Detection of stable secondary structure at the 3' terminus of dengue virus type 2 RNA.
The 3'-terminal sequences of flavivirus genomes within approx. 100 nucleotides (nt) have been suggested to have a highly conserved secondary structure, as based on the known nt sequence data and free-energy calculations using computer programs. To test the existence of a secondary structure in solution, we devised a strategy to generate truncated RNA molecules from about 0.3-1.4 kb in length, having the same polarity and nt sequence as dengue virus type 2 (DEN-2) RNA (New Guinea-C strain). When these labeled RNA molecules were digested by RNase A, and analyzed by denaturing polyacrylamide-gel electrophoresis, three resistant fragments of 16, 20 and 23 nt in length were reproducibly obtained. To examine whether these RNase A-resistant (RNaseR) fragments emerged from a stable secondary structure formed in solution consisting of 3'-terminal sequences, hybridization of the RNaseR fragments to four chemically synthesized oligodeoxyribonucleotides (oligos), complementary to nt 1-24, 25-48, 49-72, and 73-96 from the 3' terminus of DEN-2 RNA, followed by RNase H digestion were carried out. Oligos complementary to nt 25-48 and 49-72 from the 3' end of DEN-2 RNA were sufficient to render all three RNaseR fragments susceptible to RNase H digestion. These data indicate that a stable secondary structure is formed in solution involving nt 18-67 from the 3' terminus. The potential use of these unique transcripts to identify the viral and/or host proteins which might interact at the 3' terminus of DEN-2 RNA during initiation of replication is discussed.
Mohan
P M
PM
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66103.
Padmanabhan
R
R
D001482:Base Composition / Q000235:genetics; D001483:Base Sequence; D003716:Dengue Virus / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics*; D016914:Ribonuclease H / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral; D016914:Ribonuclease H; D012259:Ribonuclease, Pancreatic