Hybridization of DNA and PNA Molecular Beacons to Single-Stranded and Double-Stranded DNA Targets

Abstract

Molecular beacons are sensitive fluorescent probes hybridizing selectively to designated DNA and RNA targets. They have recently become practical tools for quantitative real-time monitoring of single-stranded nucleic acids. Here, we comparatively study the performance of a variety of such probes, stemless and stem-containing DNA and PNA (peptide nucleic acid) beacons, in Tris-buffer solutions containing various concentrations of NaCl and MgCl(2). We demonstrate that different molecular beacons respond differently to the change of salt concentration, which could be attributed to the differences in their backbones and constructions. We have found that the stemless PNA beacon hybridizes rapidly to the complementary oligodeoxynucleotide and is less sensitive than the DNA beacons to the change of salt thus allowing effective detection of nucleic acid targets under various conditions. Though we found stemless DNA beacons improper for diagnostic purposes due to high background fluorescence, we believe that use of these DNA and similar RNA constructs in molecular-biophysical studies may be helpful for analysis of conformational flexibility of single-stranded nucleic acids. With the aid of PNA "openers", molecular beacons were employed for the detection of a chosen target sequence directly in double-stranded DNA (dsDNA). Conditions are found where the stemless PNA beacon strongly discriminates the complementary versus mismatched dsDNA targets. Together with the insensitivity of PNA beacons to the presence of salt and DNA-binding/processing proteins, the latter results demonstrate the potential of these probes as robust tools for recognition of specific sequences within dsDNA without denaturation and deproteinization of duplex DNA.

Peptide nucleic acids on microarrays and other biosensors

“…Molecular beacons represent a versatile tool in DNA diagnostics [24]. Although these molecules initially had a hairpin structure with a stem, it was later found, especially for PNA molecules, that a stem is not required for their functioning [25,26]. In contrast to DNA molecular beacons, stemless PNA beacons are less sensitive to ionic strength, and the quenched fluorescence of PNA is not affected by DNAbinding proteins.…”

Kinetics and mechanism of the DNA double helix invasion by pseudocomplementary peptide nucleic acids

“…It may also have various biotechnological applications, including the PNA-assisted DNA rare cleavage (10,(13)(14)(15)(16)(17), duplex DNA capture (8,18,19), topological DNA labeling (8,(20)(21)(22)(23), direct DNA sequencing (8,17,24), and hybridization of molecular beacons to dsDNA (8,25,26). Although the previous studies have provided convincing evidence for the double-duplex invasion mode of pcPNA binding to dsDNA (9,10), the mechanism of this process is still not well characterized, and key factors controlling the formation of pcPNA-dsDNA complexes have yet to be revealed.…”

New Ideas for in Vivo Detection of RNA

“…To increase in vivo stability and prevent Rnase H degradation of the probe, oligonucleotides with modified backbone have been introduced, including 2'-O-methyl modified MBs, locked nucleic acid MBs (LNA-MBs) and peptide nucleic acid MBs (PNA-MBs). For further information regarding these modifications, the following publications should be consulted (Tsourkas et al, 2002;Wang et al, 2005, Kuhn et al, 2002. A number of innovations have been proposed to improve the signal-to-noise ratio of molecular beacons such as dual fluorescence resonance energy transfer (FRET) MBs and quenched autoligation (QUAL) probes (Santangelo et al, 2004;Santangelo et al, 2006;Satterfield et al, 2007).…”