A ribonuclease H-oligo DNA conjugate that specifically cleaves hepatitis B viral messenger RNA.
Ribonuclease H (RNaseH) recognizes and efficiently cleaves the RNA strand of DNA-RNA hybrids, but has no inherent sequence selectivity. However, the formation of DNA-RNA hybrids does require specific sequence recognition. On the basis of this concept, we wondered whether antisense oligonucleotides complementary to target RNA covalently linked to RNase H could be used to direct specific cleavage events mediated by RNase H. The aim of this research was to couple a DNA oligonucleotide to RNase H to confer specificity of ribonuclease activity toward hepatitis B viral (HBV) mRNA. A modified 13-base oligonucleotide that is specific for the DR1 region of HBV mRNA was conjugated to modified E. coli RNase H using a water soluble cross-linker. A 1200 base fragment of HBV RNA including the DR1 region was synthesized as a substrate using T7 RNA polymerase. Incubation of the RNase H-oligonucleotide conjugate with the RNA transcript resulted in cleavage of the HBV mRNA transcript in a concentration dependent manner. Eighty-five percent of substrate was cleaved under optimal conditions. Controls consisting of RNase H alone, oligonucleotide alone, and incubation of the conjugate with an unrelated mRNA substrate resulted in no cleavage activity. RNase H coupled to an HBV antisense oligonucleotide can specifically cleave target HBV transcripts.
Walton
C M
CM
Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Health Center, Farmington, Connecticut 06032, USA.
Wu
C H
CH
Wu
G Y
GY
D002384:Catalysis / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000737:chemistry; D015195:Drug Design; D006515:Hepatitis B virus / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000138:chemical synthesis / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D016914:Ribonuclease H / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D012367:RNA, Viral; D016914:Ribonuclease H