Difference between revisions of "Part:BBa K4140019"

Latest revision as of 04:32, 12 October 2022

Phenylalanine aptamer 2

Part Description

Our aptamer is a single-strand DNA with defined and stable tertiary structures that bind to target molecules (phenylalanine) with high affinity and specificity in physiological buffers and fluids and complex biological matrices. Aptamers have made it possible to detect small molecules, including neutral targets, in these environments in sensitive and selective ways.

Usage

Taking advantage of aptamers properties that have a very high recognition specificity, affinity, and stable tertiary structures that bind to target molecules (phenylalanine) we used a ssDNA aptamers in our LFA to consume the maximum normal level of phenylalanine in the blood leaving the excess phenylalanine to flow the next line test which contains E-coli with a reporter gene in response to phenylalanine that is above normal level as shown in figure 1.



Figure 1 illustrates the duplex-dissociation mechanism as the aptamer is bound by a weak bond to the capture probe (DNA3), waiting for the target (Phe) of interest to bind to it, and dissociate from the capture probe to be stabilized. Then the capture probe will be bound to its complementary structure (cDNA3) to shed the signal.

Aptamer Characterization

In this study, Three previously unknown DNA aptamer sequences which can directly actually recognise the amino acid phenylalanine were identified via solution-phase, in vitro systematic evolution of ligands by exponential enrichment (SELEX) (Figure 2C-E). Competitive fluorescence tests were used to calculate the dissociation constants. For Phe 1, Phe 2, and Phe 3, the solution dissociation constants (Kd) were 10 M, 7 M, and 16 M, respectively. When the three direct-detection phenylalanine aptamers were tested for selectivity using competitive florescence experiments, the responses to the endogenous aromatic amino acids tyrosine and tryptophan were reduced (Phe 1 and Phe 2) or undetectable (Phe 3). (Figure 2C-E). We also looked into the ability of the direct-detection phenylalanine aptamers to distinguish between two phenylalanine analogues that have the potential to cause hyperphenylalaninemia in animal models, para-chlorophenylalanine (PCPA) and paraethynylphenylalanine (PEPA) (Figure 2B) (vide infra). Contrary to Phe 1 and Phe 2, the Phe 3 aptamer demonstrated negligible reactions to PCPA or PEPA using competitive fluorescence assays (Figure 2E) (Figure 2C-D).

Figure 2.Phenylalanine aptamers solution dissociation constants and target concentration levels

Characterization by structural modeling

Characterization by mathematical modeling

In order to simulate the kinetics of aptamer binding with phenylalanine on lateral flow assay, we have used a set of ordinary differential equations (ODEs) and plotted them in order to model the binding on the lateral flow assay as shown in figure (3) and graph (1).
Figure (3) illustrates the kinetics of the aptamer binding with its target on LFA

Graph (1) represents plotting both analyte in free state (A) and aptamer analyte complex (RA) after the binding occurs between them. It’s shown that the analyte (phenylalanine) is loaded to the test strip through the sample at the beginning. (A) shows gradual decrease of analyte over the time units. On the other hand, There’s a gradual increase in the aptamer-analyte complex over the time units via binding between the aptamer with its target.This model was beneficial in the optimization of aptamer to analyte concentration that is essential in constructing the consumption line in our lateral flow assay (LFA).

Experimental validation

Optimization of the ratio of aptamer-AuNPs able to consume the normal level of phe: Colorimetric detection with aptamer-gold nanoparticle conjugates coupled to a paper strip, showing gradual change of color based on different concentrations of phenylalanine added at each drop, We started by titrating a different concentration of aptamer in 20 mg/dL phe and cDNA3. till no color changes, and the results show that at concentration 3ng of aptamer all the 20 ml/dL of phe are consumed and the excess of phe will be passed to the next line (WCB).

This figure shows colourimetric change based on the concentration of phenylalanine added at each trial, showing Red color reflecting maximum saturation of the 3ng of aptamers when phenylalanine is added at the given concentration of 20 mg/dL.

References

1. Cheung, K. M., Yang, K. A., Nakatsuka, N., Zhao, C., Ye, M., Jung, M. E., ... & Andrews, A. M. (2019). Phenylalanine monitoring via aptamer-field-effect transistor sensors. ACS sensors, 4(12), 3308-3317. Sequence and Features