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

Crey-Desbiolles, Caroline; Ahn, Dae-Ro; Leumann, Christian J. (2005). Molecular beacons with a homo-DNA stem: improving target selectivity. Nucleic acids research, 33(8), e77. Oxford University Press 10.1093/nar/gni076

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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

Item Type:

Journal Article (Original Article)

Division/Institute:

08 Faculty of Science > Departement of Chemistry and Biochemistry

UniBE Contributor:

Desbiolles, Caroline and Leumann, Christian

Subjects:

500 Science > 570 Life sciences; biology
500 Science > 540 Chemistry

ISSN:

0305-1048

Publisher:

Oxford University Press

Language:

English

Submitter:

Christian Leumann

Date Deposited:

26 May 2014 15:43

Last Modified:

04 Nov 2018 01:49

Publisher DOI:

10.1093/nar/gni076

PubMed ID:

15879349

Uncontrolled Keywords:

ACID, Acids, analog, base, BASE-PAIR, binding, BUILDING-BLOCKS, chemistry, conformation, DNA, DNA probes, fluorescence, hairpin, Homo-DNA, Hoogsteen, In Vitro, IN-VIVO, molecular, molecular beacons, Nucleic Acids, NUCLEIC-ACIDS, reversed, Selectivity, synthesis

BORIS DOI:

10.7892/boris.53102

URI:

https://boris.unibe.ch/id/eprint/53102

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