Hybridization of two or more DNA or RNA strands is well documented for the process taking place with all strands free in solution or when one strand is immobilized on a substrate. This study contributes to the investigation of the hybridization process when two single DNA strands (ssDNA) are in close proximity. We took advantage of an X sensor in which hybridization of four DNA strands enables the formation of a DNA four-way junction (crossover or X) structure. We immobilized multiple layers of crossover structures to study its hybridization being triggered by short ssDNA coming from solution and further investigate how many layers of these structures can hybridize by the addition of only one ssDNA (called input). Using a molecular beacon as reporter, we combined crossover DNA strands that recognize the reporter sequence at one side and at the other, the sequence of its input or downward crossover layer. Fluorescent signal was detected by separation of the molecular beacon’s fluorophore and quencher, as it hybridizes with the system of layers. Immobilization of the X structures into the scaffold proved to increase their communication, in comparison to being free in solution. This evidence gives us significant information for the communication of hybridized layers in a localized system, showing a promising standard for development of multilayered logic gates. The potential of these crossover DNA strands using X structure include applications in the future of biological systems, nanotechnology, and target DNA recognition for its ability to quickly recognize a signal and propagate it through extended DNA nanostructure in a controlled manner.

Due to nucleic acid’s programmability, it is possible to realize DNA structures with computing functions, and thus a new generation of molecular computers is evolving to solve biological and medical problems. There is evidence that genetic heredity diseases and cancer can be the result of genetic heterogeneity, thus there is a need for diagnostics and therapeutic tools with multiplex and smart components to compute all the molecular drivers. DNA molecular computers mimics electronic computers by programming synthetic nucleic acids to perform similarly to central processing units. Considering how the evolution of integrated circuits made possible the revolution of silicon-based computers, integrated DNA molecular circuits can be developed to allow modular designing and scale to complex DNA nano-processors. This dissertation covers the development of four-way junction (4J) DNA logic gates that can be wired to result in functionally complete gates, and their immobilization on a modular DNA board that serves as a scaffold for logic gate integration, fast signal processing, and cascading. Connecting 4J DNA logic gates YES and NOT resulted in OR, NAND, and IMPLY logic circuits; the three circuits can operate under the input of miRNAs, either oncogenic or/and tumor-suppressors, and give two possible diagnoses: healthy or cancerous. The DNA board can expand as the DNA circuit grows in the number of integrated 4J units. Signal propagation across a wired of 4J YES logic gates showed signal completion in < 3 min, accounting for a signal propagation rate of 4.5 nm/min and that up to 6 units can be cascaded before the signal dissipates. Lastly, an approach to chemically ligate all oligonucleotide components of the DNA molecular device is presented, in which we also found a route for the bioconjugation of 5’ to 5’ and 3’ to 3’ oligonucleotides.