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Design of LNA probes that improve mismatch discrimination.

ABSTRACT: Locked nucleic acids (LNA) show remarkable affinity and specificity against native DNA targets. Effects of LNA modifications on mismatch discrimination were studied as a function of sequence context and identity of the mismatch using ultraviolet (UV) melting experiments. A triplet of LNA residues centered on the mismatch was generally found to have the largest discriminatory power. An exception was observed for G-T mismatches, where discrimination decreased when the guanine nucleotide at the mismatch site or even the flanking nucleotides were modified. Fluorescence experiments using 2-aminopurine suggest that LNA modifications enhance base stacking of perfectly matched base pairs and decrease stabilizing stacking interactions of mismatched base pairs. LNAs do not change the amount of counterions (Na+) that are released when duplexes denature. New guidelines are suggested for design of LNA probes, which significantly improve mismatch discrimination in comparison with unmodified DNA probes.

SUBMITTER: You Y

PROVIDER: S-EPMC1456327 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Design of LNA probes that improve mismatch discrimination.

You Yong Y Moreira Bernardo G BG Behlke Mark A MA Owczarzy Richard R

Nucleic acids research 20060502 8

Locked nucleic acids (LNA) show remarkable affinity and specificity against native DNA targets. Effects of LNA modifications on mismatch discrimination were studied as a function of sequence context and identity of the mismatch using ultraviolet (UV) melting experiments. A triplet of LNA residues centered on the mismatch was generally found to have the largest discriminatory power. An exception was observed for G-T mismatches, where discrimination decreased when the guanine nucleotide at the mis ...[more]

PMID: 16670427

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Project description:BackgroundDetection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci.ResultsIn this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA) substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers) while the other a secondary endosymbiont Arsenophonus (and present in less numbers). Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe.ConclusionBy employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction about their specific distribution within samples.

Dataset Information

Design of LNA probes that improve mismatch discrimination.

Publications

Design of LNA probes that improve mismatch discrimination.

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