Surface-Initiated Enzymatic Polymerization of DNA Nanostrutcures for Highly Amplified Sensing

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The overall goal of the proposed research is to use an enzyme ?terminal deoxynucleotidyl transferase (TdT)? to grow long single stranded (ss) DNA chains from a surface that incorporate a diverse range of unnatural ?chemically or optically functional nucleotides? in the growing DNA chain; we call this technology surface initiated enzymatic polymerization (SIEP) of DNA. The outcome of this research is that it will provide a new and versatile, on-chip signal detection and amplification scheme that is broadly applicable to on-chip sensors, heterogeneous immunoassays, protein and DNA microarrays, and for the detection of microRNA. To fulfill the goal of the proposed research, a systematic, mechanistic investigation and optimization of SIEP of linear and branched DNA structures will be carried out with a set of natural and unnatural nucleotides that embed chemically reactive nucleotides, or optically or electrochemically reactive detection moieties into the polymerized DNA chains. The intellectual merit of the research is that it will yield rationally designed, DNA-based nanostructures that can be employed in versatile and multiplexed molecular detection schemes. The technically simple and experimentally convenient approaches for signal amplification developed herein will: (1) allow analysis of dilute or weakly binding samples and thus increase array sensitivity; (2) allow reduction of the feature size without sacrificing signal intensity, which would enable more information to be encoded on a single substrate; and (3) provide robust strategies for signal detection for point-of-care diagnostics. The proposed research is transformative because of its broad utility, as it will lead to the development of a novel and versatile detection and amplification platform technology that is broadly applicable to a broad range of on-chip sensors, heterogeneous immunoassays, protein and DNA microarrays, and for the emerging field of microRNA detection. The broader impact of this research in terms of education and outreach is that: (1) it will engage two PhD students, a Duke undergraduate student (through the Pratt fellows program) and a hearing-impaired student (through an existing REU program) to engage in team science; (2) it will target 4th-6th grade female students from Durham public schools through the FEMMES program at Duke University, by providing hands-on laboratory experiences to foster their excitement and understanding of science and engineering; (3) it will engage highly motivated, selected senior high school students in the project directly through the mentorship program of the North Carolina School of Science and Mathematics (NCSSM) in Durham.