8-17 DNAzyme Silencing Gene Expression in Cells via Cleavage and Antisense

Abstract

Gene silencing is an important biological strategy for studying gene functions, exploring disease mechanisms and developing therapeutics. 8-17 DNAzyme is of great potential for gene silencing, due to its higher RNA-cleaving activity. However, it is not generally used in practice, due to its divalent cation dependence and poor understanding of its cellular mechanisms. To address these issues, we have explored its activity in vitro and in cells and found that it can cleave RNA substrates under the simulated physiological conditions, and its gene-silencing activity is additionally enhanced by its RNase H compatibility, offering both cleavage and antisense activities in cells. Further, chemical modifications can facilitate its stability, substrate binding affinity and gene-silencing activity. Our research results suggest that this DNAzyme can demonstrate high levels of activities for both actions in cells, making it a useful tool for exploring biomedical applications.

Keywords: 8–17 DNAzyme; antisense; catalysis; chemical modifications; gene silencing.

Figure 1

8–17 DNAzyme exerts high gene-silencing effect via catalytic cleavage and antisense activities.

Figure 2

Finding the DNAzyme with gene-silencing activity in cells. (a) In vitro cleavage study of twelve Dz8–17 DNAzymes via agarose gel analysis. All reactions were carried out under simulated physiological conditions (150 mM KCl, 0.5 mM MgCl₂, pH 7.5) at 37 °C for 2 h. s: substrate; P1: product 1; P2: product 2. (b) EGFP gene-silencing study with the DNAzymes in cells. The fluorescent data were collected 48 h after transfection and reported as mean value ± SD of three repeats. The transfection concentrations of the DNAzymes were 400 nM, and Mock was a negative control using ultrapure water instead of a DNAzyme. NSP: negative DNAzyme control with nonspecific arm sequences. Each DNAzyme and control were labeled with different colors.

Figure 3

Identification the DNAzyme (Dz04) gene-silencing activity in cells. (a) The mismatched arm sequences of Dz04 compromised the EGFP gene silencing, and the data significance was determined by a Student’s t-test (*** p < 0.001, **** p < 0.0001); Each DNAzyme and mock control were labeled with different colors. (b) The gene-silencing activities of Dz04 at different concentrations.

Figure 4

The DNAzyme (Dz04) with both cleavage and antisense activities in cells. (a,b) are secondary structures of Dz04, Dz4M and Dz04-4s. (c) The in vitro cleavage activities of Dz04-4s and Dz4M-4s under simulated physiological conditions. All reactions carried out in 1.0 μM Dz8–17, 1.0 μM substrates, 150 mM KCl and 0.5 mM MgCl₂ at 37 °C for 2 h. (d) The cellular mRNA degradation by Dz04-4s and Dz4M-4s was analyzed by RT-qPCR, 24 h after the DNAzyme transfection. Stu-dent’s t-tests were used to assess significance of the data according to mean values and SDs of three repeats (* p < 0.05). Dz04, Dz4M and mock control were labeled with different colors.

Figure 5

In vitro studies of stability, catalysis and RNase H compatibility of the DNAzymes with arm modifications. (a) Phosphorothioate linkage (PS), 2′-O-methyl ribonucleotide (OMe), 2′-O and 4′-C LNA (LNA) are represented by “s”, “underlined” and “box” labels, respectively. (b) Stability study of the modified DNAzymes under FBS serum treatment. (c) In vitro cleavage study of the modified DNAzymes under physiological conditions. S: substrate; P: product. (d) Modified DNAzyme compatibility with RNase H.

Figure 6

The modified DNAzyme (Dz04) with high gene-silencing effect in cells. (a) Effect of Dz04 PS modifications on gene silencing. Statistical significance was determined by a Student’s t-test of three repeats (** p < 0.01). (b) Gene silencing of Dz04 with different chemical modifications. The fluorescent data significance was determined by a Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001). Dz04-4s, Dz04-4OMe and Dz04-4LNA contained two PS, OMe and LNA modifications at both 5′ and 3′ ends of Dz04, respectively. Dz04-4s(2LNA)m contained one LNA in the middle of each Dz04-4s arm. Dz04-4s(2OMe)m and Dz04-4s(4OMe)m contained one and two OMe modifications in the middle of each Dz04-4s arm, respectively; Dz4M-4LNA contained two LNAs at both 5′ and 3′ ends of Dz4M (Figure 4b). Dz4S-4s, Dz4S-4OMe and Dz4S-4LNA contained two PS, OMe and LNA modifications at both 5′ and 3′ ends of Dz4S, respectively. Dz4S-4s(2LNA)m contained one LNA in the middle of each Dz4S-4s arm. Dz4S-4s(2OMe)mand Dz4S-4s(4OMe)m contained one and two OMe modifications in the middle of each Dz4S-4s arm, respectively. Their detailed sequences are shown in Table S3. (c) Cellular fluorescent images of EGFP gene silencing with the modified DNAzymes in Figure 6b. Scale bar: 500 μm.