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. 2023 May 7;28(9):3942.
doi: 10.3390/molecules28093942.

Studies on the Effect of Lipofectamine and Cell-Penetrating Peptide on the Properties of 10-23 DNAzyme

Huanhuan Liu  1   2 , Yang Li  2 , Shanshan Du  2 , Chenhong Wang  2 , Yuexiang Li  2 , Ruiyuan Cao  2 , Weiguo Shi  2 , Shihui Liu  1 , Junlin He  2
Affiliations

Studies on the Effect of Lipofectamine and Cell-Penetrating Peptide on the Properties of 10-23 DNAzyme

Huanhuan Liu et al. Molecules. .

Abstract

Cationic polymeric materials and cell-penetrating peptides (CPPs) were often used as the delivery vectors in the evaluation of nucleic acid therapeutics. 10-23 DNAzyme is a kind of potential antisense therapeutics by catalytic cleavage of the disease-related RNAs. Here, lipofectamine 2000 and Tat peptide were evaluated for their effect on the catalytic activity of 10-23 DNAzyme, with the observed rate constant, thermal stability, CD spectra, and PAGE analysis, with a duplex DNA mimicking DNAzyme-substrate as a control. It was shown that the cationic carriers had a negative effect on the catalytic performance of the 10-23 DNAzyme. Significantly, the destabilizing effect of the cationic carriers on the duplex formation was noteworthy, as a duplex formation is an essential prerequisite in the silencing mechanisms of antisense and RNAi.

Keywords: 10-23 DNAzyme; CD spetra; PAGE analysis; Tm; cell-penetrating peptide; lipofectamine 2000; observed rate constant.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The secondary structure of 10-23 DNAzyme-substrate complex. N constitutes the two recognition arms, with base-pairing specificity for N′ of the substrate. R is purine and Y is pyrimidine residue in the substrate, and the cleavage site indicated by an arrow.
Figure 2
Figure 2
The effect of Tat on the CD spectra of the tertiary structures: (a) CD spectra of DZ01+D19 in the absence and presence of Tat at different N/P ratios; (b) CD spectra of D19+D19S in the absence and presence of Tat at different N/P ratios.
Figure 3
Figure 3
The effect of Tat on melting profiles of the tertiary structure formation: (a) the melting curve of DZ19+D19S in the absence and presence of Tat at different N/P ratios; (b) the melting curve of DZ01+D19 in the absence and presence of Tat at different N/P ratios.
Figure 4
Figure 4
PAGE analysis of DZ01+D19 and D19+D19S in the presence of Tat. (A–E) denaturing PAGE of the catalytic reaction profiles of DZ01 in the presence of Tat with N/P of 1:0.34 (A), 1:0.68 (B), 1:1.36 (C), 1:2.72 (D), and 1:5.44 (E), with sampling time point: 0, 0.5, 1, 3, 5, 7 h. (F) DZ01+D19 complex formation in the reaction buffer. (G) DZ01/Tat (0.5:1 or 1:1) + D19 with different incubation times (0, 0.5, 1, 3, 5, 7 h); (H) D19/Tat (1:0.5 or 1:1) + D19S with different incubation times (1, 0.5, 1, 3, 5, 7 h); (I) D19/Tat (1:2) + D19S with different incubation times.
Figure 4
Figure 4
PAGE analysis of DZ01+D19 and D19+D19S in the presence of Tat. (A–E) denaturing PAGE of the catalytic reaction profiles of DZ01 in the presence of Tat with N/P of 1:0.34 (A), 1:0.68 (B), 1:1.36 (C), 1:2.72 (D), and 1:5.44 (E), with sampling time point: 0, 0.5, 1, 3, 5, 7 h. (F) DZ01+D19 complex formation in the reaction buffer. (G) DZ01/Tat (0.5:1 or 1:1) + D19 with different incubation times (0, 0.5, 1, 3, 5, 7 h); (H) D19/Tat (1:0.5 or 1:1) + D19S with different incubation times (1, 0.5, 1, 3, 5, 7 h); (I) D19/Tat (1:2) + D19S with different incubation times.
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