Nucleic Acids Research, 2002, Vol. 30, No. 23 5168-5174
© 2002 Oxford University Press
Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA and 1 Integrated DNA Technologies, Inc., Coralville, IA 52241, USA
*To whom correspondence should be addressed. Tel: +1 404 385 0373; Fax: +1 404 894 4243; Email: gang.bao{at}bme.gatech.edu
Molecular beacons are stem–loop hairpin oligonucleotide probes labeled with a fluorescent dye at one end and a fluorescence quencher at the other end; they can differentiate between bound and unbound probes in homogeneous hybridization assays with a high signal-to-background ratio and enhanced specificity compared with linear oligonucleotide probes. However, in performing cellular imaging and quantification of gene expression, degradation of unmodified molecular beacons by endogenous nucleases can significantly limit the detection sensitivity, and results in fluorescence signals unrelated to probe/target hybridization. To substantially reduce nuclease degradation of molecular beacons, it is possible to protect the probe by substituting 2'-O-methyl RNA for DNA. Here we report the analysis of the thermodynamic and kinetic properties of 2'-O-methyl and 2'-deoxy molecular beacons in the presence of RNA and DNA targets. We found that in terms of molecular beacon/target duplex stability, 2'-O-methyl/RNA > 2'-deoxy/RNA > 2'-deoxy/DNA > 2'-O-methyl/DNA. The improved stability of the 2'-O-methyl/RNA duplex was accompanied by a slightly reduced specificity compared with the duplex of 2'-deoxy molecular beacons and RNA targets. However, the 2'-O-methyl molecular beacons hybridized to RNA more quickly than 2'-deoxy molecular beacons. For the pairs tested, the 2'-deoxy-beacon/DNA-target duplex showed the fastest hybridization kinetics. These findings have significant implications for the design and application of molecular beacons.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. B. Kerby, S. Freeman, K. Prachanronarong, A. W. Artenstein, S. M. Opal, and A. Tripathi Direct Sequence Detection of Structured H5 Influenza Viral RNA J. Mol. Diagn., May 1, 2008; 10(3): 225 - 235. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Pattanayak, L. K. Gifford, P. Lu, and A. M. Gewirtz Observed versus predicted structure of fluorescent self-quenching reporter molecules (SQRM): Caveats with respect to the use of "stem-loop" oligonucleotides as probes for mRNA folding RNA, April 1, 2008; 14(4): 657 - 665. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. J. Rhee, P. J. Santangelo, H. Jo, and G. Bao Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons Nucleic Acids Res., March 1, 2008; 36(5): e30 - e30. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Yang, L. Wang, Y. Wu, Y. Kim, C. D. Medley, H. Lin, and W. Tan Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons Nucleic Acids Res., June 9, 2007; 35(12): 4030 - 4041. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Silverman and E. T. Kool Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA Nucleic Acids Res., September 2, 2005; 33(15): 4978 - 4986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Crey-Desbiolles, D.-R. Ahn, and C. J. Leumann Molecular beacons with a homo-DNA stem: improving target selectivity Nucleic Acids Res., May 5, 2005; 33(8): e77 - e77. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. H. Kehlenbach In vitro analysis of nuclear mRNA export using molecular beacons for target detection Nucleic Acids Res., June 1, 2003; 31(11): e64 - e64. [Abstract] [Full Text] [PDF]
|
|