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Molecular beacon (MB) probes have been extensively used for nucleic acid analysis. However, MB probes fail to hybridize with folded DNA or RNA. Here, we demonstrate that MB probes equipped with extra sequences complementary to the analyte, named ‘tail’, can increase the signal-to-background ratio by B40- fold and hybridization rates by B800-fold compared to conventional MB probes. Tailed MB probes can be used as mismatched-tolerant alternatives to traditional hairpin probes for fast assays. 

Hybridization probes have been used to detect specific nucleic acids for the last 50 years. These probes have applications in medicine, including identifying disease-causing genes or multi-drug resistant bacteria. To be considered robust, a probe should have high selectivity at ambient or low temperatures, be able to detect folded analytes, and remain economical for use in clinical settings. This work will uncover a challenge faced by molecular beacon probes (MBP), describe an adaptation to a molecular beacon probe (MBP) that enables the hybridization of the probe to a folded target, a multicomponent DNA sensor (OWL2) that overcomes common challenges faced by hybridization probes, and a thresholding sensor (MB-Th) that allows for the quantification of microRNA. Through the use of unwinding arms, the MBP adaptation and OWL2 sensor are able to hybridize with and detect folded analytes. Additionally, the OWL2 sensor contains two analyte-binding arms to unwind folded analytes and two sequence-specific strands that bind both the analyte and a universal molecular beacon (UMB) probe to form a fluorescent ‘OWL’ structure. The sensor can differentiate single base mismatches in folded analytes in the temperature range of 5–38 °C, even when challenged with excess wild-type analytes. The MB-Th sensor consists of two gates with increasing affinity for the target, with each varying in thermodynamic stability. The gates bind to separate molecular beacons, each with a unique fluorophore, and produce distinct signals that can be measured simultaneously. Both sensor designs are cost-efficient since the same UMB probe can be used to detect any analyte sequence. These sensors have significant clinical benefits for the diagnosis of non-invasive early-stage cancer and cancers associated with miRNA dysregulation. iv 

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. 

t has been shown that active learning strategies are effective in teaching complex STEM concepts. In this study, we developed and implemented a laboratory experiment for teaching the concepts of Boolean logic gates, molecular beacon probes, molecular computing, DNA logic gates, microRNA, and molecular diagnosis of hepatocellular carcinoma, which are related to DNA molecular computing, an interdisciplinary cutting-edge research technology in biochemistry, synthetic biology, computer science, and medicine. The laboratory experience takes about 110–140 min and consists of a multiple-choice pretest (15 min), introductory lecture (20 min), wet laboratory experiment (60–90 min), and a post-test (15 min). Students are tasked to experimentally construct three molecular logic circuits made of DNA oligonucleotides and use them for the fluorescence-based detection of microRNA markers related to diagnostics of hepatocellular carcinoma. The class was taught to undergraduate students from freshman to senior academic levels majoring in chemistry, biochemistry, biotechnology, and biomedical sciences. Students were engaged during the session and motivated to learn more about the research technology. A comparison of students’ scores on the pretest and post-test demonstrated improvement in knowledge of the concepts taught. Visual observation of the fluorescence readout led to a straightforward interpretation of the results. The laboratory experiment is portable; it uses inexpensive nontoxic reagents and thus can be employed outside a laboratory room for outreach and science popularization purposes. 

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. Pioneered by Milan Stojanovic, Boolean DNA logic gates created the foundation for the development of DNA computers. Similar to electronic computers, the field is evolving towards integrating DNA logic gates and circuits by positioning them on substrates to increase circuit density and minimize gate distance and undesired crosstalk. In this minireview, we summarize recent developments in the integration of DNA logic gates into circuits localized on DNA substrates. This approach of all‐DNA integrated circuits (DNA ICs) offers the advantages of biocompatibility, increased circuit response, increased circuit density, reduced unit concentration, facilitated circuit isolation, and facilitated cell uptake. DNA ICs can face similar challenges as their equivalent circuits operating in bulk solution (bulk circuits), and new physical challenges inherent in spatial localization. We discuss possible avenues to overcome these obstacles. 

A functionally complete Boolean operator is sufficient for computational circuits of arbitrary complexity. We connected YES (buffer) with NOT (inverter) and two NOT four-way junction (4J) DNA gates to obtain IMPLY and NAND Boolean functions, respectively, each of which represents a functionally complete gate. The results show a technological path towards creating a DNA computational circuit of arbitrary complexity based on singleton NOT or a combination of NOT and YES gates, which is not possible in electronic computers. We, therefore, concluded that DNA-based circuits and molecular computation may offer opportunities unforeseen in electronics. 

Accessibility of synthetic oligonucleotides and the success of DNA nanotechnology open a possibility to use DNA nanostructures for building sophisticated enzyme-like catalytic centers. Here we used a double DNA crossover (DX) tile nanostructure to enhance the rate, the yield, and the specificity of 5'-5' ligation of two oligonucleotides with arbitrary sequences. The ligation product was isolated via a simple procedure. The same strategy was applied for the synthesis of 3'-3' linked oligonucleotides, thus introducing a synthetic route to DNA and RNA with a switched orientation that is affordable by a low-resource laboratory. To emphasize the utility of the ligation products, we synthesized a circular structure formed from intramolecular complementarity that we named "an impossible DNA wheel" since it cannot be built from regular DNA strands by enzymatic reactions. Therefore, DX-tile nanostructures can open a route to producing useful chemical products that are unattainable via enzymatic synthesis. This is the first example of the use of DNA nanostructures as a catalyst. This study advocates for further exploration of DNA nanotechnology for building enzyme-like reactive systems. 

Hybridization probes have been used in the detection of specific nucleic acids for the last 50 years. Despite the extensive efforts and the great significance, the challenges of the commonly used probes include (1) low selectivity in detecting single nucleotide variations (SNV) at low ( e.g. room or 37 °C) temperatures; (2) low affinity in binding folded nucleic acids, and (3) the cost of fluorescent probes. Here we introduce a multicomponent hybridization probe, called OWL2 sensor, which addresses all three issues. The OWL2 sensor uses two analyte binding arms to tightly bind and unwind folded analytes, and two sequence-specific strands that bind both the analyte and a universal molecular beacon (UMB) probe to form fluorescent ‘OWL’ structure. The OWL2 sensor was able to differentiate single base mismatches in folded analytes in the temperature range of 5–38 °C. The design is cost-efficient since the same UMB probe can be used for detecting any analyte sequence.