Tandem DNAzymes for mRNA cleavage: Choice of enzyme, metal ions and the antisense effect

Wang, Feng; Saran, Runjhun; Liu, Juewen

doi:10.1016/j.bmcl.2015.02.032

Cited by 35 publications

(35 citation statements)

References 37 publications

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“…This supports the concern that RNA cleavage by the 10-23 DNAzyme in biological buffer conditions is hindered due to a lack of metal ions. 25,26,57 On the other hand, the 17E…”

Section: Dna Hybridization Kineticsmentioning

confidence: 99%

DNAzyme Hybridization, Cleavage, Degradation, and Sensing in Undiluted Human Blood Serum

et al. 2015

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RNA-cleaving DNAzymes provide a unique platform for developing biosensors. However, a majority of the work has been performed in clean buffer solutions, while the activity of some important DNAzymes in biological sample matrices is still under debate. Two RNA-cleaving DNAzymes (17E and 10-23) are the most widely used. In this work, we carefully studied a few key aspects of the 17E DNAzyme in human blood serum, including hybridization, cleavage activity and degradation kinetics. Since direct fluorescence monitoring is difficult due to the opacity of serum, denaturing and non-denaturing gel electrophoresis were combined for studying the interaction between serum proteins and DNAzymes. The 17E DNAzyme retains its activity in 90% human blood serum with a cleavage rate of 0.04 min -1 , which is similar to that in the PBS buffer (0.06 min -1 ) with a similar ionic strength. The activity in serum can be accelerated to 0.3 min -1 with an additional 10 mM Ca 2+ . As compared to 17E, the 10-23 DNAzyme produces negligible cleavage in serum. Degradation of both the substrate and the DNAzyme strand is very slow in serum, especially at room temperature. Degradation occurs mainly at the fluorophore label (linked to DNA via an amide bond) instead of the DNA phosphodiester bonds. Serum proteins can bind more tightly to the 17E DNAzyme complex than to the single-stranded substrate or enzyme.The 17E DNAzyme hybridizes extremely fast in serum. With these understandings, detection of DNA using the 17E DNAzyme is demonstrated in serum.3

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“…This supports the concern that RNA cleavage by the 10-23 DNAzyme in biological buffer conditions is hindered due to a lack of metal ions. 25,26,57 On the other hand, the 17E…”

Section: Dna Hybridization Kineticsmentioning

confidence: 99%

DNAzyme Hybridization, Cleavage, Degradation, and Sensing in Undiluted Human Blood Serum

et al. 2015

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“…As shown in Figure 7, N-Ac-l-Leu-PEI/Dz and Lipofectamine™2000/ Dz groups could efficiently downregulate the expression level of target aurora kinase A, whereas the iDz transfection showed a relatively weak inhibition on the expression of aurora kinase A with 87% of original expression level. The decreased expression induced by iDz transfection was mainly caused by the simple antisense effect, 37,38 accounting for the slight inhibition of cell proliferation and colony formation described earlier. Meanwhile, DNAzyme delivery mediated by N-Ac-l-Leu-PEI could inhibit the expression level of aurora kinase A in a higher level than Lipofectamine™2000 did.…”

mentioning

confidence: 99%

“…The apoptosis induction mediated by iDz transfection could also be attributed to the partial cleavage of aurora kinase A mRNA owing to the simple antisense effect. 37,38 Furthermore, the cell cycle analysis was performed via flow cytometry to elucidate whether the antiproliferative effect of DNAzyme transfection was associated with cell cycle arrest ( Figure 9). Compared with the control, DNAzyme delivery would lead to a significant increase in the G2 phase, indicating that downregulating the expression level of aurora kinase A could cause the disruption of cell cycle transition and an arrest in G2 phase.…”

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confidence: 99%

“…The phenomenon was probably caused by iDz's cleavage of target mRNA due to a simple antisense effect, especially at a high concentration of DNAzyme, which was consistent with previous reports. 37,38 To elucidate whether the inhibition of cell proliferation was triggered by DNAzyme-induced gene knockout, the expression level of aurora kinase A was evaluated at the protein level through Western blotting. As shown in Figure 7, N-Ac-l-Leu-PEI/Dz and Lipofectamine™2000/ Dz groups could efficiently downregulate the expression level of target aurora kinase A, whereas the iDz transfection showed a relatively weak inhibition on the expression of aurora kinase A with 87% of original expression level.…”

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confidence: 99%

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Delivery of DNAzyme targeting aurora kinase A to inhibit the proliferation and migration of human prostate cancer

Xing¹,

Gao²,

Duan³

et al. 2015

IJN

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Herein, a polyethylenimine derivative N -acetyl- l -leucine-polyethylenimine ( N -Ac- l -Leu-PEI) was employed as a carrier to achieve the delivery of DNAzyme targeting aurora kinase A using PC-3 cell as a model. Flow cytometry and confocal laser scanning microscopy demonstrated that the derivative could realize the cellular uptake of nanoparticles in an energy-dependent and clathrin-mediated pathway and obtain a high DNAzyme concentration in the cytoplasm through further endosomal escape. After DNAzyme transfection, expression level of aurora kinase A would be downregulated at the protein level. Meanwhile, the inhibition of cell proliferation was observed through 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cell colony formation assay, attributing to the activation of apoptosis and cell cycle arrest. Through flow cytometric analysis, an early apoptotic ratio of 25.93% and G2 phase of 22.58% has been detected after N -Ac- l -Leu-PEI-mediated DNAzyme transfection. Finally, wound healing and Transwell migration assay showed that DNAzyme transfection could efficiently inhibit the cell migration. These results demonstrated that N -Ac- l -Leu-PEI could successfully mediate the DNAzyme delivery and downregulate the expression level of aurora kinase A, triggering a significant inhibitory effect of excessive proliferation and migration of tumor cells.

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“…[2][3][4][5] While most studies were focused on environmental metal analysis, in recent years we see a growing trend of using DNAzymes for biological samples, such as serum, 6,7 cells, [8][9][10][11][12][13][14] and cell extracts. 15 Ca 2+ and Mg 2+ are the most abundant metal ions in biological fluids, and they are critical in cell signaling, protein folding, and catalysis. Therefore, their detection has been a longstanding topic of bioanalytical chemistry.…”

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confidence: 99%

In Vitro Selection in Serum: RNA-Cleaving DNAzymes for Measuring Ca²⁺ and Mg²⁺

et al. 2016

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RNA-cleaving DNAzymes have been attempted as in vivo analytical probes and gene silencing reagents over the past two decades. Despite progress already achieved, concerns still exist regarding the activity of DNAzymes in biological fluids. An example is the low activity of the 10–23 DNAzyme in intracellular Mg2+ concentrations. To obtain DNAzymes that work optimally in biological samples, we herein report the first DNAzyme in vitro selection in undiluted human blood serum. The selection starts with a large DNA library containing 50 random nucleotides, and sequences that can be cleaved in serum were isolated and amplified. After deep sequencing analysis, 80% of the final library are a variant of the 8–17 DNAzyme (named 17EV1). The main difference between 17E and 17EV1 is a single mutation at the N12 position of the catalytic core. 17EV1 is ∼6-fold faster in serum than 17E, since 17EV1 is preferentially activated by Ca2+ and serum is rich in Ca2+ over Mg2+. On the other hand, 17E has a similar activity with Ca2+ or Mg2+. With this observation, a method for measuring the Ca2+/Mg2+ ratio was developed by combining the 17E and 17EV1 DNAzymes. This study demonstrates the feasibility of selecting DNAzymes in biological fluids and will facilitate the application of DNAzymes in bioanalytical chemistry and gene therapy.

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Tandem DNAzymes for mRNA cleavage: Choice of enzyme, metal ions and the antisense effect

Cited by 35 publications

References 37 publications

DNAzyme Hybridization, Cleavage, Degradation, and Sensing in Undiluted Human Blood Serum

DNAzyme Hybridization, Cleavage, Degradation, and Sensing in Undiluted Human Blood Serum

Delivery of DNAzyme targeting aurora kinase A to inhibit the proliferation and migration of human prostate cancer

In Vitro Selection in Serum: RNA-Cleaving DNAzymes for Measuring Ca²⁺ and Mg²⁺

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Tandem DNAzymes for mRNA cleavage: Choice of enzyme, metal ions and the antisense effect

Cited by 35 publications

References 37 publications

DNAzyme Hybridization, Cleavage, Degradation, and Sensing in Undiluted Human Blood Serum

DNAzyme Hybridization, Cleavage, Degradation, and Sensing in Undiluted Human Blood Serum

Delivery of DNAzyme targeting aurora kinase&nbsp;A to inhibit the proliferation and migration of human prostate cancer

In Vitro Selection in Serum: RNA-Cleaving DNAzymes for Measuring Ca2+ and Mg2+

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Product

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Delivery of DNAzyme targeting aurora kinase A to inhibit the proliferation and migration of human prostate cancer

In Vitro Selection in Serum: RNA-Cleaving DNAzymes for Measuring Ca²⁺ and Mg²⁺