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Superior structure stability and selectivity of hairpin nucleic acid probes with an L-DNA stem.

ABSTRACT: Hairpin nucleic acid probes have been highly useful in many areas, especially for intracellular and in vitro nucleic acid detection. The success of these probes can be attributed to the ease with which their conformational change upon target binding can be coupled to a variety of signal transduction mechanisms. However, false-positive signals arise from the opening of the hairpin due mainly to thermal fluctuations and stem invasions. Stem invasions occur when the stem interacts with its complementary sequence and are especially problematic in complex biological samples. To address the problem of stem invasions in hairpin probes, we have created a modified molecular beacon that incorporates unnatural enantiomeric l-DNA in the stem and natural d-DNA or 2'-O-Me-modified RNA in the loop. l-DNA has the same physical characteristics as d-DNA except that l-DNA cannot form stable duplexes with d-DNA. Here we show that incorporating l-DNA into the stem region of a molecular beacon reduces intra- and intermolecular stem invasions, increases the melting temperature, improves selectivity to its target, and leads to enhanced bio-stability. Our results suggest that l-DNA is useful for designing functional nucleic acid probes especially for biological applications.

SUBMITTER: Kim Y

PROVIDER: S-EPMC2175343 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Superior structure stability and selectivity of hairpin nucleic acid probes with an L-DNA stem.

Kim Youngmi Y Yang Chaoyong James CJ Tan Weihong W

Nucleic acids research 20071024 21

Hairpin nucleic acid probes have been highly useful in many areas, especially for intracellular and in vitro nucleic acid detection. The success of these probes can be attributed to the ease with which their conformational change upon target binding can be coupled to a variety of signal transduction mechanisms. However, false-positive signals arise from the opening of the hairpin due mainly to thermal fluctuations and stem invasions. Stem invasions occur when the stem interacts with its compleme ...[more]

PMID: 17959649

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Project description:Background: CRISPR-Cas12a has been integrated with nanomaterial-based optical techniques, such as surface-enhanced Raman scattering (SERS), to formulate a powerful amplification-free nucleic acid detection system. However, nanomaterials impose steric hindrance to limit the accessibility of CRISPR-Cas12a to the narrow gaps (SERS hot spots) among nanoparticles (NPs) for producing a significant change in signals after nucleic acid detection. Methods: To overcome this restriction, we specifically design chimeric DNA/RNA hairpins (displacers) that can be destabilized by activated CRISPR-Cas12a in the presence of target DNA, liberating excessive RNA that can disintegrate a core-satellite nanocluster via toehold-mediated strand displacement for orchestrating a promising "on-off" nucleic acid biosensor. The core-satellite nanocluster comprises a large gold nanoparticle (AuNP) core surrounded by small AuNPs with Raman tags via DNA hybridization as an ultrabright Raman reporter, and its disassembly leads to a drastic decrease of SERS intensity as signal readouts. We further introduce a magnetic core to the large AuNPs that can facilitate their separation from the disassembled nanostructures to suppress the background for improving detection sensitivity. Results: As a proof-of-concept study, our findings showed that the application of displacers was more effective in decreasing the SERS intensity of the system and attained a better limit of detection (LOD, 10 aM) than that by directly using activated CRISPR-Cas12a, with high selectivity and stability for nucleic acid detection. Introducing magnetic-responsive functionality to our system further improves the LOD to 1 aM. Conclusion: Our work not only offers a platform to sensitively and selectively probe nucleic acids without pre-amplification but also provides new insights into the design of the CRISPR-Cas12a/SERS integrated system to resolve the steric hindrance of nanomaterials for constructing biosensors.

Dataset Information

Superior structure stability and selectivity of hairpin nucleic acid probes with an L-DNA stem.

Publications

Superior structure stability and selectivity of hairpin nucleic acid probes with an L-DNA stem.

Similar Datasets

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