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 U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2'-NH2 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 approximately 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.
Beigelman
L
L
Department of Chemistry and Biochemistry, Ribozyme Pharmaceuticals Inc., Boulder, Colorado 80301, USA.
McSwiggen
J A
JA
Draper
K G
KG
Gonzalez
C
C
Jensen
K
K
Karpeisky
A M
AM
Modak
A S
AS
Matulic-Adamic
J
J
DiRenzo
A B
AB
Haeberli
P
P
D000818:Animals; D001483:Base Sequence; D001769:Blood / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D006207:Half-Life; D006801:Humans; D008969:Molecular Sequence Data; D009705:Nucleosides / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism* / Q000493:pharmacokinetics; D012260:Ribonucleases / Q000378:metabolism; D013329:Structure-Activity Relationship
D009705:Nucleosides; D016337:RNA, Catalytic; D012260:Ribonucleases