Enhanced recognition of non-complementary hybridization by single-LNA-modified oligonucleotide probes.
ABSTRACT Locked nucleic acid (LNA) is a deoxyribonucleotide analogue with an unusual 'locked' furanose conformation. LNA-modified oligonucleotide probes have demonstrated an enhanced binding affinity towards their complementary strands; however, their potential to discriminate non-complementary hybridization of mismatches has not been explored. In this study, we investigated the effect of the chemical nature of LNA nucleobases on the hybridization stability and the capability of LNA-modified oligonucleotides to discriminate the LNA:DNA mismatched base pairs. It was observed that LNA modification indeed improves the discrimination capability of oligonucleotides by increasing the melting temperature differences between the complementary duplexes and hybrids containing mismatches. Particularly, LNA purines offer a greater potential to recognize the mismatches than LNA pyrimidines and DNA purines. Real-time PCR experiments further confirmed that LNA modifications at the 3'-end are more effective. The results and conclusions in this study provide useful information for hybridization-based nucleic acid analysis where designing sound oligonucleotide probes is crucial to the success of the analyses.
- Journal of The American Chemical Society - J AM CHEM SOC. 12/1998; 120(50).
- [show abstract] [hide abstract]
ABSTRACT: Locked nucleic acid (LNA) is a new class of bicyclic high affinity DNA analogs. LNA-containing oligonucleotides confer significantly increased affinity against their complementary DNA targets, increased mismatch discrimination (delta Tm) and allow full control of the melting point of the hybridization reaction. LNA chemistry is completely compatible with the traditional DNA phosphoramidite chemistry and therefore LNA-DNA mixmer oligonucleotides can be designed with complete freedom for optimal performance. These properties render LNA oligonucleotides very well suited for SNP genotyping and have enabled several approaches for enzyme-independent SNP genotyping based on allele-specific hybridization. In addition, allele-specific PCR assays relying on enzymatically-enhanced discrimination can be improved using LNA-modified oligonucleotides. The use of LNA transforms enzyme-independent genotyping approaches into experimentally simple, robust and cost-effective assays, which are highly suited for genotyping in clinical and industrial settings.Expert Review of Molecular Diagnostics 02/2003; 3(1):27-38. · 4.09 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: We report the development of new software, OligoDesign, which provides optimal design of LNA (locked nucleic acid) substituted oligonucleotides for functional genomics applications. LNAs constitute a novel class of bicyclic RNA analogs having an exceptionally high affinity and specificity toward their complementary DNA and RNA target molecules. The OligoDesign software features recognition and filtering of the target sequence by genome-wide BLAST analysis in order to minimize cross-hybridization with non-target sequences. Furthermore it includes routines for prediction of melting temperature, self-annealing and secondary structure for LNA substituted oligonucleotides, as well as secondary structure prediction of the target nucleotide sequence. Individual scores for all these properties are calculated for each possible LNA oligonucleotide in the query gene and the OligoDesign program ranks the LNA capture probes according to a combined fuzzy logic score and finally returns the top scoring probes to the user in the output. We have successfully used the OligoDesign tool to design a Caenorhabditis elegans LNA oligonucleotide microarray, which allows monitoring of the expression of a set of 120 potential marker genes for a variety of stress and toxicological processes and toxicologically relevant pathways. The OligoDesign program is freely accessible at http://lnatools.com/.Nucleic Acids Research 08/2003; 31(13):3758-62. · 8.28 Impact Factor