Synthesis and application of electrochemically active oligonucleotides

Abstract

Modified oligonucleotides with redox-active functional groups could emerge as attractive tools for sensor development. In principle, changes in oligonucleotide hybridization or conformation may be read out as a change in an electrochemical signal. Monitoring this signal might allow for a direct interface between biology and electronics. This dissertation describes efforts devoted to creating redox active oligonucleotide derivatives designed to allow these application goals to be pursued. The focus is primarily on the synthesis, characterization, and application of oligonucleotides bearing on of two electroactive moieties, namely ferrocene and methylene blue. Chapter 1 provides a brief overview of electrochemically modified oligonucleotides and is designed to provide an historical perspective. Synthetic methodology, fabrication of electrode system, and current applications are introduced. Chapter 2 describes the synthesis of a ferrocene-modified oligonucleotide and its use as a multiplexing signal probe. Included in this chapter are syntheses of a ferrocene subunit bearing alkynes, as well as modified nucleoside phosphoramidites and the oligonucleotide syntheses they permit. A synopsis of electrochemical studies are also provided. Chapter 3 describes a ratiometric electrochemical DNA sensor (a so-called E-Sensor) based on the ferrocene-modified oligonucleotide described in Chapter 2 and its used in the detection of specific genes with greatly improved reproducibility. Oligonucleotide syntheses achieved through enzyme ligation, the fabrication of an E-sensor, and the results of electrochemical assays are provided in this chapter. Chapter 4 describes the design and fabrication of possible wearable devices with the modified electrochemically active oligonucleotides toward real diagnostic applications. This work is being done in collaboration with Dr. Nanshu Lu’s group in the Dept. of Aerospace Engineering and Engineering Mechanics at the University of Texas at Austin. Chapter 5 the details the synthetic procedures, provides characterization of all new products, and contains electrochemical analytical data discussed in this dissertation.