Cooperativity can be used to manipulate binding affinities of DNA biosensors – improving specificity without sacrificing sensitivity; examples include tentacle probes (TPs) and cooperative primers (CPs). This thesis body of work: (1) used TPs to develop a rapid, low-cost diagnostic for detecting the point mutation leading to Navajo Neurohepatopathy (NNH) and (2) used CPs to amplify a symmetric bowtie-barcoded origami with captured t-cell receptor (TCR) α and β mRNA of a single cell.

NNH (affecting 1-in-1600 Navajo babies) is a fatal genetic disorder often caused by 149G>A mutation and is characterized by brain damage and liver disease/failure. Phoenix Children’s Hospital currently uses gene sequencing to identify the 149G>A mutation. While this process is conclusive, there are limitations, as it requires both time (3-4 weeks) and money (>$700). Ultimately, these factors create barriers that can directly impact a patient’s quality of life. Assessment of the developed TP diagnostic, using genomic DNA derived from FFPE patient liver samples, suggests nearly 100% specificity and sensitivity while reducing cost to ~$250 (including cost of labor) and providing a diagnosis within 48 hours.

TCR specificity is dependent on V(D)J recombination as well as pairing of the αβ chains. Drs. Schoettle and Blattman have developed a solution in which a bowtie-barcoded origami strand nanostructure is transfected into individual cells of a heterogeneous cell population to capture and protect αβ mRNA. When PCR of the origami template is performed with Vα, X, Vβ, and Y primers, the α and β gene segments cannot be tied back to a barcode – and paired. Assessment of the developed CPs for PCR suggests correct individual amplification using (1) Va + Xcp and (2) Vβ + Ycp primers, whereas combination of all the primers (Va, Xcp, Vb, and Ycp) suggests hybridization of the Vα + Xcp and Vβ + Ycp products due to the origami target symmetry.

Microvillus Inclusion disease is a fatal disease found in the Navajo population caused by a single nucleotide polymorphism. It is characterized by intractable diarrhea and is often fatal early in life.1 The current method of diagnosis is sending duodenal biopsies for histopathological examination and confirmatory testing through genomic sequencing. The purpose of this experiment was to create a more simple and cost-effective diagnostic method for detecting Microvillus Inclusion disease. Three methods were explored (RFLP2, ARMS3,4, and Tentacle Probes5,6) and two methods were tested to determine their ability and their efficiency in detecting the SNP that causes the disease.2 Tests using the RFLP2 method and synthetic DNA resulted in 9% false-positive rate and 11% false-negative rate in a blind trial for detecting both target (mutation present) and non-target (mutation absent) DNA when gel analyzing software was used to compare Rf values after gel electrophoresis. Using the ARMS method3, a nine-sample randomized test was run that ended up with 22% false-positive rate and 19% false-negative rate from a blind trial when using a gel analyzing software to determine presence of the SNP by band intensity. Disclaimer: No DNA from human patients was used in this study. Only synthetic DNA used.

MPV17-related hepatocerebral mitochondrial DNA depletion syndrome, previously known as Navajo Neurohepatopathy (NNH), is a rare genetic disease affecting Navajo children of the American Southwest. These children can suffer from several severe symptoms like brain damage and liver disease, and a diagnosis leads to death by age 10, on average. The only known effective therapy for NNH is a liver transplant. Currently, the disease is diagnosed through a lengthy and expensive process of gene sequencing, but oftentimes patients with the most severe forms of NNH deteriorate quickly; thus a rapid diagnostic would be beneficial to beginning the transplant process as early as possible. Here, Tentacle Probes, a novel technology to detect genetic mutations, were proposed to rapidly and accurately diagnose NNH. Because of Tentacle Probes' double binding site kinetics, they can detect mutations more accurately than other types of genetic probes. Probes specific to the NNH mutation were designed for use with a real-time polymerase chain reaction (PCR) detection platform. Initial synthetic DNA testing of Tentacle Trobes showed capable differentiation between mutated and non-mutated samples. However, experiments to validate those results at Phoenix Children's Hospital before moving to patient samples showed that test viability decreased over time. Efforts to diagnose the issues that led to decreased viability suggested four possible explanations that are as follows (in order of decreasing likelihood): first, undesired products from improper PCR primer design was supported by double bands in DNA gel electrophoresis; second, DNA may have degraded over time or due to repeated cycles of freezing and thawing stock solutions, and this was supported by smeared DNA gel electrophoresis; third, probe degradation, specifically of the fluorescent reporter, is possible; finally, contaminants that inhibit the PCR reaction may have been introduced. A combination of these factors may also have caused the change in assay viability. As a result of these most likely possibilities, new primers were designed and steps suggested to return viability to the assay. Thus, the various limitations and requirements for this Tentacle Probe diagnostic have been identified, and as assay development continues following the promising initial results achieved, we are confident that a rapid method if diagnosing NNH is on its way to help the children afflicted with this devastating disease receive timely access to treatment.