Susan Grimm

Chemical modification of hammerhead ribozymes. Catalytic activity and nuclease resistance.

A systematic study of selectively modified, 36-mer hammerhead ribozymes has resulted in the identification of a generic, catalytically active and nuclease stable ribozyme motif containing 5 ribose residues, 29-30 2′-O-Me nucleotides, 1-2 other 2′-modified nucleotides at positions U4 and U7, and a 3′-3′-linked nucleotide “cap.” Eight 2′-modified uridine residues were introduced at positions U4 and/or U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2′-NH substitutions at U4 and U7, or 2′-C-allyl substitutions at U4, retain most of their catalytic activity when compared to the all-RNA parent. Their serum half-lives were 5-8 h in a variety of biological fluids, including human serum, while the all-RNA parent ribozyme exhibits a stability half-life of only 0.1 min. The addition of a 3′-3′-linked nucleotide “cap” (inverted T) did not affect catalysis but increased the serum half-lives of these two ribozymes to >260 h at nanomolar concentrations. This represents an overall increase in stability/activity of 53,000-80,000-fold compared to the all-RNA parent ribozyme.

Three cytoplasmic DNA polymerases that utilize poly(rA)-oligo(dT)

Three DNA polymerases that use poly(rA).oligo(dT) were partially purified from cytoplasmic extracts of cultured mouse cells (after removal of mitochondria), and characterized. One is similar to, and may be the same as, the mitochondrial DNA polymerase gamma. The other two enzymes, one 7.5 S and the other 3.6 S, share some properties with DNA polymerases beta and gamma, e.g. their responses to certain inhibitors; however, they are not clearly identified with any previously well-characterized mammalian DNA polymerases. It is also demonstrated that the response of DNA polymerase gamma to N-ethylmaleimide is template dependent, and that DNA polymerase alpha has an authentic (albeit small) activity with poly(rA).oligo(dT).

2′-SUBSTITUTED PHOSPHOROTHIOATE CONTAINING OLIGORIBONUCLEOTIDES: AN APPLICATION TO THE SYNTHESIS AND PURIFICATION OF HAMMERHEAD RIBOZYMES

Abstract A systematic study of the catalytic activity and nuclease stability of selectively modified hammerhead ribozymes has resulted in the identification of a generic motif containing 5 ribose residues and 31 2′- modified sugars (1). This substructure has been further elaborated to include phosphorothioate linkages. Although oligodeoxyribonucleotides containing phosphorothioate linkages have been studied extensively, similarly substituted RNA molecules or ribozymes have not been explored at-length. The synthesis and purification of these ribozymes is discussed (2).