. The use of SIM allows specific components to be selected and monitored, e.g. protonated molecule of the biotin derivatives. In this case, no peaks were observed for biotin-N-succinimidylester (m/z 342), because under the applied conditions fragmentation occurred to m/z 245. In combination with full-scan MS measurements, the molecular mass of active compounds can be determined simultaneously to the biochemical measurement. 

The assay principle shown in Fig. 5.10 has the potential of multiplexing, i.e. performing several assays in parallel, by pumping mixtures of receptors, i.e. streptavidin and anti-digoxigenin and reporter ligands, i.e. fluorescein-biotin and digoxin [29]. Clearly this approach will only be feasible for those assays... 

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