Stability and functional effectiveness of phosphorothioate modified duplex DNA and synthetic 'mini-genes'.
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.
D001483:Base Sequence; D001769:Blood; D002457:Cell Extracts; D003470:Culture Media; D004247:DNA / Q000138:chemical synthesis* / Q000187:drug effects / Q000378:metabolism*; D011088:DNA Ligases / Q000378:metabolism; D017931:DNA Primers / Q000138:chemical synthesis; D004259:DNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I / Q000494:pharmacology; D004706:Endodeoxyribonucleases / Q000494:pharmacology; D005813:Genes, Synthetic / Q000235:genetics; D008969:Molecular Sequence Data; D009124:Muscle Proteins; D016133:Polymerase Chain Reaction / Q000379:methods; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D019914:Taq Polymerase; D013873:Thionucleotides; D014158:Transcription, Genetic; D014764:Viral Proteins
D002457:Cell Extracts; D003470:Culture Media; D017931:DNA Primers; D009124:Muscle Proteins; D012333:RNA, Messenger; D013873:Thionucleotides; D014764:Viral Proteins; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D019914:Taq Polymerase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; C038407:exonuclease Bal 31; D003850:Deoxyribonuclease I; D011088:DNA Ligases