DNA Chemical Reaction Network Design Synthesis and Compilation

The advantages of biomolecular computing include 1) the ability to interface with, monitor, and intelligently protect and maintain the functionality of living systems, 2) the ability to create computational devices with minimal energy needs and hazardous waste production during manufacture and lifecycle, 3) the ability to store large amounts of information for extremely long time periods, and 4) the ability to create computation analogous to human brain function. To realize these advantages over electronics, biomolecular computing is at a watershed moment in its evolution. Computing with entire molecules presents different challenges and requirements than computing just with electric charge. These challenges have led to ad-hoc design and programming methods with high development costs and limited device performance. At the present time, device building entails complete low-level detail immersion. We address these shortcomings by creation of a systems engineering process for building and programming DNA-based computing devices. Contributions of this thesis include numeric abstractions for nucleic acid sequence and secondary structure, and a set of algorithms which employ these abstractions. The abstractions and algorithms have been implemented into three artifacts: DNADL, a design description language; Pyxis, a molecular compiler and design toolset; and KCA, a simulation of DNA kinetics using a cellular automaton discretization. Our methods are applicable to other DNA nanotechnology constructions and may serve in the development of a full DNA computing model

In Situ SiRNA Assembly in Living Cells for Gene Therapy with MicroRNA Triggered Cascade Reactions Templated by Nucleic Acids

The in situ generation of siRNAs in living cells can greatly enhance the specificity and efficiency of gene therapy. Inspired by the natural molecular machines that organize different compartments sequentially in a limited space to facilitate cellular process, this work constructs a DNA nanomachine (DNM) by alternately hybridizing two pairs of DNA/RNA hybrids to a DNA scaffold generated by rolling circle amplification for highly efficient in situ siRNA assembly in living cells. After target cell-specific delivery of DNM, intracellular specific microRNA can work as a trigger to operate the DNM by initiating DNA cascade displacement reaction between DNA/RNA hybrids along the scaffold for continuous generation of siRNAs. Using miR-21 as a model, efficient siRNAs generation is achieved via DNA templated cascade reaction, which demonstrated impressive suppressions to VEGF mRNA and protein expressions in cells and in vivo tumor growth and indicated promising application of the designed strategy in gene therapy

Compartmentalization of DNA-Based Molecular Computing Elements Using Lipid Bilayers

This dissertation will present a progression from the detection of double-stranded DNA using a combination of toehold-mediated strand displacement and DNAzyme reactions in dilute saline solutions, to the generation of separate compartments to allow standardization of DNA computing elements, by protecting from complementary strands. In well-mixed solutions complementary regions cause spurious interactions. Importantly, these compartments also provide protection from nucleases. Along the way we will also explore the use of silica microsphere supported lipid bilayers to run compartmentalized DNA reactions on a fluid surface and the design of a molecule capable of DNA-based transmembrane signal transduction

Computational Design and Study of Structural and Dynamic Nucleic Acid Systems

abstract: DNA and RNA are generally regarded as one of the central molecules in molecular biology. Recent advancements in the field of DNA/RNA nanotechnology witnessed the success of usage of DNA/RNA as programmable molecules to construct nano-objects with predefined shapes and dynamic molecular machines for various functions. From the perspective of structural design with nucleic acid, there are basically two types of assembly method, DNA tile based assembly and DNA origami based assembly, used to construct infinite-sized crystal structures and finite-sized molecular structures. The assembled structure can be used for arrangement of other molecules or nanoparticles with the resolution of nanometers to create new type of materials. The dynamic nucleic acid machine is based on the DNA strand displacement, which allows two nucleic acid strands to hybridize with each other to displace one or more prehybridized strands in the process. Strand displacement reaction has been implemented to construct a variety of dynamic molecular systems, such as molecular computer, oscillators, in vivo devices for gene expression control. This thesis will focus on the computational design of structural and dynamic nucleic acid systems, particularly for new type of DNA structure design and high precision control of gene expression in vivo. Firstly, a new type of fundamental DNA structural motif, the layered-crossover motif, will be introduced. The layered-crossover allow non-parallel alignment of DNA helices with precisely controlled angle. By using the layered-crossover motif, the scaffold can go through the 3D framework DNA origami structures. The properties of precise angle control of the layered-crossover tiles can also be used to assemble 2D and 3D crystals. One the dynamic control part, a de-novo-designed riboregulator is developed that can recognize single nucleotide variation. The riboregulators can also be used to develop paper-based diagnostic devices.Dissertation/ThesisDoctoral Dissertation Chemistry 201

CONTRAfold: RNA secondary structure prediction without physics-based models

doi:10.1093/bioinformatics/btl24

DNA-BASED SELF-ASSEMBLY AND NANOROBOTICS: THEORY AND EXPERIMENTS

We study the following fundamental questions in DNA-based self-assembly and nanorobotics: How to control errors in self-assembly? How to construct complex nanoscale objects in simpler ways? How to transport nanoscale objects in programmable manner? Fault tolerance in self-assembly: Fault tolerant self-assembly is important for nanofab-rication and nanocomputing applications. It is desirable to design compact error-resilient schemes that do not result in the increase in the original size of the assemblies. We present a comprehensive theory of compact error-resilient schemes for algorithmic self-assembly in two and three dimensions, and discuss the limitations and capabilities of redundancy based compact error correction schemes. New and powerful self-assembly model: We develop a reversible self-assembly model in which the glue strength between two juxtaposed tiles is a function of the time they have been in neighboring positions. Under our time-dependent glue model, we can rigorously study and demonstrate catalysis and self-replication in the tile assembly. We can assemble thin rectangles of size k × N using O

Characterizing the bending and flexibility induced by bulges in DNA duplexes

Advances in DNA nanotechnology have stimulated the search for simple motifs that can be used to control the properties of DNA nanostructures. One such motif, which has been used extensively in structures such as polyhedral cages, two-dimensional arrays, and ribbons, is a bulged duplex, that is, two helical segments that connect at a bulge loop. We use a coarse-grained model of DNA to characterize such bulged duplexes. We find that this motif can adopt structures belonging to two main classes: one where the stacking of the helices at the center of the system is preserved, the geometry is roughly straight, and the bulge is on one side of the duplex and the other where the stacking at the center is broken, thus allowing this junction to act as a hinge and increasing flexibility. Small loops favor states where stacking at the center of the duplex is preserved, with loop bases either flipped out or incorporated into the duplex. Duplexes with longer loops show more of a tendency to unstack at the bulge and adopt an open structure. The unstacking probability, however, is highest for loops of intermediate lengths, when the rigidity of single-stranded DNA is significant and the loop resists compression. The properties of this basic structural motif clearly correlate with the structural behavior of certain nano-scale objects, where the enhanced flexibility associated with larger bulges has been used to tune the self-assembly product as well as the detailed geometry of the resulting nanostructures. We further demonstrate the role of bulges in determining the structure of a "Z-tile," a basic building block for nanostructures

The effect of target secondary structure on microarray data quality

DNA? microarrays? have? become? an? invaluable? high? throughput? biotechnology? method,? which? allows? a? parallel? investigation? of? thousands? of? cellular? events? in? a? single?experiment.?The?principle?behind?the?technology?is?very?simple:?fluorescently? labeled? single? stranded? target? molecules? bind? to? their? specific? probes? deposited? on? the? microarray? surface.? However,? the? microarray? data? rarely? represent? a? yes? or? no? answer? to? a? biological? community,? but? rather? provide? a? direction? for? further? investigation.? There? is? a? complicated? quantitative? relationship? between? a? detected? spot? signal? and? the? amount? of? target? present? in? the? unknown? mixture.? We? hypothesize? that? physical? characteristics? of? probe? and? target? molecules? complicate? the?binding?reaction?between?target?and?probe.?To?test?this?hypothesis,?we?designed? a? controlled? microarray? experiment? in? which? the? amount? and? stability? of? the? secondary? structure? present? in? the? probe-binding? regions? of? target? as? biophysical? properties? of? nucleic? acids? varies? in? a? known? way.? ? Based? on? computational? simulations? of? hybridization,? we? hypothesize? that? secondary? structure? formation? in? the? target? can? result? in? considerable? interference? with? the? process? of? probe-target? binding.? ? This? interference? will? have? the? effect? of? lowering? the? spot? signal? intensity.?? We? simulated? hybridization? between? probe? and? target? and? analyzed? the? simulation? data? to? predict? how? much? the? microarray? signal? is? affected? by? folding? of? the? target? molecule,? for? the? purpose? of? developing? a? new? generation? of? microarray? design? and? analysis?software.

Development of microRNA triggered therapeutic oligonucleotides and gold nanoparticle conjugates to improve specificity of RNA therapeutics

RNA-targeting oligonucleotide therapeutics and their nanoparticle conjugates hold great promise in treating intractable diseases, but their clinical applications are still limited by significant barriers including the lack of tissue or cell type specificity. Current strategy to improve tissue or cell type specificity of oligonucleotides therapeutics mainly involves conjugation with ligands. However, this strategy encounters bottleneck in diseased conditions where a specific surface marker is absent. In addition to protein markers, transcriptomic techniques have revealed complex and diverse alterations of coding and non-coding transcripts in different tissues, cell types or disease conditions, which opens up opportunities to control the activity of oligonucleotide therapeutics utilizing these endogenous transcripts to improve their specificity. The overall hypothesis of the dissertation is that using specific transcripts as triggering stimulus, oligonucleotides and their nanoparticle conjugates can be activated via toehold-mediated strand displacement reaction to conditionally regulate gene expression. As a proof-of-concept, we chose miRNA as the transcript trigger, hoping to provide a foundation for future design of smart therapeutics sensing more complicated transcript inputs. In this dissertation, we demonstrated the idea of miRNA-inducible conditional gene regulation agents with two models: (1) miR-33 triggered activation of DNAzyme-gold nanoparticle (AuNP) conjugates to down regulate tumor necrosis factor α (TNFα) in pro-inflammatory macrophages; and (2) miR-122-indicible antisense to down regulate hypoxia inducible factor 1α (HIF1α) in liver cells. In addition, to gain insights on the intracellular fate of oligonucleotide-AuNP conjugates for better design of conditional gene regulatory agents, we leveraged a powerful imaging modality, fluorescence lifetime imaging (FLIM), to monitor the intracellular integrity of oligonucleotide-AuNP conjugates. Programmable therapeutics with controllability of location, timing and intensity of their activity can lead to precise medicine with minimal side effects. We envision that the design principles for conditional oligonucleotides and their AuNP conjugates discovered from this dissertation could be adopted to a variety of translatable clinical applications and improve the controllability and safety of oligonucleotide therapeutics and nanoparticle conjugates.Ph.D