Molecular beacons with a homo-DNA stem: improving target selectivity

Abstract

Molecular beacons (MBs) are stem–loop DNA probes used for identifying and reporting the presence and localization of nucleic acid targets in vitro and in vivo via target-dependent dequenching of fluorescence. A drawback of conventional MB design is present in the stem sequence that is necessary to keep the MBs in a closed conformation in the absence of a target, but that can participate in target binding in the open (target-on) conformation, giving rise to the possibility of false-positive results. In order to circumvent these problems, we designed MBs in which the stem was replaced by an orthogonal DNA analog that does not cross-pair with natural nucleic acids. Homo-DNA seemed to be specially suited, as it forms stable adenine-adenine base pairs of the reversed Hoogsteen type, potentially reducing the number of necessary building blocks for stem design to one. We found that MBs in which the stem part was replaced by homo-adenylate residues can easily be synthesized using conventional automated DNA synthesis. As conventional MBs, such hybrid MBs show cooperative hairpin to coil transitions in the absence of a DNA target, indicating stable homo-DNA base pair formation in the closed conformation. Furthermore, our results show that the homo-adenylate stem is excluded from DNA target binding, which leads to a significant increase in target binding selectivity.

An Elegant Biosensor Molecular Beacon Probe: Challenges and Recent Solutions

“…L-DNA can only form stable duplexes with complementary L-DNA, but not with D-DNA of the target. The same effect was achieved by using other artificial nucleotide analogs, nucleic acids with bis(hydroxymethyl)benzene-phosphate backbone [102], artificially expanded genetic information system (AEGIS) [103–105], DNA analog that forms adenine-adenine base pairs (Homo-DNA) [106], and potentially can be achieved using x-DNA introduced by Kool and colleagues [107]. Conceivably, the aforementioned strategies allow the design of a series of universal stems that would cover the entire useful temperature range.…”

Tuning Molecular Recognition of RNA and DNA via Sugar Modification

“…3D) readily form hairpin-loop structures with a stability that roughly matches that of an unmodified MB [23]. No interaction between the homo-DNA sequence tracts with the target DNA was observed.…”

Rapid monitoring of mRNA levels with a molecular beacon during microbial fermentation

“…A more recently developed method based on molecular beacons (MBs) (Li et al, 2008a,b) has been used to identify microorganisms, image intracellular gene expression (Crey-Desbiolles et al, 2005;Deiman et al, 2007), detect the transcriptional level of RNA in vitro (Belon and Frick, 2008;Hopkins and Woodson, 2005;Nadal et al, 2007), and measure the folding rates of RNA (Silverman and Kool, 2005). A molecular beacon is a stem-loop DNA oligonucleotide carrying a fluorophore and a quencher.…”

“…Based on reports involving MB hybridization (Crey-Desbiolles et al, 2005;Deiman et al, 2007;Belon and Frick, 2008;Hopkins and Woodson, 2005;Nadal et al, 2007;Silverman and Kool, 2005;Bonnet et al, 1999;Horejsh et al, 2005;Li et al, 2006a,b;Martí et al, 2006;Tsai et al, 2003;Tyagi and Kramer, 1996), we found that MB hybridization conditions such as hybridization buffer, temperature, and time were all arbitrarily chosen. Seven typical hybridization buffers (see Table 1) had been reported but there are no explanations why these buffers were chosen.…”