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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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Related in: MedlinePlus

Hybridization of DNA-MB and LNA-MB-E0 to 10-fold excess of loop cDNA (GCG ACC ATA GTG ATT TAG A). Both MBs shared the same sequence FAM-CCTAGCTCTAAATCACTATGGTCGCGCTA GG-DABCYL. DNA-MB was synthesized with DNA bases, while the LNA-MB-E0 was fully modified with 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 2: Hybridization of DNA-MB and LNA-MB-E0 to 10-fold excess of loop cDNA (GCG ACC ATA GTG ATT TAG A). Both MBs shared the same sequence FAM-CCTAGCTCTAAATCACTATGGTCGCGCTA GG-DABCYL. DNA-MB was synthesized with DNA bases, while the LNA-MB-E0 was fully modified with 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: Compared to DNA-MBs, MBs fully modified with LNA respond slowly to complementary target DNA or RNA sequences. Figure 2 shows the hybridization of a DNA-MB and a fully modified LNA-MB (LNA-MB-E0) with the same loop target DNA. The fluorescence signal of DNA-MB reached equilibrium within minutes. In contrast, the fluorescence intensity of the fully modified LNA-MB-E0 increased slowly over time, indicating its slow hybridization kinetics. The reaction did not reach equilibrium even after 20 h under the same conditions. The slow hybridization kinetics would compromise temporal resolution when obtaining the dynamic information of RNA in living cells. In order to take advantage of LNA-MBs for intracellular analysis, it is necessary to expedite LNA-MB hybridization kinetics.Figure 2.


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 DNA-MB and LNA-MB-E0 to 10-fold excess of loop cDNA (GCG ACC ATA GTG ATT TAG A). Both MBs shared the same sequence FAM-CCTAGCTCTAAATCACTATGGTCGCGCTA GG-DABCYL. DNA-MB was synthesized with DNA bases, while the LNA-MB-E0 was fully modified with 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 2: Hybridization of DNA-MB and LNA-MB-E0 to 10-fold excess of loop cDNA (GCG ACC ATA GTG ATT TAG A). Both MBs shared the same sequence FAM-CCTAGCTCTAAATCACTATGGTCGCGCTA GG-DABCYL. DNA-MB was synthesized with DNA bases, while the LNA-MB-E0 was fully modified with 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: Compared to DNA-MBs, MBs fully modified with LNA respond slowly to complementary target DNA or RNA sequences. Figure 2 shows the hybridization of a DNA-MB and a fully modified LNA-MB (LNA-MB-E0) with the same loop target DNA. The fluorescence signal of DNA-MB reached equilibrium within minutes. In contrast, the fluorescence intensity of the fully modified LNA-MB-E0 increased slowly over time, indicating its slow hybridization kinetics. The reaction did not reach equilibrium even after 20 h under the same conditions. The slow hybridization kinetics would compromise temporal resolution when obtaining the dynamic information of RNA in living cells. In order to take advantage of LNA-MBs for intracellular analysis, it is necessary to expedite LNA-MB hybridization kinetics.Figure 2.

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