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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 LNA molecular beacons with alternating DNA/LNA bases and different stem lengths to their shared-stem cDNA. The hybridization of MB-DNA was also included as a reference for comparison purpose. Both MB-DNA and LNA-MB-E1 had a 6-mer stem, while LNA-MB-E1-5S had a 5-mer stem and the LNA-MB-E1-4S had a 4-mer stem. 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. [MB] = 100 nM, [cDNA] = 500 nM.
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Figure 11: Hybridization of LNA molecular beacons with alternating DNA/LNA bases and different stem lengths to their shared-stem cDNA. The hybridization of MB-DNA was also included as a reference for comparison purpose. Both MB-DNA and LNA-MB-E1 had a 6-mer stem, while LNA-MB-E1-5S had a 5-mer stem and the LNA-MB-E1-4S had a 4-mer stem. 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. [MB] = 100 nM, [cDNA] = 500 nM.

Mentions: As a result of decreasing stem stability, MBs with shorter stems hybridize to their target DNA much faster. For example, in the presence of 5-fold excess of shared-stem target DNA, it took about 4 h for LNA-MB -E1 to reach its hybridization equilibrium. Under the same condition, the hybribrization of LNA-MB-E1-5S reached equilibrium after about 1 h, while the 4-mer-stem probe LNA-MB-E1-4S completely opened up in less than 15 min. As shown in Figure 11, the hybridization rate of LNA-MB-E1-4S was comparable to a regular DNA-MB. More than 80% of the 4-mer LNA-MB hybridized to the target DNA within 3 min, which makes it an excellent probe for introceullar imaging applications.Figure 11.


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 LNA molecular beacons with alternating DNA/LNA bases and different stem lengths to their shared-stem cDNA. The hybridization of MB-DNA was also included as a reference for comparison purpose. Both MB-DNA and LNA-MB-E1 had a 6-mer stem, while LNA-MB-E1-5S had a 5-mer stem and the LNA-MB-E1-4S had a 4-mer stem. 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. [MB] = 100 nM, [cDNA] = 500 nM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 11: Hybridization of LNA molecular beacons with alternating DNA/LNA bases and different stem lengths to their shared-stem cDNA. The hybridization of MB-DNA was also included as a reference for comparison purpose. Both MB-DNA and LNA-MB-E1 had a 6-mer stem, while LNA-MB-E1-5S had a 5-mer stem and the LNA-MB-E1-4S had a 4-mer stem. 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. [MB] = 100 nM, [cDNA] = 500 nM.
Mentions: As a result of decreasing stem stability, MBs with shorter stems hybridize to their target DNA much faster. For example, in the presence of 5-fold excess of shared-stem target DNA, it took about 4 h for LNA-MB -E1 to reach its hybridization equilibrium. Under the same condition, the hybribrization of LNA-MB-E1-5S reached equilibrium after about 1 h, while the 4-mer-stem probe LNA-MB-E1-4S completely opened up in less than 15 min. As shown in Figure 11, the hybridization rate of LNA-MB-E1-4S was comparable to a regular DNA-MB. More than 80% of the 4-mer LNA-MB hybridized to the target DNA within 3 min, which makes it an excellent probe for introceullar imaging applications.Figure 11.

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