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Surface Plasmon Resonance Imagery (SPRi)

SPR is a technique that measures apparent rate constants for the interaction between an immobilised ligand and an analyte in solution in order to calculate equilibrium binding constants. The general scope and utility of this approach has been covered in a number of reviews.  It is however worthwhile here to review the basic principles behind the technique in order to understand its application to studying nucleoprotein complexes. The SPR principles used by the main commercial supplier of this technology (Biacore) are illustrated below.
SPRi technique
Light from a laser source arriving through a prism at a gold surface at the angle of total internal reflection induces a non-propagative evanescent wave that penetrates into the flow cell opposite the prism. The intensity of the reflected light is continuously monitored. At a given angle dependent upon the refractive index of the solution in the flow cell, resonance between the evanescent wave and free electrons in the gold layer results in a reduction in the intensity of reflected light. The change in angle of reduced intensity reflects changes in the refractive index (n) of the solution in the flow cell immediately adjacent to the gold layer. A dextran surface coupled to the gold layer allows immobilisation of ligands within the evanescent field.In physical terms, the detection system of a typical SPR machine consists of a monochromatic, plane-polarised light source and a photo-detector that are connected optically through a glass prism. A thin gold film (50 nm) thick, deposited on one side of the prism, is in contact with the sample solution; in the Biacore configuration this gold film may in turn be covered with a long-chain hydroxyalkanethiol, which forms a monolayer (approximately 100 nm thick) at the surface. This layer essentially serves as an attachment point for carboxymethylated dextran chains that create a hydrophilic surface to which ligands can be covalently coupled. Light incident to the back side of the metal film is totally internally reflected on to the diode-array detector. A property of this situation is that a non-propagative evanescent wave penetrates into the solution side of the prism away from the light source. Free electrons in the gold layer enter into resonance with the evanescent wave. In fact, such resonance implies that the amplitude vector characterizing a transversal wave propagating along the gold surface (image3 SPRi ) is equal to the component ( image4 SPRi) of the evanescent wave. Since e = n 2 (see above), then if w is the frequency of the wave and c the speed of light then
image8 SPRi

Furthermore, given that for the evanescent wave :
image10 SPRi

When resonance occurs,  image12 SPRi=  image14 SPRiand the intensity of the reflected light decreases at a sharply defined angle of incidence, the SPR angle, given by the simple expression :
image16 SPRi
Thus the SPR angle at which a decrease in the intensity of reflected light occurs, measures the refractive index of the solution in contact with the gold surface and is dependent on several instrumental parameters, e.g. the wavelength of the light source and the metal of the film. When these parameters are kept constant, the SPR angle shifts are dependent only on changes in refractive index of a thin layer adjacent to the metal surface. Any increase of material at the surface will cause a successive increase of the SPR angle, which is detected as a shift of the position of the light intensity minimum on the diode array. This change can be monitored over time, thus allowing changes in local concentration to be accurately followed. The SPR angle shifts obtained from different proteins in solution have been correlated to surface concentrations determined from radio-labeling techniques and found to be linear over a wide range of surface concentration. The instrument output, the resonance signal, is indicated in resonance units (RU); In the Biacore configuration, 1000 RU correspond to a 0.1° shift in the SPR angle and for an average protein this corresponds to a surface concentration change of about 1 ng/mm 2 .The machine measures refractive index changes (n) at or near a surface and relates these to changes in local concentration. This relationship is given by the Clausius Mossotti form

 image18 SPRi of the Debye equation   image20 SPRi                   

Where e is the real part of the dielectric constant or permittivity constant related to the refractive index by e = n 2, N is the number density given by N a r /M a (N a = Avogadro's number, r = the density and M a the molecular mass). It is assumed that n/C is a constant.

            In general either the DNA or the protein may be immobilized on the surface and the choice will depend on the nature of the interaction to be studied. It is however technically more expedient to immobilize DNA. Whilst a variety of techniques exist for the immobilization of DNA on the dextran surface the most   efficient for the majority of protein/DNA interactions is the use of immobilized streptavidin that can then interact with a suitably end labelled DNA molecule. Whilst it is often possible to obtain useful binding data pertaining to a specific nucleoprotein complex, it is critical to recall that SPR measurements monitor a steady state rather than equilibrium, i.e. a reaction involving two components may form an initial binary complex that then evolves' through intra-molecular interactions into a final complex having different dissociation characteristics than that of intermediate(s) present during the reaction.  In many cases now, SPR has furnished useful data concerning specific nucleoprotein complexes and even in complicated cases, such as promoter recognition by RNA polymerase, SPR can furnish useful binding data that correlates well with other footprinting techniques.

            An interesting application of this technique towards a more dynamic approach was carried out by using SPR to follow the polymerization of a reverse transcriptase along an immobilized single stranded DNA template. The resulting change in SPR signal could be closely correlated with the incremental progression of the reverse transcriptase along the immobilised DNA template as it replicated the corresponding RNA or DNA strand. This approach has also been extended to observing RNA polymerases transcribing an immobilized double stranded DNA template into an RNA molecule and again could be correlated with changes in the population of polymerases on the surface and the appearance of the RNA molecule.

We have used SPR extensively to follow interactions between macromolecules notably protein-DNA, protein-RNA and protein-protein. Recently we have begun to use an emerging technology based on SPR linked to imagery in collaboration with Genoptics-spr a biotechnology company situated at Orsay in France. For details on SPRi see http://www.genoptics-spr.com/. However, grosso modo, SPRi like SPR is based on the principle of resonance between plasma waves generated at the interface of a metal and a dielectric, and an evanescent field created from a light beam arriving through the prism at an angle of total internal reflection. This resonance effect is measured by imaging the entire reflected light from a polarized electroluminescent diode using a 12 bit CCD camera linked via a dedicated optical system. Consequently this allows analysis of an entire surface upon which discrete spots of ligand have been immobilised. A microcuvette system allows material to be flowed across the surface and the SPR response at each spot can be assessed in parallel by time resolved CCD. Analysis of changes in pixel density at each spot can then be carried out as a function of time thus providing information of the kinetics of the interaction at the surface.
SPRi imagery
This technique allows immobilisation on a flat surface and we have used this to create extravidin surfaces to which we can graft DNA molecules.
SPRi imagery 3
An estimation of the apparent density of material ( G in pg/mm 2) on the surface may be obtained from the expression
image24 SPRi

Where delta R is the variation of reflectivity (expressed as %); the variation of reflectivity per unit of variation of refracted index (S P,R) is calibrated for the apparatus used, the penetration depth of the evanescent wave in the medium immediately above the gold layer is L (zc)  and the dependence of the variation in refractive index as a function of concentration ( d n/ d C) for proteins and nucleic acids is a constant.
In a typical experiment a surface containing DNA is exposed to a flow of protein that will initially bind to the DNA then following injection slowly dissociate. In the film below you can follow as a protein binds then dissociates from immobilised DNA fragments on a surface. We can thus access kinetics and the stoichiometry for this interaction.
Image film SPRi