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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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Monitor RNase H cleavage of RNA in LNA-MB-E3/RNA duplex (left) and in DNA-MB/RNA duplex (right) using Ion Exchange HPLC. The concentrations of MBs and RNA were 1 µM. All samples were incubated overnight at room temperature before analyzed by ion-exchange HPLC (Dionex DNAPac™ PA-100 column (4 × 250 mm), 30%–70%, 45 min gradient 1 M NaCl/20 mM NaOH, pH 12).
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Figure 10: Monitor RNase H cleavage of RNA in LNA-MB-E3/RNA duplex (left) and in DNA-MB/RNA duplex (right) using Ion Exchange HPLC. The concentrations of MBs and RNA were 1 µM. All samples were incubated overnight at room temperature before analyzed by ion-exchange HPLC (Dionex DNAPac™ PA-100 column (4 × 250 mm), 30%–70%, 45 min gradient 1 M NaCl/20 mM NaOH, pH 12).

Mentions: The series of DNA/LNA chimeric MBs enabled the systematic study of the effect of LNA on the RNase H activity using fluorescence measurements. Insertion of LNA bases in DNA MB sequence slowed down the degradation of RNA in the probe: target duplex as compared to the DNA-MB. As seen in Figure 9, as the DNA gap between LNA bases in a MB was shortened, the activity of RNase was inhibited more. For example, about 30% of RNA target bound to LNA-MB-E4 was immediately destroyed after the introduction of RNase H (Figure 9). No significance signal decrease was observed, when the enzyme was added to the duplexes of RNA with LAN-MB-E3, LNA-MB-E2, LNA-MB-E1 and LNA-MB-E0. Ion exchange HPLC analysis of a solution containing LNA-MB-E3, RNA, and RNase H further confirmed the protection of RNA after overnight incubation of LNA-MB-E3 with RNA. There were no small pieces of RNA cleavage product observed in HPLC, as compared to the appearance of different lengths of RNA pieces for the mixture of DNA-MB, RNA and RNAse H (Figure 10). Our results suggested that the number of DNA bases in a stretch for an LNA-MB should be less than three, in order to prevent significant cleavage of the target RNA.Figure 9.


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)

Monitor RNase H cleavage of RNA in LNA-MB-E3/RNA duplex (left) and in DNA-MB/RNA duplex (right) using Ion Exchange HPLC. The concentrations of MBs and RNA were 1 µM. All samples were incubated overnight at room temperature before analyzed by ion-exchange HPLC (Dionex DNAPac™ PA-100 column (4 × 250 mm), 30%–70%, 45 min gradient 1 M NaCl/20 mM NaOH, pH 12).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: Monitor RNase H cleavage of RNA in LNA-MB-E3/RNA duplex (left) and in DNA-MB/RNA duplex (right) using Ion Exchange HPLC. The concentrations of MBs and RNA were 1 µM. All samples were incubated overnight at room temperature before analyzed by ion-exchange HPLC (Dionex DNAPac™ PA-100 column (4 × 250 mm), 30%–70%, 45 min gradient 1 M NaCl/20 mM NaOH, pH 12).
Mentions: The series of DNA/LNA chimeric MBs enabled the systematic study of the effect of LNA on the RNase H activity using fluorescence measurements. Insertion of LNA bases in DNA MB sequence slowed down the degradation of RNA in the probe: target duplex as compared to the DNA-MB. As seen in Figure 9, as the DNA gap between LNA bases in a MB was shortened, the activity of RNase was inhibited more. For example, about 30% of RNA target bound to LNA-MB-E4 was immediately destroyed after the introduction of RNase H (Figure 9). No significance signal decrease was observed, when the enzyme was added to the duplexes of RNA with LAN-MB-E3, LNA-MB-E2, LNA-MB-E1 and LNA-MB-E0. Ion exchange HPLC analysis of a solution containing LNA-MB-E3, RNA, and RNase H further confirmed the protection of RNA after overnight incubation of LNA-MB-E3 with RNA. There were no small pieces of RNA cleavage product observed in HPLC, as compared to the appearance of different lengths of RNA pieces for the mixture of DNA-MB, RNA and RNAse H (Figure 10). Our results suggested that the number of DNA bases in a stretch for an LNA-MB should be less than three, in order to prevent significant cleavage of the target RNA.Figure 9.

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