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1. OWL2: a molecular beacon-based nanostructure for highly selective detection of single-nucleotide variations in folded nucleic acids

   Mueller B. L, Liberman M. (March 2023, Nanoscale)

   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) temp- eratures; (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. 

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2. DNA NANOTECHNOLOGY: THE DEVELOPMENT OF MULTI-FUNCTIONAL HYBRIDIZATION SENSORS

   MUELLER, Brittany L (December 2024, University of Central Florida https://stars.library.ucf.edu/etd2024/55/)

   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 

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3. The owl sensor: a ‘fragile’ DNA nanostructure for the analysis of single nucleotide variations

   https://doi.org/10.1039/c8nr01107a  

   Karadeema, Rebekah J.; Stancescu, Maria; Steidl, Tyler P.; Bertot, Sophia C.; Kolpashchikov, Dmitry M. (January 2018, Nanoscale)

   Analysis of single nucleotide variations (SNVs) in DNA and RNA sequences is instrumental in healthcare for the detection of genetic and infectious diseases and drug-resistant pathogens. Here we took advantage of the developments in DNA nanotechnology to design a hybridization sensor, named the ‘owl sensor’, which produces a fluorescence signal only when it complexes with fully complementary DNA or RNA analytes. The novelty of the owl sensor operation is that the selectivity of analyte recognition is, at least in part, determined by the structural rigidity and stability of the entire DNA nanostructure rather than exclusively by the stability of the analyte–probe duplex, as is the case for conventional hybridization probes. Using two DNA and two RNA analytes we demonstrated that owl sensors differentiate SNVs in a wide temperature range of 5 °C–32 °C, a performance unachievable by conventional hybridization probes including the molecular beacon probe. The owl sensor reliably detects cognate analytes even in the presence of 100 times excess of single base mismatched sequences. The approach, therefore, promises to add to the toolbox for the diagnosis of SNVs at ambient temperatures. 

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4. Fluorescent nanoparticle sensors with tailor-made recognition units and proximate fluorescent reporter groups

   https://doi.org/10.1039/C8NJ01139G  

   Xing, Xiaoyu; Zhao, Yan (January 2018, New Journal of Chemistry)

   The molecular recognition unit of a fluorescent sensor is its most cumbersome part to design and synthesize, but is key to the specificity of the sensor. Molecular imprinting within cross-linked micelles using easily synthesized modular templates allowed us to create analyte-specific binding sites with a nearby fluorescent probe. This strategy makes it straightforward to vary the recognition unit independent of the reporting unit, making the sensor potentially applicable to a wide range of molecular analytes. 

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5. Tailed molecular beacon probes: an approach for the detection of structured DNA and RNA analytes

   https://doi.org/10.1039/D4CC05984K  

   Mueller, Brittany L; Molden, Tatiana A; Hammock, Jordan; Kolpashchikov, Dmitry M (January 2025, Chemical Communications)

   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. 

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