ABSTRACT
Conjugate DNAzymes with polyamines and peptides were successfully prepared by solid phase fragment condensation (SPFC) and showed up to 4.2 times higher catalytic efficiency (kcat/Km). Intracellular localization of DNAzymes could be controlled by conjugated with naturally occurring signal peptides which are responsible for nuclear cytoplasmic transport of proteins. Suppression of bcr/abl chimeric gene on Philadelphia chromosome by conjugate DNA enzymes were largely enhanced in human leukemia cells.
Subject(s)
DNA, Catalytic/chemistry , Fusion Proteins, bcr-abl/genetics , Catalysis , DNA, Catalytic/chemical synthesis , DNA, Catalytic/metabolism , Fusion Proteins, bcr-abl/antagonists & inhibitors , Genes, abl , Humans , K562 Cells , Kinetics , Polyamines/chemistry , Protein Sorting Signals , RNA/metabolismABSTRACT
Conjugate DNAzymes with polyamines and peptides were successfully prepared by solid phase fragment condensation (SPFC) and showed up to 4.2 times higher catalytic efficiency (k(cat)/K(m)) and enhanced tolerance against DNase 1digestion. To be pointed out, intracellular localization of DNAzymes could be controlled by conjugated with naturally occurring signal peptides responsible for nuclear cytoplasmic transport of proteins.
Subject(s)
DNA, Catalytic/metabolism , RNA, Viral/metabolism , Base Sequence , Gene Products, gag/genetics , HIV-1/genetics , Hydrolysis , RNA, Viral/chemistryABSTRACT
Conjugate DNAzymes were synthesized by solid phase fragment condensation and their biological properties were characterized. They have increased affinity to target RNA, enhanced stability against DNase 1 digestion and comparable or higher RNA cleaving activity compared with native and also phosphorothioate DNA zymes. It was also demonstrated that conjugate DNAzymes could inhibit BCR-ABL tyrosine kinase in cellular lysis of human leukemia cell line. Consequently DNAzymes can be expected to act effectively in cellular system and also in vivo system.
Subject(s)
DNA, Catalytic/chemistry , Base Sequence , DNA, Catalytic/chemical synthesis , Fusion Proteins, bcr-abl , Humans , Leukemia/enzymology , Peptides/chemistry , Protein-Tyrosine Kinases/antagonists & inhibitors , Tumor Cells, CulturedABSTRACT
Various types of DNA-peptide conjugates were synthesized by solid phase fragment condensation (SPFC). DNA-LNS (nuclear localizing signal) peptide conjugate was proved to be delivered and localized into cellular nucleus and exhibited higher antisense inhibitory effect against telomerase than antisense phosphorothioate DNA. In contrast, DNAzymes conjugated with NES (nuclear export signal) peptide was shown to be taken up and localized in cytoplasm. Inhibitory effect of the conjugate DNAzyme against BCR-ABL tyrosine kinase was evaluated to be more significant than the native DNAzyme.
