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

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.

Figure 1.

Structure of a MB (A) and a LNA sequence (B). A LNA nucleotide contains a ribose ring that has a 2′-O, 4′-C methylene bridge.

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.

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.

Figure 4.

Blocking LNA-MB sticky-end pairing with shared-stem targets. (A) Thermal denaturation profiles of solutions containing LNA-MB-E3 in the absence of target (blue), in the presence of 5-fold excess of loop complementary DNA target (green), and in the presence of 5-fold excess of shared-stem complementary DNA target (red). (B) Schematic representation of the phases for the MB solutions in the absence of target DNA (B1), in the presence of loop target DNA (B2) and in the presence of shared-stem target DNA(B3).

Figure 5.

Hybridization of DNA-MB and LNA-MBs to shared-stem target sequences. All MBs had the same sequences. All bases in DNA-MB were DNA and LNA-MB-E0 was fully modified by LNA bases. Every other base in LNA-MB-E1 was a LNA base and every other third base in LNA-MB-E3 was a LNA base. The concentrations of the probes were 100 nM, and the concentration of the target sequence (CCT AGC GCG ACC ATA GTG ATT TAG A) was 500 nM. 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.

Figure 6.

Interactions between MBs and SSBs. (Left) Gel electrophoresis (3% agarose gel) of SSB solutions containing no MB (lane 1), MB-DNA (lane 2), LNA-MB-E5 (lane 3), LNA-MB-E4 (lane 4), LNA-MB-E3 (lane 5), LNA-MB-E2 (lane 6), LNA-MB-E1 (lane 7) and LNA-MB-E0 (lane 8). (Right) Signal enhancement of MBs to the addition of the same concentration of SSB. [MB] = [SSB] = 5 μM.

Figure 7.

Response of MBs to DNase I. To 100 nM of MB in 20 mM Tris-HCl (pH 7.5, 5 mM MgCl₂, 50 mM NaCl), one unit of ribonuclease-free DNase I was added and the subsequent change in fluorescence was recorded at room temperature.

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.

Figure 9.

Effect of LNA composition in a MB on the activity of RNase H. To 100 nM LNA-MB in 20 mM Tris-HCl (5 mM MgCl₂, 50 mM NaCl), RNA target was added to reach a final concentration of 100 nM. After the hybridization reached its steady-state, 12 units of RNase H was added.

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).

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.