Title

Molecular-Beacon-Based Tricomponent Probe For Snp Analysis In Folded Nucleic Acids

Keywords

DNA; fluorescent probe; hybridization; real-time assay; RNA; sensors

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 °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. SNP detection in complicated DNA: A molecular-beacon-based tricomponent probe can be used for the analysis of mutations that are located in stem or loop regions of a folded nucleic acid. The approach can be used at ambient temperatures for real-time analysis of nucleic acids containing very stable secondary structures (see scheme). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Publication Date

11-11-2011

Publication Title

Chemistry - A European Journal

Volume

17

Issue

46

Number of Pages

13052-13058

Document Type

Article

Personal Identifier

scopus

DOI Link

https://doi.org/10.1002/chem.201101987

Socpus ID

80555144145 (Scopus)

Source API URL

https://api.elsevier.com/content/abstract/scopus_id/80555144145

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