Publication Date

12-2016

Date of Final Oral Examination (Defense)

9-23-2016

Type of Culminating Activity

Dissertation

Degree Title

Doctor of Philosophy in Materials Science and Engineering

Department

Materials Science and Engineering

Supervisory Committee Chair

William L. Hughes, Ph.D.

Supervisory Committee Member

Elton Graugnard, Ph.D.

Supervisory Committee Member

Bernard Yurke, Ph.D.

Supervisory Committee Member

Peter B. Allen, Ph.D.

Abstract

Nucleic acids are information-dense, programmable polymers that can be engineered into primers, probes, molecular motors, and signal amplification circuits for computation, diagnostic, and therapeutic purposes. Signal amplification circuits increase the signal-to-noise ratio of target nucleic acids in the absence of enzymes and thermal cycling. Amplification is made possible via toehold mediated strand displacement – a process where one nucleic acid strand binds to a nucleation site on a complementary helix, which then displaces one of the two strands in a nucleic acid complex. When compared to polymerase chain reactions (PCR), the sensitivity and stability of toehold-mediated strand displacement reactions suffer from circuit leakage – reactions of the system in the absence of an initiator. Presented here, from a materials science and engineering perspective, defect engineering has improved the leakage performance of model strand displacement systems made from DNA. Engineered defects used in this study included mismatched base pairs and alternative nucleic acids – both of which are known to impact the stability of hybridization.

To identify sources of leakage in a model signal amplification circuit, availability was defined as the probability that a DNA base (A.T.C.G) was unpaired at equilibrium. This design metric was calculated using NUPACK, a thermodynamic modeling tool. To further understand the relationship between leakage rates and secondary structures, mutual availability was defined as the sum of all pairwise products of the availabilities of the corresponding bases in solution. This thermodynamic analysis yielded rational design principles for how to minimize leakage by as much as 4-fold by site-specifically introducing mismatched base pairs into DNA duplex regions. To further reduce leakage, chemically modified locked nucleic acids (LNAs) were site-specifically introduced into a model DNA strand displacement system. Briefly described, LNAs are geometrically restricted RNA analogues with enhanced thermo-mechanical stability towards their complement base. When compared to a DNA control with identical sequences, the leakage exhibited by a hybrid DNA/LNA system was reduced from 1.48 M-1s-1 (for the DNA system) to 0.03 M-1s-1. In addition, the signal-to-noise ratio increased ~50-fold for a similar hybrid system.

This research provides insight into the sources of leakage in DNA strand-displacement systems, as well as how to maximize strand-displacement performance via the selective introduction of hybridization defects. Rational design of future nucleic acid signal amplification circuits will lead to broader applications in a variety of fields that range from DNA computation to point-of-care diagnostics and therapeutics.

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