Efficient silencing of gene expression by an ASON-bulge-DNAzyme complex

Abstract

Background: DNAzymes are DNA molecules that can directly cleave cognate mRNA, and have been developed to silence gene expression for research and clinical purposes. The advantage of DNAzymes over ribozymes is that they are inexpensive to produce and exhibit good stability. The "10-23 DNA enzyme" is composed of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of approximately eight nucleotides in each direction, which provides the complementary sequence required for specific binding to RNA substrates. However, these eight nucleotides might not afford sufficient binding energy to hold the RNA substrate along with the DNAzyme, which would interfere with the efficiency of the DNAzyme or cause side effects, such as the cleavage of non-cognate mRNAs.

Methodology: In this study, we inserted a nonpairing bulge at the 5' end of the "10-23 DNA enzyme" to enhance its efficiency and specificity. Different sizes of bulges were inserted at different positions in the 5' end of the DNAzyme. The non-matching bulge will avoid strong binding between the DNAzyme and target mRNA, which may interfere with the efficiency of the DNAzyme.

Conclusions: Our novel DNAzyme constructs could efficiently silence the expression of target genes, proving a powerful tool for gene silencing. The results showed that the six oligo bulge was the most effective when the six oligo bulge was 12-15 bp away from the core catalytic domain.

Figure 1. Schematic of the structure of ASON-Bulge-DNAzyme.

A. Regular DNAzyme. B. ASON-Bulge-DNAzyme. A 15 base AS-ON was inserted at the 5′ end of “10-23”deoxyribozyme to target the mRNA substrate and an unmatched loop between the DNAzyme sequence and ASON sequence was designed.

Figure 2. Transfection of ASON-Bulge-DNAzyme resulted in significantly reduced eGFP expression.

BHK21 cells were co-transfected with 0.5 µg pECFP-C1 plasmid and 0.25 µg of DNAzymes A. pEGFP control; B. Regular DNAzyme-1; C. ASON-Bulge-DNAzyme-1; D. Regular DNAzyme-2; E.ASON-Bulge-DNAzyme-2. Calibration bar for A-E, 100 µm.

Figure 3. The effect of different bulge size on DNAzyme efficiency.

Cells were cotransfected with 0.5 µg pECFP-C1 plasmid and 0.25 µg of DNAzymes. 1. pEGFP control; 2. Regular DNAzyme; 3. 3nt-Bulge-DNAzyme; 4. 6nt-Bulge-DNAzyme; 5. 9nt-Bulge-DNAzyme; 6. 12nt-Bulge-DNAzyme. The error bars denote Standard deviation (SD).

Figure 4. The effect of different bulge positions on DNAzyme efficiency.

Cells were co-transfected with 0.5 µg pECFP-C1 plasmid and 0.25 µg of DNAzymes. 1. pEGFP control; 2. Regular DNAzyme; 3. 6nt; 4. 9nt; 5. 12nte; 6.15 nt. The error bars denote Standard deviation (SD).

Figure 5. Quantitative assessment of the silencing efficiency of ASON-Bulge-DNAzyme by GFP fluorescence assay.

BHK21 cells were harvested and lysed in 200 µl lysis buffer (0.1 M Tris-HCl, 0.1% Triton X-100, 2 mM EDTA, pH 7.8). Thefluorescence of eGFP was measured using a luminometer. The error bars denote Standard deviation (SD).

Figure 6. Semi-quantitative RT-PCR and Western blot analysis of the mechanism of ASONBulge-DNAzyme.

A. RT-PCR of eGFP. B. RT-PCR of β-actin. C. Western blot of eGFP.

Figure 7. Quantitative assessment of the silencing efficiency of ASON-Bulge-DNAzyme against luciferase.

BHK21 cells were harvested and lysed by in 200 µl lysis buffer (0.1 M Tris-HCl, 0.1% Triton X-100, 2 mM EDTA, pH 7.8). The luciferase activity was measured using a luminometer. The error bars denote Standard deviation (SD).

Figure 8. ASON-Bulge-DNAzyme can efficiently silence the expression of endogenous cellular genes.

BHK21 cells were transfected with 0.25 µg of DNAzyme against integrin β1 in each well of a 24-well plate. RT-PCR was performed at 36 h after transfection. ASON-DNAzyme significantly reduced the mRNA level of integrin β1, but not integrin β3.