Published online 5 May 2005
Methods Online |
Molecular beacons with a homo-DNA stem: improving target selectivity
Department of Chemistry and Biochemistry, University of Bern Freiestrasse 3, CH-3012 Bern, Switzerland
*To whom correspondence should be addressed. Tel: +41 0 31 631 4355; Fax: +41 0 31 631 3422; Email: leumann{at}ioc.unibe.ch
Received March 16, 2005. Revised April 20, 2005. Accepted April 20, 2005.
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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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