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Superior structure stability and selectivity of hairpin nucleic acid probes with an L-DNA stem.

Kim Y, Yang CJ, Tan W - Nucleic Acids Res. (2007)

Bottom Line: Stem invasions occur when the stem interacts with its complementary sequence and are especially problematic in complex biological samples.Here we show that incorporating l-DNA into the stem region of a molecular beacon reduces intra- and intermolecular stem invasions, increases the melting temperature, improves selectivity to its target, and leads to enhanced bio-stability.Our results suggest that l-DNA is useful for designing functional nucleic acid probes especially for biological applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and UF Genetics Institute, Shands Cancer Center, Center for Research at Bio/nano Interface and McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, USA.

ABSTRACT
Hairpin nucleic acid probes have been highly useful in many areas, especially for intracellular and in vitro nucleic acid detection. The success of these probes can be attributed to the ease with which their conformational change upon target binding can be coupled to a variety of signal transduction mechanisms. However, false-positive signals arise from the opening of the hairpin due mainly to thermal fluctuations and stem invasions. Stem invasions occur when the stem interacts with its complementary sequence and are especially problematic in complex biological samples. To address the problem of stem invasions in hairpin probes, we have created a modified molecular beacon that incorporates unnatural enantiomeric l-DNA in the stem and natural d-DNA or 2'-O-Me-modified RNA in the loop. l-DNA has the same physical characteristics as d-DNA except that l-DNA cannot form stable duplexes with d-DNA. Here we show that incorporating l-DNA into the stem region of a molecular beacon reduces intra- and intermolecular stem invasions, increases the melting temperature, improves selectivity to its target, and leads to enhanced bio-stability. Our results suggest that l-DNA is useful for designing functional nucleic acid probes especially for biological applications.

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Melting temperature of DS and LS MBs. (a) Melting temperature profiles of DS and LS MB 1 and (b) comparisons of stem melting temperatures of MBs. LS MBs generally showed much higher melting temperature, about 5–10°C, compared to their counterparts.
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Figure 3: Melting temperature of DS and LS MBs. (a) Melting temperature profiles of DS and LS MB 1 and (b) comparisons of stem melting temperatures of MBs. LS MBs generally showed much higher melting temperature, about 5–10°C, compared to their counterparts.

Mentions: To evaluate the stem stability of MBs, the Tm of each probe was determined and compared. For all MB sequences prepared, LS MBs showed higher melting temperatures than their counterparts. For example, the LS MB 1 has a Tm of about 62°C while that for the DS MB 1 is about 58°C (Figure 3a). This difference is well above the errors in Tm measurement in our instrument (<0.5°C). The other sequences, MB 2 and MB 3, also had consistent higher Tm, ∼5–10°C, in the case of l-DNA stem design (Figure 3b). As l-DNA:l-DNA base pairs have comparable stability to that of their d-DNA counterparts, such an increase in Tm is probably due to a more stable hairpin conformation of the LS MB than that of the DS MB rather than stronger base paring between l-DNA bases (19). The improved stability of the l-DNA stem is consistent with the enhanced sensitivity of the probe observed in our hybridization experiments. In an earlier report, the partial replacement of d-DNA bases with l-DNA bases in DNA duplexes caused lower Tm (19). Our results do not contradict this work since the double-stranded stem of the LS MBs is made entirely of l-DNA and therefore does not have to accommodate both helicities simultaneously. Thus, the l-DNA stems are strong enough to maintain the stable hairpin structure, and, actually, show better stability than the pure d-DNA probes.Figure 3.


Superior structure stability and selectivity of hairpin nucleic acid probes with an L-DNA stem.

Kim Y, Yang CJ, Tan W - Nucleic Acids Res. (2007)

Melting temperature of DS and LS MBs. (a) Melting temperature profiles of DS and LS MB 1 and (b) comparisons of stem melting temperatures of MBs. LS MBs generally showed much higher melting temperature, about 5–10°C, compared to their counterparts.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175343&req=5

Figure 3: Melting temperature of DS and LS MBs. (a) Melting temperature profiles of DS and LS MB 1 and (b) comparisons of stem melting temperatures of MBs. LS MBs generally showed much higher melting temperature, about 5–10°C, compared to their counterparts.
Mentions: To evaluate the stem stability of MBs, the Tm of each probe was determined and compared. For all MB sequences prepared, LS MBs showed higher melting temperatures than their counterparts. For example, the LS MB 1 has a Tm of about 62°C while that for the DS MB 1 is about 58°C (Figure 3a). This difference is well above the errors in Tm measurement in our instrument (<0.5°C). The other sequences, MB 2 and MB 3, also had consistent higher Tm, ∼5–10°C, in the case of l-DNA stem design (Figure 3b). As l-DNA:l-DNA base pairs have comparable stability to that of their d-DNA counterparts, such an increase in Tm is probably due to a more stable hairpin conformation of the LS MB than that of the DS MB rather than stronger base paring between l-DNA bases (19). The improved stability of the l-DNA stem is consistent with the enhanced sensitivity of the probe observed in our hybridization experiments. In an earlier report, the partial replacement of d-DNA bases with l-DNA bases in DNA duplexes caused lower Tm (19). Our results do not contradict this work since the double-stranded stem of the LS MBs is made entirely of l-DNA and therefore does not have to accommodate both helicities simultaneously. Thus, the l-DNA stems are strong enough to maintain the stable hairpin structure, and, actually, show better stability than the pure d-DNA probes.Figure 3.

Bottom Line: Stem invasions occur when the stem interacts with its complementary sequence and are especially problematic in complex biological samples.Here we show that incorporating l-DNA into the stem region of a molecular beacon reduces intra- and intermolecular stem invasions, increases the melting temperature, improves selectivity to its target, and leads to enhanced bio-stability.Our results suggest that l-DNA is useful for designing functional nucleic acid probes especially for biological applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and UF Genetics Institute, Shands Cancer Center, Center for Research at Bio/nano Interface and McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, USA.

ABSTRACT
Hairpin nucleic acid probes have been highly useful in many areas, especially for intracellular and in vitro nucleic acid detection. The success of these probes can be attributed to the ease with which their conformational change upon target binding can be coupled to a variety of signal transduction mechanisms. However, false-positive signals arise from the opening of the hairpin due mainly to thermal fluctuations and stem invasions. Stem invasions occur when the stem interacts with its complementary sequence and are especially problematic in complex biological samples. To address the problem of stem invasions in hairpin probes, we have created a modified molecular beacon that incorporates unnatural enantiomeric l-DNA in the stem and natural d-DNA or 2'-O-Me-modified RNA in the loop. l-DNA has the same physical characteristics as d-DNA except that l-DNA cannot form stable duplexes with d-DNA. Here we show that incorporating l-DNA into the stem region of a molecular beacon reduces intra- and intermolecular stem invasions, increases the melting temperature, improves selectivity to its target, and leads to enhanced bio-stability. Our results suggest that l-DNA is useful for designing functional nucleic acid probes especially for biological applications.

Show MeSH
Related in: MedlinePlus