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Future Laboratory Work

After a screen with the nucleotide library for a peptide aptamer against a target the next step would be to examine features of the peptide aptamer.

The peptide aptamers affinity and specificity towards its target are two important parameters to know, especially if it is meant for use in diagnostics and treatment.

The affinity is especially important to know, if we should get more than one positive in a screen. There are several ways to test this, here among competitive Enzyme-Linked Immunosorbent Assay (ELISA). The ELISA wells will be coated with the target protein, to this we will add the peptide aptamer and the unbound peptide aptamer will be washed out. A detection molecule that binds to the peptide aptamer though anti-FLAG will be added and excess detection molecule will be washed out. An enzyme that converts the detections molecule to a color will be added. The color change or the amount of light emitted is proportional to the level of peptide aptamer bound to target. Reference: Technical Guide for ELISA. Available from KPL, Kirkegaard & Perry Laboratories (Link) Accessed 17 September 2015 Thus the amount of peptide aptamer bound to target protein, provides a measurement for the affinity. That the ELISA is competitive, means that the amount of peptide aptamer are greater than target protein, thus the peptide aptamers will have to “compete” for binding the target protein.

Figure 1: Illustrates the detector with the Sensor chip, as the aqueous solution containing the target proteins flows by (2)

Surface Plasmon Resonance (SPR) is a technology designed by Biacore and could be used to investigate the affinity and the specificity of the peptide aptamer. Central for the SPR method is the movable sensor chip. This chip monitor the interaction between the peptide aptamer and the target. The operation of the instrument, the data and the analyze of the data will be handled by a software. The sensor chip consist of a glass surface covered with a thin layer of gold, which forms a good basis to optimize the binding of a variety of molecules. To this chip the peptide aptamer would be bound. The target proteins will pass over the surface of the chip in a continuous, pulse-free and controlled flow, maintaining constant target protein concentration at the sensor chip surface. SPR measures changes in refractive index, thus senses changes in mass in the aqueous layer close to the sensor chip. Reference: An Introduction to BiaCore's SPR Technology. Available from BiaCore.com (Link) Accessed 17 September 2015 Figure 1 illustrates the principle. This method would besides providing quantitative information of the peptide aptamers affinity towards the target, also provides information of its specificity.

Since we have been working with E. coli BTH101, a β-Galactosidase Assay would also be an option for examine the affinity of the peptide aptamer towards the target. The assay exploits β –Galactosidase’s ability to recognize the synthetic compound o-nitrophenyl-β-D-galactoside (ONPG). The enzyme cleaves ONPG to galactose and o-nitrophenol, which has a yellow color. Instead of using X-gal as we have done in our previous experiments, we will in such assay use ONPG. The production of the yellow color can be used to determine the concentration of the enzyme, since when ONPG is in excess to the enzyme, the production of o-nitrophenol is proportional to the concentration of β-Galactosidase. From the assay, the miller unit can be determined. The miller unit for a known interaction in the system, e.g. our control with the Leuzine Zipper, could be used as a stand that peptide aptamers-target interaction could be compared to. Reference: Open WetWare contributors, "Beta-Galactosidase Assay (A better Miller)," Open WetWare27 August 2012, 23:20 UTC. http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller) Accessed 17 September 2015.

The peptide aptamer’s specificity could be examined with protein microarray. Many different proteins would be immoblized to the microarry chip, the peptid aptamer taged with flourecent dye though anti-FLAG, would be added. Thus the fewer proteins towards the peptid aptamer can bind, the higher specificity.