Detection of stable secondary structure at the 3′ terminus of dengue virus type 2 RNA

• Published in: Gene Vol. 108; no. 2; pp. 185 - 191
• Main Authors: Mohan, P.Maruthi, Padmanabhan, R.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 15.12.1991; Amsterdam Elsevier; New York, NY
• Subjects: Base Composition - genetics; Base Sequence; Biological and medical sciences; Dengue Virus - genetics; flavivirus; Fundamental and applied biological sciences. Psychology; Genetics; in vitro transcription; Microbiology; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides - genetics; Polymerase Chain Reaction; Recombinant DNA; Ribonuclease H - metabolism; Ribonuclease, Pancreatic - metabolism; RNA, Viral - genetics; RNase A cleavage sites; RNase H mapping; Virology; ORF; DTT; nt; dNTP; RNase H mapping; Recombinant DNA; DEN-2; RNase R; YF; bp; Taq polymerase; ds; TBE; kb; WN; in vitro transcription; cDNA; oligo; NGS-C; polymerase chain reaction; flavivirus; ss; SSC; RNase A cleavage sites; r; PolIk; JE; MVE; PCR; Virus; Hairpin loop; RNA; 3' terminal-Sequence; Dengue virus 3; Dengue virus 2; Flaviviridae; Secondary structure; Flavivirus
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(91)90433-C

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 (RNase R) fragments emerged from a stable secondary structure formed in solution consisting of 3'-terminal sequences, hybridization of the RNase R 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 RNase R 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.