Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method

Levy, Matan; Lotfian, Pantea; O’Kennedy, Ronan; Lo-Yim, M.Y.A.; Shamlou, P. Ayazi

doi:10.1093/nar/28.12.e57

Cited by 47 publications

(23 citation statements)

References 9 publications

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“…PicoGreen ® binds double-stranded DNA and forms a highly fluorescent complex. Thus, PicoGreen ® is used to follow DNA denaturation due to a decreasing fluorimetric signal intensity, corresponding to the production of ssDNA and mononucleotide content, which can be indicative of DNA damage and possible cell death [33]. At lower doses of Pentostatin, we observed decreased levels of dsDNA relative to uninfected controls, indicative of cell survival (Fig.…”

Section: Effect Of Cordycepin and Pentostatin Treatment On Renal And mentioning

confidence: 75%

Dose finding of 3′deoxyadenosine and deoxycoformycin for the treatment of Trypanosoma evansi infection: An effective and nontoxic dose

Rosa

Silva

Oliveira

et al. 2015

Microbial Pathogenesis

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The aim of this study was to evaluate the therapeutic efficacy and safety of using 3'deoxyadenosine (Cordycepin - adenosine analogue) combined with deoxycoformycin (Pentostatin - an adenosine deaminase inhibitor) in mice infected with Trypanosoma evansi. We show that the combination of Cordycepin (2.0 mg kg(-1)) and Pentostatin (0.2, 0.5, 1.0, 2.0 mg kg(1)) is effective in the clearance of T. evansi, although at the higher concentrations of Pentostatin 2 mg kg(-1) some toxicity was observed in the liver and kidney. Since the Cordycepin 2.0 mg kg(-1) and Pentostatin 0.2 mg kg(-1) combination was effective and had low toxicity, we recommend this as a therapeutic option for a T. evansi mouse model.

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Section: Effect Of Cordycepin and Pentostatin Treatment On Renal And mentioning

confidence: 75%

Dose finding of 3′deoxyadenosine and deoxycoformycin for the treatment of Trypanosoma evansi infection: An effective and nontoxic dose

Rosa

Silva

Oliveira

et al. 2015

Microbial Pathogenesis

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“…Agarose gel electrophoresis of the CEdG‐modified and unmodified plasmid showed, for the latter, a higher prevalence of the intact supercoiled structure. Both heat treatment and CEdG modification have been shown previously to diminish the amount of supercoiled DNA [11,31]. As CEdG‐modified and unmodified plasmids have been heated in parallel, the observed changes in the DNA structure are most likely caused by the CEdG adducts.…”

Section: Discussionmentioning

confidence: 99%

Analysis and biological relevance of advanced glycation end‐products of DNA in eukaryotic cells

et al. 2008

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Advanced glycation end‐products (AGEs) of DNA are formed spontaneously by the reaction of carbonyl compounds such as sugars, methylglyoxal or dihydroxyacetone in vitro and in vivo. Little is known, however, about the biological consequences of DNA AGEs. In this study, a method was developed to determine the parameters that promote DNA glycation in cultured cells. For this purpose, the formation rate of N2‐carboxyethyl‐2′‐deoxyguanosine (CEdG), a major DNA AGE, was measured in cultured hepatic stellate cells by liquid chromatography (LC)‐MS/MS. In resting cells, a 1.7‐fold increase of CEdG formation rate was observed during 14 days of incubation. To obtain insights into the functional consequences of DNA glycation, CEdG was introduced into a luciferase reporter gene vector and transfected into human embryonic kidney (HEK 293 T) cells. Gene activity was determined by chemiluminescence of the luciferase. Thus, CEdG adducts led to a dose‐dependent and highly significant decrease in protein activity, which is caused by loss of functionality of the luciferase in addition to reduced transcription of the gene. When the CEdG‐modified vector was transformed into Escherichia coli, a loss of ampicillin resistance was observed in comparison to transformation with the unmodified plasmid. These results indicate that CEdG accumulates in the genomic DNA of resting cells, which could lead to diminished protein activity.

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“…At predetermined time intervals, the CMCS-CLDPD suspensions were centrifuged (15,000 rpm for 30 minutes) and the amount of DNA released in the supernatant was analyzed using fluorospectrophotometry with PicoGreen™ double-stranded DNA quantitative reagent. 22 Background readings were obtained using the supernatants from PBS at pH 5.5. Fluorescence was measured by fluorescence spectrophotometer (model 850; Hitachi High-Technologies, Tokyo, Japan) at excitation and emission wavelengths of 480 nm and 520 nm, respectively.…”

Section: Ph Sensitivity Analysis Of Cmcs-cldpdmentioning

confidence: 99%

A pH-sensitive multifunctional gene carrier assembled via layer-by-layer technique for efficient gene delivery

Liu²,

Miao³

et al. 2012

IJN

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Background:The success of gene therapy asks for the development of multifunctional vectors that could overcome various gene delivery barriers, such as the cell membrane, endosomal membrane, and nuclear membrane. Layer-by-layer technique is an efficient method with easy operation which can be used for the assembly of multifunctional gene carriers. This work describes a pH-sensitive multifunctional gene vector that offered long circulation property but avoided the inhibition of tumor cellular uptake of gene carriers associated with the use of polyethylene glycol. Methods: Deoxyribonucleic acid (DNA) was firstly condensed with protamine into a cationic core which was used as assembly template. Then, additional layers of anionic DNA, cationic liposomes, and o-carboxymethyl-chitosan (CMCS) were alternately adsorbed onto the template via layer-by-layer technique and finally the multifunctional vector called CMCS-cationic liposome-coated DNA/protamine/DNA complexes (CLDPD) was constructed. For in vitro test, the cytotoxicity and transfection investigation was carried out on HepG2 cell line. For in vivo evaluation, CMCS-CLDPD was intratumorally injected into tumor-bearing mice and the tumor cells were isolated for fluorescence determination of transfection efficiency. Results: CMCS-CLDPD had ellipsoidal shapes and showed "core-shell" structure which showed stabilization property in serum and effective protection of DNA from nuclease degradation. In vitro and in vivo transfection results demonstrated that CMCS-CLDPD had pH-sensitivity and the outermost layer of CMCS fell off in the tumor tissue, which could not only protect CMCS-CLDPD from serum interaction but also enhance gene transfection efficiency. Conclusion: These results demonstrated that multifunctional CMCS-CLDPD had pH-sensitivity, which may provide a new approach for the antitumor gene delivery.

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Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method

Cited by 47 publications

References 9 publications

Dose finding of 3′deoxyadenosine and deoxycoformycin for the treatment of Trypanosoma evansi infection: An effective and nontoxic dose

Dose finding of 3′deoxyadenosine and deoxycoformycin for the treatment of Trypanosoma evansi infection: An effective and nontoxic dose

Analysis and biological relevance of advanced glycation end‐products of DNA in eukaryotic cells

A pH-sensitive multifunctional gene carrier assembled via layer-by-layer technique for efficient gene delivery

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