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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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Comparisons of selectivity of LS MB 1 and DS MB 1. (a) A final concentration of 100 nM MB was incubated with 1 μM of each target for 1 h and the fluorescence signal was measured. The experiment was repeated five times and the average value and SD were calculated. (b) The calculated melting temperature of each target with its complementary sequence.
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Figure 5: Comparisons of selectivity of LS MB 1 and DS MB 1. (a) A final concentration of 100 nM MB was incubated with 1 μM of each target for 1 h and the fluorescence signal was measured. The experiment was repeated five times and the average value and SD were calculated. (b) The calculated melting temperature of each target with its complementary sequence.

Mentions: The other major advantage of using l-DNA stem in MB construction is to prevent false reporting coming from the stem and non-target sequence interactions (Figure 1b). This is an intrinsic problem of all DS MBs. Presumably, a short sequence with only 5–6 bp might not have significant contribution in stem invasion, but the one with the extended sequences matched to partial loop can have high potential to cause non-selective opening of MB and false-positive signal. In order to evaluate the extent of such non-specific interactions, we designed oligo- d-DNA with different lengths that had six bases complementary to the stem of the MB while the remaining sequence matched the loop region: 5′-CCTAGC-3′, 5′-CCTAGCGC-3′, 5′-CCTAGCGCGA-3′ and 5′-CCTAGCGCGACC-3′ (underline is complementary to the 3′ stem sequence of the MB). The calculated thermodynamic stabilities of each target with its complementary sequence are shown in Figure 5b. The loop complementary DNA target, 5′-GCGACCATAGTGATTTAGA-3′ was also prepared as a reference. These sequences were separately incubated with both LS and DS MB 1 for 1 hr. The responses of the MBs were recorded and the fluorescence signal of each sample was compared based on the fluorescence signal of MB with loop-target DNA mixture (Figure 5a). For DS MB 1, incubation with 10-fold excess of the 6mer DNA target failed to open the hairpin structure. This is expected since the intramolecular-binding constant between the stem sequences is far greater than the intermolecular interaction between one arm of the stem and the 6mer DNA target. Similar results were obtained for the 8mer sequence. On the contrary, in the presence of the 10mer DNA, which was complementary to one arm of the stem and four adjacent bases in the loop, about 55% of the DS MB 1 opened. The 12-mer sequence, in 10-fold excess, was able to fully open the DS MB 1 (Figure 5a). This result clearly demonstrates how severely the stem invasion sequence can compromise the MB's function and its selectivity in biological applications. There are tremendously diversified sequences in a real sample matrix, and the copy number of this type of short complementary sequences could be very high. The chance of such a stem invasion is thus equally high and this is one of the reasons that limit the usage of MBs in quantitative analysis. In contrast, l-DNA MB does not have such a problem. None of the mock target sequences except the full-length target sequence was able to open up the LS MB 1, indicating superior selectivity and stability. This excellent selectivity coming from the l-DNA stem will be useful when performing quantitative analysis on raw biological samples as well as for intracellular studies.Figure 5.


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)

Comparisons of selectivity of LS MB 1 and DS MB 1. (a) A final concentration of 100 nM MB was incubated with 1 μM of each target for 1 h and the fluorescence signal was measured. The experiment was repeated five times and the average value and SD were calculated. (b) The calculated melting temperature of each target with its complementary sequence.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
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Figure 5: Comparisons of selectivity of LS MB 1 and DS MB 1. (a) A final concentration of 100 nM MB was incubated with 1 μM of each target for 1 h and the fluorescence signal was measured. The experiment was repeated five times and the average value and SD were calculated. (b) The calculated melting temperature of each target with its complementary sequence.
Mentions: The other major advantage of using l-DNA stem in MB construction is to prevent false reporting coming from the stem and non-target sequence interactions (Figure 1b). This is an intrinsic problem of all DS MBs. Presumably, a short sequence with only 5–6 bp might not have significant contribution in stem invasion, but the one with the extended sequences matched to partial loop can have high potential to cause non-selective opening of MB and false-positive signal. In order to evaluate the extent of such non-specific interactions, we designed oligo- d-DNA with different lengths that had six bases complementary to the stem of the MB while the remaining sequence matched the loop region: 5′-CCTAGC-3′, 5′-CCTAGCGC-3′, 5′-CCTAGCGCGA-3′ and 5′-CCTAGCGCGACC-3′ (underline is complementary to the 3′ stem sequence of the MB). The calculated thermodynamic stabilities of each target with its complementary sequence are shown in Figure 5b. The loop complementary DNA target, 5′-GCGACCATAGTGATTTAGA-3′ was also prepared as a reference. These sequences were separately incubated with both LS and DS MB 1 for 1 hr. The responses of the MBs were recorded and the fluorescence signal of each sample was compared based on the fluorescence signal of MB with loop-target DNA mixture (Figure 5a). For DS MB 1, incubation with 10-fold excess of the 6mer DNA target failed to open the hairpin structure. This is expected since the intramolecular-binding constant between the stem sequences is far greater than the intermolecular interaction between one arm of the stem and the 6mer DNA target. Similar results were obtained for the 8mer sequence. On the contrary, in the presence of the 10mer DNA, which was complementary to one arm of the stem and four adjacent bases in the loop, about 55% of the DS MB 1 opened. The 12-mer sequence, in 10-fold excess, was able to fully open the DS MB 1 (Figure 5a). This result clearly demonstrates how severely the stem invasion sequence can compromise the MB's function and its selectivity in biological applications. There are tremendously diversified sequences in a real sample matrix, and the copy number of this type of short complementary sequences could be very high. The chance of such a stem invasion is thus equally high and this is one of the reasons that limit the usage of MBs in quantitative analysis. In contrast, l-DNA MB does not have such a problem. None of the mock target sequences except the full-length target sequence was able to open up the LS MB 1, indicating superior selectivity and stability. This excellent selectivity coming from the l-DNA stem will be useful when performing quantitative analysis on raw biological samples as well as for intracellular studies.Figure 5.

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