This year our team is developing a novel biosensor which is designed on two integral approaches and can shed signals responding to the concentration of phenylalanine (Phe).

How does our Lateral flow assay work?

First, the sample passes thorugh the Jury Line, which consumes the normal levels of phenylalanine. Then, it moves on to the Fugitive Line, which captures the excess levels of phenylalanine, producing blue color.

Our Sensor consists of 3 parts:

(i) the signal input:
-based on an Aptamer conjugate AuNPs detector fixed on the control line corresponding to the normal concentration of phenylalanine in blood. -DNA binding protein TyrR sensing the excess level of phenylalanine in the WCB on the test line.

(ii) the regulatory:
consisting of signal input module-dependent parts that regulate the downstream expression (ParoF and L7ae).

(iii) the signal output (detector):
a reporter gene (LacZ) that is used for the quantification of the signal.

1-Aptamer:

Aptamers are single stranded DNA or RNA oligonucleotides as shown in figure 1, that possess functional properties arising from their ability to bind to target molecules with high affinity. As they have a very high recognition specificity, they can effectively replace antibodies, minimizing undesired immune responses, and are therefore useful in a great range of domains, such as biosensing and therapeutics. Aptamers are also naturally found in the triggering process of riboswitches, where the DNA or RNA sequence is generated by SELEX-probing against the molecule of interest.

Figure 1 illustrates the structure of the ssDNA aptamer and how it changes 3D configuration once it binds to its target.

-What is the duplex dissociation mechanism ? and why do we use it ?

The duplex-dissociation mechanism (Figure 2) maintains a rapid, sensitive sensor for small molecule detection. It works through the unique ability of nucleic acids to form Watson-Crick-Franklin base pairs, forming a 3D configuration only with the target analytes of interest by high affinity and specificity. which uses a short oligonucleotide "complementary strand" that is hybridized to the aptamer and can be displaced by the conformation change induced upon the introduction of a ligand. A signal response can be produced by dissociating the short complementary strand.

Figure 2 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.

-The Aptamer part of biosensor:

Our sensing consumption line (Jury line) this year is based on a duplex dissociation mechanism through a competitive binding (figure 3).
This approach consumes the normal level of phenylalanine in blood which is between 2-20 milligrams per deciliter (mg/dL), and allows the excess concentration to flow to the test line.

Figure 3 illustrates our Jury line that consumes the normal concentration of Phe depending on the duplex-dissociation mechanism by selecting the best aptamer for our target by directed evolution method.

-A quick view to our approach:

A capture aptamer strand (DNA3) attached to Gold nanoparticles AuNPs by a linker sequence 20T (DNA1), to which DNA3 can attach by binding to the free region of the aptamer sequence forming a sensor probe with tunable thermodynamic stability (Figure 4).

Figure 4 illustrates the thermodynamic stability of the aptamer in bound and unbound states.

And this is how our sensor probe was designed.
As shown in (figure 5). In the absence of the target, the two strands are hybridized (DNA3 capture probe and the aptamer), while in the presence of the target there is a conformational change of the aptamer sequence leading to dissociation from the capture probe(DNA3), so the capture probe (DNA3) will be unbound and waiting to bind to a sequence complementary to it immobilized on the lateral flow assay (LFA). Accordingly when the sample contains phenylalanine it will interact with the aptamer and dissociate from the capture probe(DNA3), then the capture probe (DNA3) will hybridize to its complementary strand on the LFA and a red band will be produced.

Figure 5. illustrates the duplex-dissociation mechanism on the consumption line (Jury line).

Sequences

name	sequence	bp
Aptamer	5’ GGA CGC TAA TCT TAC AAG GGC GTA GTG TAT 3’	30
Capture probe = DNA3	5’ TT TTT TTT TTT TTT TTT TTT AAG ATT AGC GTC C 3’	33
cDNA3	5’ GG ACG CTA ATC TTA A 3’	15
Control	5’ AAA AAA AAA AAA AAA AAA AA 3’	20

Table 1 shows the sequences aptamer, capture probe and its complementary.

-Lateral flow assay results:

To double-check that the test is as convenient as realizable. We made a specific design for our LFA to be user-friendly through adding the sample onto sample pad and waiting for the window (figure 1) to represent the result, and also to make sure that the design eco-friendly we used a recyclable materials also protecting the reagents and cells on test strip from environment for future implementations.

Figure 6 illustrates different results in our LFA. 1- positive if all the lines are present. 2- negative if the fugitive line is absent. 3,4- invalid and must be repeated if the control line is absent.

-Whole cell biosensor :

Another step in designing our diagnostic tool for PKU is adding a whole cell biosensor that senses Phenylalanine (Phe) and Tyrosine (Tyr), Figure 6,7.

TyrR-Regulon:

-TyrR acts as an activator in its dimer form and as a suppressor in its hexamer form. And a regulator protein, binds to two distinct operons known as the weak and strong TyrR boxes to control the activity of seven promoters, two of which are the paroF and TyrP promoters.
-When phenylalanine is present, it exerts its excitatory function by forming a dimer structure and attaching to the strong box, which solely serves to increase RNAP activity by interacting with the alpha subunit (alphaCTD) of RNA polymerase (RNAP).
-But the other. TyrR changes to a hexamer shape when tyrosine is present and binds to both the weak and strong boxes simultaneously to change the activity of RNAP and suppress the production of L7Ae and PAH.
-As we need it to sense extracellular Phenylalanine and Tyrosine, that’s why we designed a parallel circuit that produces Permease, an enzyme that makes the cell membrane permeable to Phe, Tyr, and the linker KS-PS allow extracellular transport of the produced B-Galactosidase.

This figure illustrate the role of TyrR in regulating the activity of paroF and TyrP promoter to control the expression of L7Ae and PAH.

In the presence of Phe, the TyrR turns on each Parof and TyrP, resulting in transcription of B-galactosidase enzyme that makes use of X-gal that produces blue color indicating the presence of Phe.

Figure 7 illustrates our whole cell biosensor design and how it works. As it sense the Phe or Tyr by relation between the TyrR and PtyrP regulated by the input (Phe), resulting in turning on the reporter gene (lacZ), or a relation between Parof and PtyrP regulated by Tyr resulting in turning off LacZ gene.

Plasmid Vector/Backbone: pUC19 Restriction Sites: Pst1 & Ecor1 Diagnostic Part Inserted/Cloned: part(1) T7P - TyrR RBS - TyrR - ParoF promoter - P2A - L7Ae - TyrPromoter - KpSp signal - LacZ Alpha

Plasmid Vector/Backbone: pUC19 Restriction Sites: Pst1 & Ecor1 Diagnostic Part Inserted/Cloned: part(2) Lac Promoter - Permease

Plasmid Vector/Backbone: pUC19 Restriction Sites: Ecor1 & Ecor1 Diagnostic Part Inserted/Cloned: part(3) Human u6 Promoter - guidescaffold - cRNA -Kinkturn - CMV Promoter - Cas12g

-In the presence of tyrosine (figure 8), TyrR suppresses parof, which in turn cuts the circuit at this point,
inhibiting the transcription of B-galactosidase and no blue color is produced.

Figure 8 illustrates the whole cell biosensor design and how it works. during sensing the Tyr by relation between Parof and PtyrP regulated by the input (Tyr) resulting in turning off LacZ gene

-The reporter gene:

The lacZ gene encodes beta-galactosidase, which catalyzes the cleavage of lactose to form galactose and glucose that utilizes X-gal to change the color to blue.

-decrease the background noise:

-background noise refer to the leakage of the system or presence of output signal to non input which is need to be suppressed, we found 2 way to ensure that the noise at low level through (figure 9):
-CRISPR is the regulatory system for our biosensor which is designed to interfere with the over expression of the reporter gene.
-And also to ensure that the system is working in the presence of phenylalanine we must remove or inhibit the effect of the CRISPR by regulating it by riboswitch L7Ae.

In our mission to create a suitable solution to PKU patients, we put all our thoughts on one of the roots of the problem “ the deficiency of the phenylalanine hydroxylase (PAH) enzyme“ (figure 10). This enzyme is responsible for the processing of phenylalanine, which is responsible for the conversion of phenylalanine to another amino acid, tyrosine.

We are developing a tunable therapeutic approach to give the body the ability to produce phenylalanine hydroxylase (PAH) by implementing our designed therapeutic circuit in a cell-based system that produces PAH according to phenylalanine and tyrosine levels.

And to increase the safety of our project we added a CRISPR system that regulates the production of PAH.

In the presence of phenylalanine (Phe), the TyrR will activate tyrP, accordingly tyrP will enhance the production of PAH gene. While the inhibitory effect of the ParoF will not be initiated, thus L7Ae will be expressed, preventing the expression of the dcas12g.

In the presence of tyrosine (Tyr), the TyrR will inhibit ParoF, therefore L7Ae will not be expressed. This will lead to free expression of the dcas12g to prevent the synthesis of the PAH enzyme. At the same time, tyrP will not be activated and PAH will not be expressed by its promoter.

Plasmid Vector/Backbone: pCDNA3.1(+) Restriction Sites: Pst1 & Ecor1 Therapeutic Part Inserted/Cloned: part(1) T7P - TyrR RBS - TyrR - TyrPromoter

Plasmid Vector/Backbone: pCDNA3.1(+) Restriction Sites: Bgl2 & Pst1 Therapeutic Part Inserted/Cloned: part(3) ParoF promoter - P2A - L7Ae

Plasmid Vector/Backbone: pCDNA3.1(+) Restriction Sites: Eco1 & Bgl2 Therapeutic Part Inserted/Cloned: part(5) KpSp-PAH

Plasmid Vector/Backbone: pCDNA3.1(+) Restriction Sites: Pst1 & Pst1 Therapeutic Part Inserted/Cloned: part(4) Lac promoter - Permease

Plasmid Vector/Backbone: pCDNA3.1(+) Restriction Sites: Bgl2 & Bgl2 Therapeutic Part Inserted/Cloned: part(2) Human u6 Promoter - guidescaffold - cRNA -Kinkturn - CMV Promoter - Cas12g

In order to achieve our goal after the detection of the excess Phe, and to give the patients the best care that improves their quality of life we take the advantage of the novel platform “Selective Endogenous eNcapsidation for cellular Delivery (SEND)”, which is composed of targeted gene delivery that are produced naturally in the body without triggering any adverse effects or initiate any immune response. This promising gene delivery system (SEND) will enable the human liver cells / hepatocytes to produce the deficient phenylalanine hydroxylase (PAH) enzyme.

And the easiest way to explain it is with this figure .

Here you can see that there are three main systems constituting a tailored send.

-First Component :

The first thing is PEG10 which is a common type of retroelement called a long-terminal repeat retrotransposons and this is the protein that forms the final virus-like particles that binds to its own RNA sequence and form capsids for the RNA cargo that can be used as a way to transfer RNA from one cell to another.

-Second Component :

In our case, PAH resembles the cargo RNA and we call it cargo because it's going to be carried in the PEG10 capsid. Knowing that PEG10 binds to its own RNA, Cargo RNA is flanked with 5’ and 3’ UTR of MmPeg10 and those segments are added to the RNA of choice that makes up your cargo RNA (PAH).

-Third Component :

The last thing that's needed called fusogen, the vesicular stomatitis virus envelope protein (VSVg) and this is to allow the MmPEG10 VLPs pseudotyped with VSVg-fusogen to be secreted within the envelope protein (EVs) that mediate transfer of the cargo RNA to the target cells through specific-cellular tropism mechanism.

and this is achieved by engineering PEG10 which, integrated with our therapeutic system, will enable the human cells to package, secrete, and deliver “PAH RNAs” ,not protein, to hepatocyte cells without interfering with the child’s DNA.

This approach is considered as an efficient and specific therapeutic delivery system, based on the fact that Eukaryotic genomes contain domesticated genes that are originally introduced to human cells from integrating certain RNA parts of viruses (known as Gag). Gag are long terminal repeat (LTR) retrotransposons that form virus-like particles (VLPs). One of the LTR retrotransposon homologs is PEG10, that preferentially binds and facilitates vesicular secretion of its own mRNA.

We are reprogramming MmPEG10 to bind and package the cargo mRNA consisting of both the 5’ and 3’ UTR of MmPeg10 flanking our gene of interest which is phenylalanine hydroxylase (PAH).