Title

Thermodynamic Design Optimization of DNA-Based Chemical Reaction Networks with Shielded Internal Toeholds

Document Type

Student Presentation

Presentation Date

4-21-2014

Faculty Sponsor

Elton Graugnard

Abstract

Promising biomarkers for use in early-stage disease detection are microRNAs (miRNAs), small non-coding strands of ribonucleic acid ~20 nucleotides (nt) in length. Specific miRNAs found in the blood have been linked to a variety of diseases, including a number of types of cancer. One barrier to using miRNAs for diagnosis is their low concentrations in the blood. Currently miRNA detection is performed with techniques such as quantitative reverse-transcription polymerase chain reaction (qRT-PCR), which is complex and time-intensive. Amplification via DNA-based catalytic reaction networks provides a potential alternative to qRT-PCR for detecting small concentrations of miRNA. A translator network was designed using synthetic DNA to convert a target miRNA input into a DNA output strand of arbitrary sequence, which triggers a separate amplifier module. This network consists of a three-stranded substrate complex, and a single-stranded fuel. The substrate incorporates a unique design of the miRNA binding site – a “shielded” internal toehold, which is designed to allow only the target miRNA sequence to bind. Sequences for the shield strand were generated using thermodynamic modeling software for various toehold lengths. These sequence designs were tested against a series of performance criteria important for proper functionality of the translator network. Select sequences for each toehold length were tested experimentally. The shielded internal toehold was also compared to toeholds of equivalent length located at the end of the substrate, which is a more common design currently used in many DNA reaction networks. Preliminary results indicate a trend of increasing network performance as the length of the shielded toehold increases.

Acknowledgments: This project was supported in part by the: (1) W.M. Keck Foundation Award, (2) NIH Grant No. K25GM093233 from the National Institute of General Medical Sciences, (3) NIH Grant No. P20 RR016454 from the INBRE Program of the National Center for Research Resources. We also thank the students and staff within the Nanoscale Materials & Device Research Group (nano.boisestate.edu).

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