Highly active low magnesium hammerhead ribozyme.
Hammerhead (HH) ribozymes can be used for highly specific inhibition of gene expression through the degradation of target mRNA. In vitro experiments with minimal HH domains demonstrated that the efficiency of catalysis is highly dependent on concentration of magnesium ions. Optimal ion requirements for HH-catalysed RNA cleavage are far from these found in the cell. Recently, it has been proposed that the efficiency of HH ribozymes can be increased at low magnesium concentration through stabilization of a catalytically active conformation by tertiary interactions between helices I and II. We designed a ribozyme stabilized by GAAA tetraloop and its receptor motifs and demonstrated that it can efficiently catalyse target RNA hydrolysis at submillimolar Mg(2+) concentrations in vitro as well as in cultured cells. Both unmodified and locked nucleic acid-modified extended ribozymes proved superior to the minimal core ribozyme and DNAzyme against the same target sequence.
Fedoruk-Wyszomirska
Agnieszka
A
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, Poland.
Szymański
Maciej
M
Wyszko
Eliza
E
Barciszewska
Mirosława Z
MZ
Barciszewski
Jan
J
D055162:Biocatalysis / Q000187:drug effects; D021881:DNA, Catalytic / Q000737:chemistry / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015700:HIV Envelope Protein gp41 / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
D021881:DNA, Catalytic; D015700:HIV Envelope Protein gp41; D016337:RNA, Catalytic; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C427204:hammerhead ribozyme; D049452:Green Fluorescent Proteins; D008274:Magnesium