An official website of the United States government
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.
more »
« less
This content will become publicly available on December 20, 2025
DNA NANOTECHNOLOGY: THE DEVELOPMENT OF MULTI-FUNCTIONAL HYBRIDIZATION SENSORS
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
more »
« less
- PAR ID:
- 10639291
- Publisher / Repository:
- University of Central Florida https://stars.library.ucf.edu/etd2024/55/
- Date Published:
- Format(s):
- Medium: X Size: 6MB
- Size(s):
- 6MB
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
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.more » « less
-
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.more » « less
-
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.more » « less
-
The spatial arrangement of target and probe molecules on the biosensor is a key aspect of the biointerface structure that ultimately determines the properties of interfacial molecular recognition and the performance of the biosensor. However, the spatial patterns of single molecules on practical biosensors have been unknown, making it difficult to rationally engineer biosensors. Here, we have used high-resolution atomic force microscopy to map closely spaced individual probes as well as discrete hybridization events on a functioning electrochemical DNA sensor surface. We also applied spatial statistical methods to characterize the spatial patterns at the single molecule level. We observed the emergence of heterogeneous spatiotemporal patterns of surface hybridization of hairpin probes. The clustering of target capture suggests that hybridization may be enhanced by proximity of probes and targets that are about 10 nm away. The unexpected enhancement was rationalized by the complex interplay between the nanoscale spatial organization of probe molecules, the conformational changes of the probe molecules, and target binding. Such molecular level knowledge may allow one to tailor the spatial patterns of the biosensor surfaces to improve the sensitivity and reproducibility.more » « less
