G. Lautnera, R. Gyurcsanyia, K. Mercierb, E. Ly-Morinb
(a) Budapest University of Technology and Economics, Department of Inorganic and Analytical Chemistry, Budapest, Hungary
(b) HORIBA Scientific, Palaiseau, France

Aptamers are in vitro-generated, short, single-stranded nucleic acids that selectively bind to target compounds such as small molecules or proteins1, 2. Their main advantage over monoclonal antibodies is their resistance to denaturation and degradation. Being very robust and easy to synthesize, they are becoming widely used for the development of aptamer-based biosensors in the diagnostics field3.
This application note shows that the HORIBA Scientific  SPRi platform  is suitable for the analysis of aptamer-based molecular interactions.  For this purpose, the interaction  between human IgE protein, an antibody involved in allergic reactions, and a human IgE specific  aptamer4   is presented as proof-of-concept.

Key words:
Aptamer - Aptamer, protein interactions - Human IgE - Kinetics analysis - Affinity - Surface Plasmon Resonance imaging

Material and Method

DNA aptamers

The IgE specific  DNA aptamer  sequence  (5’-GGG GCA CGT TTA TCC GTC CCT CCT AGT GGC GTG CCC C-3’) and the negative control DNA (5’-GGG GCA CGT TTA TTT TTT TTT TTT TGT GGC GTG CCC C-3’) were ordered with TTT TTT T-(CH ) –SH spacer at the 3’ end from Sigma-Aldrich.

Immobilization  of the human IgE (hIgE) aptamer on the  SPRi-Biochip™                                                                  

The thiol modified IgE aptamer and reference DNA was immobilized on the planar gold SPRi-Biochip™ sensor through thiol-Au chemisorption.The aptamer solutions were prepared at 10 µM in 20 mM PBS pH 7.4. Four replicate spots were printed simultaneously for each aptamer using the SPRi-CFM (Figure 1) during  a 15-minute  back-and-forth  cycling. The SPRi-CFM uses flow  deposition  to immobilize  48 molecules  in a single run (up to 144  spots per chip). Immobilizing with flow generates increased spot homogeneity and higher immobilization levels. After the immobilization procedure, the SPRi-Biochip™ surface was blocked with a 0.1 mM MUTEG solution  prepared  in 10 mM PBS pH 7.4. While MUTEG has good resistance against non-specific adsorption, it is sterically short enough not to hinder the specific binding event.

SPRi experiment

The spotted biochip was inserted into the SPRi-PlexII™ instrument from HORIBA Scientific.  The system  is equipped  with  a 200  µL sample loop, a continuous flow pump and an in-line degasser. The flow rate was set to 50 µL/min, and the working temperature was fixed at 25°C. Different concentrations  of hIgE prepared in a two-fold dilution in the running buffer (10 mM PBS + 1 mM MgCl ) were injected in order to determine the affinity between hIgE and its aptamer (see Table 1). The SPRi-Biochip™ surface was regenerated  using a 12 mM NaOH with 1.2 % EtOH solution after each hIgE injection.
 

	c0	c1	c2	c3	c4	c5
hlgE concentration (ng/mL)	0	63	125	250	500	1000

Table 1: Concentrations of hIgE passed over the SPRi-Biochip™ surface

Results and Discussion

Specific aptamer-hIgE interaction

Figure 2 shows a SPRi difference image obtained after the injection of 1000  ng/mL hIgE (concentration c5). Only hIgE aptamer spots become white, which confirms that the aptamer-hIgE interaction is highly specific. The SPRi difference image provides a quick confirmation of the presence of binding and its specificity.

The SPRi signals obtained on the hIgE aptamer spots after the subtraction of the negative control signal were compared for the 5 different concentrations  of hIgE (see Table 1). Figure 3 shows the calibration curve obtained; the plot was fitted with a linear model (R² = 0.9842).

Figure 3: Calibration curve obtained for the aptamer – hIgE interaction

Kinetics analysis of the aptamer-hIgE interaction

The SPRi data was then processed using ScrubberGen software in order to determine the kinetic parameters (association k
 
and dissociation krates) and the affinity (K ) of the aptamer-hIgE interaction. The data was fitted using a 1:1 interaction model (Figure 4). The affinity between the aptamer and hIgE was about 730 pM.
 
References

1. C. Tuerk and L. Gold, Science, 1990,  249,  505-510.
2. A. D. Ellington and J. W. Szostak, Nature, 1990,  346, 818-822.
3. S. D. Jayasena, Clinical Chemistry, 1999,  45, 1628-1650.
4. T. Wiegand, P. Williams, S. Dreskin, M. Jouvin, J. Kinet and D. Tasset,The Journal of Immunology, 1996, 157, 221-230.

Conclusion

This application note demonstrates  that the SPR imaging platform from HORIBA Scientific  is well  adapted  to  the  characterization  of  aptamer- protein interactions. The SPRi difference image gives meaningful information regarding the presence of binding and its specificity. The real- time monitoring of the interaction gives access to the kinetics (association and dissociation rates) and the affinity of the interaction. The multiplexing capabilities of the platform will bring very promising applications for the development of aptamer-based biochips in biomedical diagnostics.
 
Figure 4: Kinetics analysis of the aptamer – hIgE interaction using ScrubberGen