Team:Queens Canada/Approaches

Approaches

We have taken two approaches to the development of biological sensors, or biosensors, for the measurement of Cortisol: Firstly, we have constructed a reagentless, and continuous glucocorticoid sensor which utilizes changes in Fluorescence Resonance Energy Transfer (FRET) to detect hormones. Secondly, we have begun developing a novel, and portable biological sensor which utilizes intein splicing to produce a luminescent signal, for hormone quantification. We will explore both of these methods in depths, as well as their advantages and disadvantages.

Approach 1: Fluorescence Resonance Energy Transfer

Fluorescence Resonance Energy Transfer (FRET), is a mechanism of energy transfer between light-sensitive molecules, such as fluorescent proteins. Fluorescent proteins work by absorbing light at a peak wavelight, called the excitation wavelength, and emitting light at a higher wavelength, called the emission wavelength. When two fluorescent proteins are within close proximity of each other, it is possible to excite the first fluorescent protein at its excitation wavelength, and produce emission of the wavelength from the second fluorescent protein. For example, the excitation of Cyan Fluorescent Protein is 436 nm, and its emission is 488 nm, while the excitation of Yellow Fluorescent Protein is 517 nm and its Emission is 528 nm. If Cyan Fluorescent Protein, and Yellow Fluorescent Protein are in close proximity, excitation at 436 nm will result in emissions at both 488 nm and 528 nm. The efficiency of this energy transfer process is dependent on the proximity of the two fluorophores, and can therefore be used to quantify minor changes in the structure and activity of proteins.

Our FRET Biosensor

red-green FRET Pairing FRET FRET F R E T

Approach 2: Inteins

Found in organisms from all domains, inteins (intervening proteins) are auto-processing proteins that function both in endogenous and exogenous contexts [3]. These proteins are involved in the cleavage and formation of peptide bonds during a unique process where they excise themselves from a polypeptide and ligate the flanking extein (external protein) [3]. This spontaneous splicing process occurs post-translationally and is most commonly observed in proteins involved in DNA transcription, replication and maintenance processes within a cell [3]. Inteins have such great potential in protein engineering because of their rapidness, the induced splicing and joining of the exteins to create a functional extein protein is much quicker than the typical transcription and translation process.

Small Molecule Triggered Intein Splicing

The mycobacterium tuberculosis RecA intein was selected for use as it has been shown to splice in a wide variety of protein contexts [6]. Small molecule triggered intein splicing allows the production of a “molecular switch” which is only activated in the presence of the designated ligand. To function, this system requires that the intein is able to bind with a high affinity to its specific ligand, and that the resulting conformational change initiates the process of protein splicing [6]. For the initial application of our technology, we will be using the binding of cortisol to the human glucocorticoid receptor as the initiating reaction that triggers intein splicing.

Split NanoLuc Luciferase Domain

Upon producing cortisol-dependent intein splicing, we can then swap out the flanking extein sequence with a quantitative reporter. We therefore chose NanoLuc® Luciferase as the extein and quantitative reporter for our construct. NanoLuc® by Promega is a luciferase derived via directed evolution from the luminous shrimp, Oplophorus gracilirostris [4]. The enzyme was obtained from deep-sea shrimp and optimized following the discovery of a novel substrate, furimazine, which allows for the production of visible light with less background activity than other luciferases [4,5]. NanoLuc® is a 19.1 kDa monomeric protein that is both soluble and ATP-independent [4]. Compared to firefly (Lampyridae) and sea pansy (Renilla) luciferases, this novel protein offers many advantages reflected by its increased stability, smaller size, and >150-fold increase in luminescence [5]. The unique characteristics of this enzyme construct combined with its high luminescence activity allow for the production of a very sensitive diagnostic assay. The split site for NanoLuc® Luciferase was chosen between the amino acids 52 and 53, as this split was previously determined to produce the highest luminescence upon successful rejoining of the N and C termini [7].

Comparing and Constrasting the two Biosensor

FRET - reagentless, continuous, small, but not portable, requires a fluorometer or a confocal laser imaging microscope. Intein - Small, portable, but irreversible reaction, and requires a luciferin substrate to produce a signal

Application of the Intein Biosensor in a Diagnostic Pacifier

The intein biosensor design would work through our engineered protein construct binding to salivary analyte, cortisol, at its glucocorticoid binding domain, splicing together the halves of the split NanoLuc, producing luminescnece. The resulting luminescent signal would be detected by our specialized built-in luminometer and the resulting signal would then be transmitted to a smartphone application. The ability to easily quantify and detect changes in cortisol can reveal critical health information.