Molecular-Beacon-Based Tricomponent Probe for SNP Analysis in Folded Nucleic Acids

    Authors

    C. Nguyen; J. Grimes; Y. V. Gerasimova;D. M. Kolpashchikov

    Comments

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    Abbreviated Journal Title

    Chem.-Eur. J.

    Keywords

    DNA; hybridization; fluorescent probe; real-time assay; RNA; sensors; IN-SITU HYBRIDIZATION; 16S RIBOSOMAL-RNA; REAL-TIME PCR; SECONDARY-STRUCTURE; DNA HYBRIDIZATION; OLIGONUCLEOTIDE MICROARRAYS; PNA; HYBRIDIZATION; ENERGY-TRANSFER; MESSENGER-RNA; LIVE CELLS; Chemistry, Multidisciplinary

    Abstract

    Hybridization probes are often inefficient in the analysis of single-stranded DNA or RNA that are folded in stable secondary structures. A molecular beacon (MB) probe is a short DNA hairpin with a fluorophore and a quencher attached to opposite sides of the oligonucleotide. The probe is widely used in real-time analysis of specific DNA and RNA sequences. This study demonstrates how a conventional MB probe can be used for the analysis of nucleic acids that form very stable (T(m) > 80 degrees C) hairpin structures. Here we demonstrate that the MB probe is not efficient in direct analysis of secondary structure-folded analytes, whereas a MB-based tricomponent probe is suitable for these purposes. The tricomponent probe takes advantage of two oligonucleotide adaptor strands f and m. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to a MB probe. In the presence of a specific analyte, the two adaptor strands hybridize to the analyte and the MB probe, thus forming a quadripartite complex. DNA strand f binds to the analyte with high affinity and unwinds its secondary structure. Strand m forms a stable complex only with the fully complementary analyte. The MB probe fluorescently reports the formation of the quadripartite associate. It was demonstrated that the DNA analytes folded in hairpin structures with stems containing 5, 6, 7, 8, 9, 11, or 13 base pairs can be detected in real time with the limit of detection (LOD) lying in the nanomolar range. The stability of the stem region in the DNA analyte did not affect the LOD. Analytes containing single base substitutions in the stem or in the loop positions were discriminated from the fully complementary DNA at room temperature. The tricomponent probe promises to simplify nucleic acid analysis at ambient temperatures in such applications as in vivo RNA monitoring, detection of pathogens, and single nucleotide polymorphism (SNP) genotyping by DNA microarrays.

    Journal Title

    Chemistry-a European Journal

    Volume

    17

    Issue/Number

    46

    Publication Date

    1-1-2011

    Document Type

    Article

    Language

    English

    First Page

    13052

    Last Page

    13058

    WOS Identifier

    WOS:000297737900030

    ISSN

    0947-6539

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