The hypothesis of this research is that hairpin polyamide-fluorescence resonance energy transfer (FRET) conjugates can be used to provide a high-throughput method for gene detection and their viability proven when used to signal a genetic predisposition for Crohn’s disease.
The polymerase chain reaction (PCR) is currently the standard technique used for DNA amplification, a crucial step in gene detection. Yet, despite its widespread use, PCR has inherent disadvantages. PCR involves numerous time-dependent steps in DNA amplification, where errors can occur, and it only amplifies the DNA sample. In fact, the presence of the gene of interest is only determined following tedious analysis of the PCR products.
A chemical-based approach to gene detection offers the potential to avoid the time-consuming aspects of DNA amplification, errors in associated PCR amplification of DNA, and the analysis of PCR products.
This approach requires two essential components–the ability to recognize and bind a DNA sequence of interest (with high specificity and affinity) and to generate an observable signal upon binding. The recognition of a specific DNA sequence can be achieved with the use of heterocyclic rings, covalently bound by repeating amide bonds, forming crescent-shaped molecules known as hairpin polyamides. These polyamides are structured to bind to the minor groove of DNA and can be engineered to exhibit high specificity and affinity for specific DNA sequences. To facilitate chemical signaling, Förster (fluorescence) energy transfer (FRET) is used. FRET is a spectroscopic phenomenon that occurs when a relaxing donor fluorophore transfers its energy to an acceptor fluorophore. In biological systems, FRET has been employed to investigate DNA structure and DNA-drug/protein interactions.
To establish hairpin polyamide-FRET conjugates (HPFCs) as a viable means for gene detection, we seek to target the clbR gene, a gene that is implicated in Crohn’s disease. The clbR gene operator is located within the clb gene cluster and has been recently identified as the key transcriptional activator responsible for controlling the production of colibactin, a molecule linked to inflammation in Crohn's disease patients. It can be inferred that if this 32-base pair sequence within the clbR gene operon is detected, the patient possesses a genetic predisposition for Crohn’s disease.

Carson Shivers

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