Browsing by Subject "DNA self-assembly"

The use of DNA as a polymeric building material transcends its function in biology and is exciting in bionanotechnology for applications ranging from biosensing, to diagnostics, and to targeted drug delivery. These applications are enabled by DNA’s unique structural and chemical properties, embodied as a directional polyanion that exhibits molecular recognition capabilities. Hence, the efficient and precise synthesis of high molecular weight DNA materials has become key to advance DNA bionanotechnology. Current synthesis methods largely rely on either solid phase chemical synthesis or template-dependent polymerase amplification. The inherent step-by-step fashion of solid phase synthesis limits the length of the resulting DNA to typically less than 150 nucleotides. In contrast, polymerase based enzymatic synthesis methods (e.g., polymerase chain reaction) are not limited by product length, but require a DNA template to guide the synthesis. Furthermore, advanced DNA bionanotechnology requires tailorable structural and self-assembly properties. Current synthesis methods, however, often involve multiple conjugating reactions and extensive purification steps.

The research described in this dissertation aims to develop a facile method to synthesize high molecular weight, single stranded DNA (or polynucleotide) with versatile functionalities. We exploit the ability of a template-independent DNA polymerase−terminal deoxynucleotidyl transferase (TdT) to catalyze the polymerization of 2’-deoxyribonucleoside 5’-triphosphates (dNTP, monomer) from the 3’-hydroxyl group of an oligodeoxyribonucleotide (initiator). We termed this enzymatic synthesis method: TdT catalyzed enzymatic polymerization, or TcEP.

Specifically, this dissertation is structured to address three specific research aims. With the objective to generate high molecular weight polynucleotides, Specific Aim 1 studies the reaction kinetics of TcEP by investigating the polymerization of 2’-deoxythymidine 5’-triphosphates (monomer) from the 3’-hydroxyl group of oligodeoxyribothymidine (initiator) using in situ 1H NMR and fluorescent gel electrophoresis. We found that TcEP kinetics follows the “living” chain-growth polycondensation mechanism, and like in “living” polymerizations, the molecular weight of the final product is determined by the starting molar ratio of monomer to initiator. The distribution of the molecular weight is crucially influenced by the molar ratio of initiator to TdT. We developed a reaction kinetics model that allows us to quantitatively describe the reaction and predict the molecular weight of the reaction products.

Specific Aim 2 further explores TcEP’s ability to transcend homo-polynucleotide synthesis by varying the choices of initiators and monomers. We investigated the effects of initiator length and sequence on TcEP, and found that the minimum length of an effective initiator should be 10 nucleotides and that the formation of secondary structures close to the 3’-hydroxyl group can impede the polymerization reaction. We also demonstrated TcEP’s capacity to incorporate a wide range of unnatural dNTPs into the growing chain, such as, hydrophobic fluorescent dNTP and fluoro modified dNTP. By harnessing the encoded nucleotide sequence of an initiator and the chemical diversity of monomers, TcEP enables us to introduce molecular recognition capabilities and chemical functionalities on the 5’-terminus and 3’-terminus, respectively.

Building on TcEP’s synthesis capacities, in Specific Aim 3 we invented a two-step strategy to synthesize diblock amphiphilic polynucleotides, in which the first, hydrophilic block serves as a macro-initiator for the growth of the second block, comprised of natural and/or unnatural nucleotides. By tuning the hydrophilic length, we synthesized the amphiphilic diblock polynucleotides that can self-assemble into micellar structures ranging from star-like to crew-cut morphologies. The observed self-assembly behaviors agree with predictions from dissipative particle dynamics simulations as well as scaling law for polyelectrolyte block copolymers.

In summary, we developed an enzymatic synthesis method (i.e., TcEP) that enables the facile synthesis of high molecular weight polynucleotides with low polydispersity. Although we can control the nucleotide sequence only to a limited extent, TcEP offers a method to integrate an oligodeoxyribonucleotide with specific sequence at the 5’-terminus and to incorporate functional groups along the growing chains simultaneously. Additionally, we used TcEP to synthesize amphiphilic polynucleotides that display self-assemble ability. We anticipate that our facile synthesis method will not only advance molecular biology, but also invigorate materials science and bionanotechnology.

Recently, solution-based systems for DNA computation have demonstrated the enormous potential of DNA nanosystems to do computation at the molecular-scale. These use DNA strands to encode values and use DNA hybridization reactions to perform computations. But most of these prior DNA computation systems relied on the diffusion of DNA strands to transport values during computations. During diffusion, DNA molecules randomly collide and interact in a three-dimensional fluidic space. At low concentrations and temperatures, diffusion can be quite slow and could impede the kinetics of these systems whereas at higher concentrations and temperature, unintended spurious interactions during diffusion can hinder the computations. Hence, increasing the concentration of DNA strands to speed up DNA hybridization reactions has the unfortunate side effect of increasing leaks, which are undesired hybridization reactions in the absence of input strands. Also, diffusion-based systems possess global states encoded via concentration of various species and hence exhibit only limited parallel ability.

To address these challenges, this dissertation describes a novel design for DNA computation called a localized hybridization network, where diffusion of DNA strands does not occur. Instead all of the DNA strands are localized by attaching them to an addressable substrate such as DNA nanotrack and DNA origami. This localization increases the relative concentration of the reacting DNA strands thereby speeding up the kinetics. This dissertation demonstrated a localized hybridization network that executed a chain reaction of five DNA hybridizations which executes faster than non-localized DNA reactions.

Another advantage of this approach is that each copy of the localized hybridization network operates independently of each other, allowing for a high level of parallelism. Localized hybridization networks also allow one to reuse the same DNA sequence to perform different actions at distinct location on the addressable substrate, increasing the scalability of such systems by exploiting the limited sequence space. An advantage of localized hybridization computational circuit is sharper switching behavior as information is encoded over the state of a single molecule. This also eliminates the need for thresholding as computation is performed locally eliminating the need for a global consensus.

There are many applications for localized hybridization networks. These include counting the number of disease marker molecules in a patient, detecting various cancer DNA sequences, and detecting and distinguishing bacteria by their distinguishing DNA. The results from localized DNA hybridization reactions may also be of practical use in performing surface computation on cellular membranes for disease detection and prevention.

A RET network consists of a network of photo-active molecules called chromophores that can participate in inter-molecular energy transfer called resonance energy transfer (RET). RET networks are used in a variety of applications including cryptographic devices, storage systems, light harvesting complexes, biological sensors, and molecular rulers. In this dissertation, we focus on creating a RET device called closed-diffusive exciton valve (C-DEV) in which the input to output transfer function is controlled by an external energy source, similar to a semiconductor transistor like the MOSFET. Due to their biocompatibility, molecular devices like the C-DEVs can be used to introduce computing power in biological, organic, and aqueous environments such as living cells. Furthermore, the underlying physics in RET devices are stochastic in nature, making them suitable for stochastic computing in which true random distribution generation is critical.

In order to determine a valid configuration of chromophores for the C-DEV, we developed a systematic process based on user-guided design space pruning techniques and built-in simulation tools. We show that our C-DEV is 15x better than C-DEVs designed using ad hoc methods that rely on limited data from prior experiments. We also show ways in which the C-DEV can be improved further and how different varieties of C-DEVs can be combined to form more complex logic circuits. Moreover, the systematic design process can be used to search for valid chromophore network configurations for a variety of RET applications.

We also describe a feasibility study for a technique used to control the orientation of chromophores attached to DNA. Being able to control the orientation can expand the design space for RET networks because it provides another parameter to tune their collective behavior. While results showed limited control over orientation, the analysis required the development of a mathematical model that can be used to determine the distribution of dipoles in a given sample of chromophore constructs. The model can be used to evaluate the feasibility of other potential orientation control techniques.

This dissertation hypothesizes,

"It is possible to design a self-assembled, nanoscale, high-speed, resonance energy transfer device exhibiting non-linear gain with a few molecules."

The report recognizes DNA self-assembly, a relatively inexpensive and a massively parallel fabrication process, as a strong candidate for self-assembled RET systems. It successfully investigates into the design and simulations of a novel sequential self-assembly process employed to realize the goal of creating large, scalable, fully-addressable DNA nanostructure-substrate for future molecular circuitry.

As a pre-cursor to the final device modeling various RET wire designs for interconnecting nanocircuits are presented and their modeling and simulation results are discussed. A chromophore RET system using a biomolecular sensor as a proof-of-concept argument that shows it is possible to model and characterize chromophore systems as a first step towards device modeling is also discussed.

Finally, the thesis report describes in detail the design, modeling, characterization, and fabrication of the Closed-Diffusive Exciton Valve: a self-assembled, nanoscale (area of 17.34 nm2), high-speed (3.5 ps to 6 ps) resonance energy transfer device exhibiting non-linear gain using only 10 molecules, thus confirming the hypothesis. It also recognized improvements that can be made in the future to facilitate better device operation and suggested various applications.

The manufacturing of molecular-scale computing systems requires a scalable, reliable, and economic approach to create highly interconnected, dense arrays of devices. As a candidate substrate for nanoscale logic circuits, DNA self-assembled nanostructures have the potential to fulfill these requirements. However, a number of open challenges remain, including the scalability of DNA self-assembly, long-range signal propagation, and precise patterning of functionalized components. These challenges motivate the development of theory and experimental techniques to illuminate the connections among the physical, optical, and thermodynamic properties of DNA self-assembled nanostructures.

In this thesis, three tools are developed, validated, and applied to study the thermo-mechanical properties of DNA nanostructures: 1) a method to quantitatively measure the quality of DNA grid self-assembly, 2) a spectrofluorometer capable of capturing fluorescence and absorbance data under simultaneous multi-wavelength excitation, and 3) a Monte Carlo simulator that models the ensemble response of DNA nanostructures as simple harmonic oscillators.

The broad contributions of this dissertation are as follows: 1) insight into the thermo-mechanical properties of DNA grid nanostructures, and 2) a categorization of self-assembly defects and their impact on proposed logic circuits.

The results of the work presented in this dissertation show that: 1) the quality of self-assembly of DNA grid nanostructures can be quantitatively calculated to demonstrate the impact of changes in temperature or structure, 2) the optical absorbance of complex DNA nanostructures can be modeled to capture their thermo-mechanical properties (i.e., worst case within 10% of experimental melting temperatures and 70% of experimental thermodynamic parameters), 3) the structural resilience of DNA nanostructures can be quantifiably improved by chemical cross-linking with up to 60% retaining their original structure, and 4) DNA self-assembly introduces structural defects which create new fault models with respect to conventional technologies for logic circuits.