Correlation of selective modifications to a 2',5'-oligoadenylate-3',5'-deoxyribonucleotide antisense chimera with affinity for the target nucleic acid and with ability to activate RNase L.
The use of an antisense oligonucleotide to address a specific targeted RNA sequence and subsequent localized activation of the 2-5A-dependent RNase (RNase L) to effect selective RNA degradation is a new approach to the control of gene expression called 2-5A-antisense. The previously reported biological activity of the 2-5A:AS chimeric oligonucleotide [p5'(A2'p)3A-antiPKR1], directed against nucleotides 55-73 of the coding sequence of the PKR mRNA, has been used as a point of reference to examine the effect of introducing mismatches into the chimeric oligonucleotide, altering the chain length of the antisense domain of the chimeras, removal of the 5'-monophosphate moiety, shortening the 2',5'-oligoadenylate domain, and substitution of 3',5'-linked 2'-deoxyadenosine nucleotides for the 2-5A domain. The general formula for the novel chimeric oligonucleotides is p5'(A2'p)3A2'p(CH2)4p(CH2)4p(5'N3'p)mN, where N is any nucleoside and m is any integer. When the biological activity of these new chimeric oligonucleotides was compared to that of the parent chimera, 2-5A-aPKR, for their ability to effect target PKR RNA cleavage in a cell-free and in an intact cell assay, it was determined that there was a close correlation between the activity of 2-5A-antisense chimeras and their affinity (Tm) for a targeted nucleic acid. In addition, there was also a close correlation between activity of the 2-5A-antisense chimeras and their ability to activate the 2-5A-dependent RNase L.
Xiao
W
W
Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0815, USA.
Li
G
G
Maitra
R K
RK
Maran
A
A
Silverman
R H
RH
Torrence
P F
PF
D000227:Adenine Nucleotides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000378:metabolism; D009843:Oligoribonucleotides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013873:Thionucleotides / Q000737:chemistry* / Q000378:metabolism; D019892:eIF-2 Kinase
D000227:Adenine Nucleotides; D009692:Nucleic Acid Heteroduplexes; D016376:Oligonucleotides, Antisense; D009843:Oligoribonucleotides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; C023505:2',5'-oligoadenylate; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease