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Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons.

Yang CJ, Wang L, Wu Y, Kim Y, Medley CD, Lin H, Tan W - Nucleic Acids Res. (2007)

Bottom Line: It was found that the LNA bases in a MB stem sequence had a significant effect on the stability of the hair-pin structure.It was found that only MB sequences with DNA/LNA alternating bases or all LNA bases were able to resist nonspecific protein binding and DNase I digestion.These findings have implications on the design of LNA molecular probes for intracellular monitoring application, disease diagnosis and basic biological studies.

View Article: PubMed Central - PubMed

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

ABSTRACT
To take full advantage of locked nucleic acid (LNA) based molecular beacons (LNA-MBs) for a variety of applications including analysis of complex samples and intracellular monitoring, we have systematically synthesized a series of DNA/LNA chimeric MBs and studied the effect of DNA/LNA ratio in MBs on their thermodynamics, hybridization kinetics, protein binding affinity and enzymatic resistance. It was found that the LNA bases in a MB stem sequence had a significant effect on the stability of the hair-pin structure. The hybridization rates of LNA-MBs were significantly improved by lowering the DNA/LNA ratio in the probe, and most significantly, by having a shared-stem design for the LNA-MB to prevent sticky-end pairing. It was found that only MB sequences with DNA/LNA alternating bases or all LNA bases were able to resist nonspecific protein binding and DNase I digestion. Additional results showed that a sequence consisting of a DNA stretch less than three bases between LNA bases was able to block RNase H function. This study suggested that a shared-stem MB with a 4 base-pair stem and alternating DNA/LNA bases is desirable for intracellular applications as it ensures reasonable hybridization rates, reduces protein binding and resists nuclease degradation for both target and probes. These findings have implications on the design of LNA molecular probes for intracellular monitoring application, disease diagnosis and basic biological studies.

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Hybridization of 100 nM MB-LNA-E3 to 500 nM loop cDNA(GCG ACC ATA GTG ATT TAG A) (blue) and shared-stem cDNA(CCT AGC GCG ACC ATA GTG ATT TAG A) (red). The sequence of MB-LNA-E3 was FAM-CCTAGC TCTAAATCACTATGGTCGCGCTAGG-DABCYL, with red letters representing LNA bases. The hybridization experiments were performed at room temperature in 20 mM Tris-HCl (pH 7.5) buffer containing 5 mM MgCl2 and 50 mM NaCl.
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Figure 3: Hybridization of 100 nM MB-LNA-E3 to 500 nM loop cDNA(GCG ACC ATA GTG ATT TAG A) (blue) and shared-stem cDNA(CCT AGC GCG ACC ATA GTG ATT TAG A) (red). The sequence of MB-LNA-E3 was FAM-CCTAGC TCTAAATCACTATGGTCGCGCTAGG-DABCYL, with red letters representing LNA bases. The hybridization experiments were performed at room temperature in 20 mM Tris-HCl (pH 7.5) buffer containing 5 mM MgCl2 and 50 mM NaCl.

Mentions: The hybridization of the 6-mer-stem MBs to their target DNA sequence that is complementary to the MBs’ loop sequence was investigated. A very slow increase of fluorescence signal was observed, when target DNA was introduced into the LNA-MB-E0 solution. Lowering the percentage of LNA in the probe sequences significantly improved the initial hybridization rate. For LNA-MB-E2, E3 and E4, interesting fluorescence traces were observed. The fluorescence of the solution initially had a sharp increase and then suddenly began to decay. The blue trace in Figure 3 shows a typical response of LNA-MB-E3 to the addition of target DNA. Similar trace was observed for DNA-MBs at higher [Mg2+] in previous studies and was attributed to SEP of MBs in the presence of target (31). Incorporation of LNA bases in MBs significantly enhanced the affinity of the stem, as indicated by the elevated melting temperature of the LNA-MBs. Consequently, intermolecular hybridization of LNA-MBs was evident for LNA-MB-E2, LNA-MB-E3 and LNA-MB-E4. The traces of LNA-MB-E1 and LNA-MB-E0, however, were somewhat different, with no clear drop in fluorescence after the initial signal increase. The signal of these two beacon solutions increased much slower because of more LNAs in the stem, resulting in a higher tendency to form sticky-end pairs at the initial hybridization state.Figure 3.


Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons.

Yang CJ, Wang L, Wu Y, Kim Y, Medley CD, Lin H, Tan W - Nucleic Acids Res. (2007)

Hybridization of 100 nM MB-LNA-E3 to 500 nM loop cDNA(GCG ACC ATA GTG ATT TAG A) (blue) and shared-stem cDNA(CCT AGC GCG ACC ATA GTG ATT TAG A) (red). The sequence of MB-LNA-E3 was FAM-CCTAGC TCTAAATCACTATGGTCGCGCTAGG-DABCYL, with red letters representing LNA bases. The hybridization experiments were performed at room temperature in 20 mM Tris-HCl (pH 7.5) buffer containing 5 mM MgCl2 and 50 mM NaCl.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919502&req=5

Figure 3: Hybridization of 100 nM MB-LNA-E3 to 500 nM loop cDNA(GCG ACC ATA GTG ATT TAG A) (blue) and shared-stem cDNA(CCT AGC GCG ACC ATA GTG ATT TAG A) (red). The sequence of MB-LNA-E3 was FAM-CCTAGC TCTAAATCACTATGGTCGCGCTAGG-DABCYL, with red letters representing LNA bases. The hybridization experiments were performed at room temperature in 20 mM Tris-HCl (pH 7.5) buffer containing 5 mM MgCl2 and 50 mM NaCl.
Mentions: The hybridization of the 6-mer-stem MBs to their target DNA sequence that is complementary to the MBs’ loop sequence was investigated. A very slow increase of fluorescence signal was observed, when target DNA was introduced into the LNA-MB-E0 solution. Lowering the percentage of LNA in the probe sequences significantly improved the initial hybridization rate. For LNA-MB-E2, E3 and E4, interesting fluorescence traces were observed. The fluorescence of the solution initially had a sharp increase and then suddenly began to decay. The blue trace in Figure 3 shows a typical response of LNA-MB-E3 to the addition of target DNA. Similar trace was observed for DNA-MBs at higher [Mg2+] in previous studies and was attributed to SEP of MBs in the presence of target (31). Incorporation of LNA bases in MBs significantly enhanced the affinity of the stem, as indicated by the elevated melting temperature of the LNA-MBs. Consequently, intermolecular hybridization of LNA-MBs was evident for LNA-MB-E2, LNA-MB-E3 and LNA-MB-E4. The traces of LNA-MB-E1 and LNA-MB-E0, however, were somewhat different, with no clear drop in fluorescence after the initial signal increase. The signal of these two beacon solutions increased much slower because of more LNAs in the stem, resulting in a higher tendency to form sticky-end pairs at the initial hybridization state.Figure 3.

Bottom Line: It was found that the LNA bases in a MB stem sequence had a significant effect on the stability of the hair-pin structure.It was found that only MB sequences with DNA/LNA alternating bases or all LNA bases were able to resist nonspecific protein binding and DNase I digestion.These findings have implications on the design of LNA molecular probes for intracellular monitoring application, disease diagnosis and basic biological studies.

View Article: PubMed Central - PubMed

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

ABSTRACT
To take full advantage of locked nucleic acid (LNA) based molecular beacons (LNA-MBs) for a variety of applications including analysis of complex samples and intracellular monitoring, we have systematically synthesized a series of DNA/LNA chimeric MBs and studied the effect of DNA/LNA ratio in MBs on their thermodynamics, hybridization kinetics, protein binding affinity and enzymatic resistance. It was found that the LNA bases in a MB stem sequence had a significant effect on the stability of the hair-pin structure. The hybridization rates of LNA-MBs were significantly improved by lowering the DNA/LNA ratio in the probe, and most significantly, by having a shared-stem design for the LNA-MB to prevent sticky-end pairing. It was found that only MB sequences with DNA/LNA alternating bases or all LNA bases were able to resist nonspecific protein binding and DNase I digestion. Additional results showed that a sequence consisting of a DNA stretch less than three bases between LNA bases was able to block RNase H function. This study suggested that a shared-stem MB with a 4 base-pair stem and alternating DNA/LNA bases is desirable for intracellular applications as it ensures reasonable hybridization rates, reduces protein binding and resists nuclease degradation for both target and probes. These findings have implications on the design of LNA molecular probes for intracellular monitoring application, disease diagnosis and basic biological studies.

Show MeSH
Related in: MedlinePlus