Several gene transfer techniques that employ 'naked DNA' molecules have recently been developed and numerous gene therapy protocols that make use of 'naked-DNA' have been proposed. We studied the possibility of enhancing the stability of 'naked DNA vectors' and thus also gene transfer and expression efficiencies, by constructing phosphorothioate (PS-) double strand DNA molecules and functional transcription units. We first synthesized short PS-double strand DNA molecules by the annealing of two complementary, 35 nt long, oligonucleotides. The accessibility of DNA modifying enzymes to this molecule was significantly decreased: T4-ligase and kinase activity were respectively reduced up to 1/2 and to 1/6, as compared to the normal phosphodiester molecule. Nucleolytic stability was increased either to purified enzymes (DNase I and Bal31) or to incubations in fresh serum, cell culture medium or in muscle protein extract. Phosphorothioate end-capped complete eukaryotic transcription units (obtained by Taq polymerase amplification with PS-primers) were not significantly protected from nucleolytic attack. On the contrary, synthetic transcription units, 'mini genes', obtained by Taq amplification with 1, 2 or 3 PS-dNTP substitutions, were resistant to DNase I and Bal31 nucleolytic activity. Transcription efficiency, driven by the T7 promoter, was 96.5, 95 and 33.5% (respectively with 1, 2 or 3 substitutions), as compared to the normal phosphodiester molecules.

Ciafre', S.a., Rinaldi, M., Gasparini, P., Seripa, D., Bisceglia, L., Zelante, L., et al. (1995). Stability and functional effectiveness of phosphorothioate modified duplex DNA and synthetic 'mini-genes'. NUCLEIC ACIDS RESEARCH, 23(20), 4134-4142.

Stability and functional effectiveness of phosphorothioate modified duplex DNA and synthetic 'mini-genes'

CIAFRE', SILVIA ANNA;FARACE, MARIA GIULIA;
1995-10-25

Abstract

Several gene transfer techniques that employ 'naked DNA' molecules have recently been developed and numerous gene therapy protocols that make use of 'naked-DNA' have been proposed. We studied the possibility of enhancing the stability of 'naked DNA vectors' and thus also gene transfer and expression efficiencies, by constructing phosphorothioate (PS-) double strand DNA molecules and functional transcription units. We first synthesized short PS-double strand DNA molecules by the annealing of two complementary, 35 nt long, oligonucleotides. The accessibility of DNA modifying enzymes to this molecule was significantly decreased: T4-ligase and kinase activity were respectively reduced up to 1/2 and to 1/6, as compared to the normal phosphodiester molecule. Nucleolytic stability was increased either to purified enzymes (DNase I and Bal31) or to incubations in fresh serum, cell culture medium or in muscle protein extract. Phosphorothioate end-capped complete eukaryotic transcription units (obtained by Taq polymerase amplification with PS-primers) were not significantly protected from nucleolytic attack. On the contrary, synthetic transcription units, 'mini genes', obtained by Taq amplification with 1, 2 or 3 PS-dNTP substitutions, were resistant to DNase I and Bal31 nucleolytic activity. Transcription efficiency, driven by the T7 promoter, was 96.5, 95 and 33.5% (respectively with 1, 2 or 3 substitutions), as compared to the normal phosphodiester molecules.
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/13
English
Con Impact Factor ISI
Muscle Proteins; DNA; Endodeoxyribonucleases; Polynucleotide 5'-Hydroxyl-Kinase; Genes, Synthetic; Base Sequence; Thionucleotides; Blood; Deoxyribonuclease I; Culture Media; DNA-Directed RNA Polymerases; Cell Extracts; DNA-Directed DNA Polymerase; RNA, Messenger; Polymerase Chain Reaction; DNA Primers; Taq Polymerase; Molecular Sequence Data; Viral Proteins; Transcription, Genetic; DNA Ligases
Ciafre', S.a., Rinaldi, M., Gasparini, P., Seripa, D., Bisceglia, L., Zelante, L., et al. (1995). Stability and functional effectiveness of phosphorothioate modified duplex DNA and synthetic 'mini-genes'. NUCLEIC ACIDS RESEARCH, 23(20), 4134-4142.
Ciafre', Sa; Rinaldi, M; Gasparini, P; Seripa, D; Bisceglia, L; Zelante, L; Farace, Mg; Fazio, V
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/9173
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