G-Quadruplex Reporters: Structural Studies and Application for Visual and Fluorescent Detection of Point Mutations in Nucleic Acid Sequences

DNA-based diagnostics traditionally utilize hybridization probes: strands of DNA complimentary to a target sequence that, upon binding, generate a signal to indicate the presence of the target. The classic hybridization probes are the molecular beacon (MB) and TaqMan probes, both single DNA strands with a fluorophore and quencher at opposing ends. Despite their widespread use in applications such as qPCR due to their ability to multiplex with a variety of bound fluorophores, these probes have several shortcomings: temperature limitations for selective target recognition and differentiating single-nucleotide polymorphisms, intrinsic design limitations to interrogate some target sequences, and a high relative cost for synthesis and purification. A split probe design in combination with label-free reporters overcomes these shortcomings. Split probes break the target-recognition sequence into two shorter pieces, each equipped with a portion of a signal reporting unit. The shorter length both allows for the probes to efficiently function at ambient temperatures, opposed to the elevated temperatures generally required for monomer probes, and provides greater ability to discriminate single-nucleotide polymorphisms. These structures will only generate a signal if complete, which may only occur when both halves of the probe are properly bound to the target. By utilizing label-free reporters such as light-up aptamers and/or (deoxy)ribozymes, split probes offer cost-efficiency advantages over other fluorescent probes. In this dissertation, advances in the usage of split probes with G-quadruplex-based signal transducing units are detailed. An alphanumeric display comprised of tandem probes utilizing the peroxidase-like deoxyribozyme for colorimetric output demonstrates the instrument-free usage of these systems. Additionally, the promiscuous activity of the dapoxyl light-up aptamer with a variety of arylmethane, offers a label-free fluorescent split probe that is capable of discerning single-nucleotide polymorphisms without expensive chemical modifications