Using double-stranded DNA probes to promote specificity in target capture

Baker, Bryan A.; Mahmoudabadi, Gita; Milam, Valeria T.

doi:10.1016/j.colsurfb.2012.09.020

Cited by 8 publications

(6 citation statements)

References 45 publications

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“…The slow displacement activity of this 15 base-long, mismatched target by P15 is attributed to the lack of a toehold region. While considerably lower than primary hybridization rates, these k d values are comparable in magnitude to values reported for related 44 and unrelated 43,55 sequences in DNAfunctionalized colloidal systems involving the exchange of one partner for a stronger partner strand. For comparable temperature and secondary target concentrations, however, the observed displacement rate constants in the current work are about 2 orders of magnitude higher than rate constants reported by Reynaldo for oligonucleotide solutions.…”

Section: ■ Introductionsupporting

confidence: 74%

Measuring in Situ Primary and Competitive DNA Hybridization Activity on Microspheres

Hardin

Milam

2013

Biomacromolecules

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Microspheres serve as convenient substrates for studying DNA activity on surfaces. Here, in addition to employing conventional sample preparation involving multiple wash and resuspension steps prior to flow cytometry measurements, we also directly sampled the reaction volume to acquire in situ measurements of primary and competitive hybridization events. Even in the absence of post hybridization wash steps, nonspecific binding events were negligible and thus allowed for direct, quantitative assessment of hybridization events as they occurred on colloidal surfaces. The in situ results indicate that primary duplex formation between immobilized probes and soluble targets on microsphere surfaces is less favorable than predicted by solution models. The kinetics of competitive displacement of primary hybridization partners by secondary targets measured in situ or post washing also deviate from expectations based on theoretical solution thermodynamics, but are consistent with predicted kinetic trends stemming from differences in either the toehold base length or branch migration.

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Section: ■ Introductionsupporting

confidence: 74%

Measuring in Situ Primary and Competitive DNA Hybridization Activity on Microspheres

Hardin

Milam

2013

Biomacromolecules

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“…Therefore, screening for miRNA requires the use of methods with high sensitivity, such as quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), , microarrays, ,,, and Northern blotting. ,− These methods are ideal for cell lysates but not in situ cellular analysis. Current biosensors for in situ cellular analysis include: molecular beacons, − dual molecular beacons, − dual linear probes, − intercalator biosensors, − double-strand displacement biosensors, , NanoFlares, and molecular sentinels. , In general, these methods provide selectivity and high sensitivity but suffer from slow analysis, false positives, or a lack of high selectivity. A majority of oligonucleotide-based fluorescent biosensors demonstrate an increase of fluorescence intensity upon target binding by disrupting a quenching mechanism.…”

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confidence: 99%

Detection of miRNA Using a Double-Strand Displacement Biosensor with a Self-Complementary Fluorescent Reporter

et al. 2014

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Design of rapid, selective, and sensitive DNA and ribonucleic acid (RNA) biosensors capable of minimizing false positives from nuclease degradation is crucial for translational research and clinical diagnostics. We present proof-of-principle studies of an innovative micro-ribonucleic acid (miRNA) reporter-probe biosensor that displaces a self-complementary reporter, while target miRNA binds to the probe. The freed reporter folds into a hairpin structure to induce a decrease in the fluorescent signal. The self-complementarity of the reporter facilitates the reduction of false positives from nuclease degradation. Nanomolar limits of detection and quantitation were capable with this proof-of-principle design. Detection of miRNA occurs within 10 min and does not require any additional hybridization, labeling, or rinsing steps. The potential for medical applications of the reporter-probe biosensor is demonstrated by selective detection of a cancer regulating microRNA, Lethal-7 (Let-7a). Mechanisms for transporting the biosensor across the cell membrane will be the focus of future work.

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“…5(a) later experimental studies were carried out by the Milam group using DNA-functionalized polystyrene microspheres and nanoparticles to facilitate visual inspection of individual colloidal particles in suspension using phase contrast and confocal microscopy techniques as well as to provide quantitative assessment of primary and competitive hybridization activity on colloidal surfaces using ow cytometry. 109,115,[117][118][119][120] Displacement scenarios have been adapted to gold nanoparticle suspensions to demonstrate mismatch discrimination based on analysis of the disassembly kinetics. 121 Other studies featuring metallic nanoparticles have demonstrated that more subtle structural changes could be induced by strand displacement.…”

Section: Strand Displacement Cues In Responsive Materials Systemsmentioning

confidence: 99%

Strand displacement in DNA-based materials systems

2013

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Hybridization activity between single-stranded DNA has been well-studied for decades and employed in numerous schemes ranging from nucleic acid detection to oligonucleotide-linked nanostructures. The related, but distinct activity involving strand exchange in duplexes, however, has received less attention.The recent emergence of DNA-based technologies in which strand exchange or displacement events play key functional roles warrants a deeper understanding of how these secondary hybridization events can uniquely enable sequence recognition-based reaction cascades and reversible or reconfigurable assembly schemes in materials systems. The following review discusses the development and implementation of DNA strand exchange events for a range of nanoscale and mesoscale structures in responsive materials. Prior to his Ph.D. studies, he received a B.S. in Physics (with highest honors) from Furman University in South Carolina. He is currently a National Research Council Postdoctoral Fellow at the National Institute of Standards and Technology working with Dr Carl Simon investigating the effects of tissue engineering scaffolds on gene expression of adult stem cells. Gita Mahmoudabadi obtained her B.S. degree in Biomedical Engineering with highest honors from the Georgia Institute of Technology in 2011. As an Undergraduate Research Scholar recipient (awarded by the Institute of Bioengineering and Biosciences) her research in Dr Valeria Milam's lab at Georgia Tech focused on designing and characterizing microparticles functionalized with doublestranded probes as nucleic acid detection platforms. In 2011 she won an NSF Graduate Fellowship and is now pursuing a Ph.D. in Bioengineering at the California Institute of Technology. Her doctoral research with Dr Rob Phillips involves environmental phage detection and evolution.

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Using double-stranded DNA probes to promote specificity in target capture

Cited by 8 publications

References 45 publications

Measuring in Situ Primary and Competitive DNA Hybridization Activity on Microspheres

Measuring in Situ Primary and Competitive DNA Hybridization Activity on Microspheres

Detection of miRNA Using a Double-Strand Displacement Biosensor with a Self-Complementary Fluorescent Reporter

Strand displacement in DNA-based materials systems

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