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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

Degradation of mRNA by RNase H. (Left)The hybridization of DNA-MB to its target mRNA forms MB/mRNA duplex, initiating RNase H action. The enzymatic cleavage breaks RNA in the duplex into pieces, releasing MB to restore to hair-pin structure and participate in next cycle of hybridization and cleavage until all mRNA sequences are cleaved. (Right)The response of 100 nM DNA-MB to sequential addition of 100 nM RNA targets, 12 units of RNase H and 100 nM of DNA target.
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Figure 8: Degradation of mRNA by RNase H. (Left)The hybridization of DNA-MB to its target mRNA forms MB/mRNA duplex, initiating RNase H action. The enzymatic cleavage breaks RNA in the duplex into pieces, releasing MB to restore to hair-pin structure and participate in next cycle of hybridization and cleavage until all mRNA sequences are cleaved. (Right)The response of 100 nM DNA-MB to sequential addition of 100 nM RNA targets, 12 units of RNase H and 100 nM of DNA target.

Mentions: RNase H (Ribonuclease H) is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA when it is hybridized to DNA. This enzyme does not digest single or double-stranded RNA. RNase H is found in both the nucleus and the cytoplasm of all cells and its normal function is to remove RNA primers from Okazaki fragments during DNA replication. Antisense RNase H activation has proved not only to be a powerful weapon in assessing gene function but is emerging as the method of choice for antisense therapeutics as well. While desirable in antisense technology, RNase H activation is not favored in RNA monitoring. The action of RNase H on MB: RNA duplexes will lead to destruction of the target RNA and loss of signal due to reformation of MB hair-pin structure (Figure 8, left).Figure 8.


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)

Degradation of mRNA by RNase H. (Left)The hybridization of DNA-MB to its target mRNA forms MB/mRNA duplex, initiating RNase H action. The enzymatic cleavage breaks RNA in the duplex into pieces, releasing MB to restore to hair-pin structure and participate in next cycle of hybridization and cleavage until all mRNA sequences are cleaved. (Right)The response of 100 nM DNA-MB to sequential addition of 100 nM RNA targets, 12 units of RNase H and 100 nM of DNA target.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Degradation of mRNA by RNase H. (Left)The hybridization of DNA-MB to its target mRNA forms MB/mRNA duplex, initiating RNase H action. The enzymatic cleavage breaks RNA in the duplex into pieces, releasing MB to restore to hair-pin structure and participate in next cycle of hybridization and cleavage until all mRNA sequences are cleaved. (Right)The response of 100 nM DNA-MB to sequential addition of 100 nM RNA targets, 12 units of RNase H and 100 nM of DNA target.
Mentions: RNase H (Ribonuclease H) is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA when it is hybridized to DNA. This enzyme does not digest single or double-stranded RNA. RNase H is found in both the nucleus and the cytoplasm of all cells and its normal function is to remove RNA primers from Okazaki fragments during DNA replication. Antisense RNase H activation has proved not only to be a powerful weapon in assessing gene function but is emerging as the method of choice for antisense therapeutics as well. While desirable in antisense technology, RNase H activation is not favored in RNA monitoring. The action of RNase H on MB: RNA duplexes will lead to destruction of the target RNA and loss of signal due to reformation of MB hair-pin structure (Figure 8, left).Figure 8.

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