Difference between revisions of "Part:BBa K4140019"

Revision as of 13:53, 7 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 properities that has a very high recognition specificity,affinity and stable tertiary structures that binds 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 stabilised. Then the capture probe will be bound to its complementary structure (cDNA3) to shed the signal.

Literature 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 1C-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 1C-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 1B) (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 1E) (Figure 1C-D).

Figure 1.Phenylalanine aptamers solution dissociation constants and target concentration levels

Characterization by structural modeling

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