Chemical probing studies of variants of the genomic hepatitis delta virus ribozyme by primer extension analysis

• Published in: Biochemistry (Easton) Vol. 33; no. 2; pp. 583 - 592
• Main Authors: Kumar, P. K. R, Taira, Kazunari, Nishikawa, Satoshi
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 18.01.1994
• Subjects: Analytical, structural and metabolic biochemistry; Aniline Compounds - pharmacology; Base Composition; Base Sequence; Biological and medical sciences; Borohydrides - pharmacology; Carbodiimides - pharmacology; Fundamental and applied biological sciences. Psychology; hepatitis D virus; Hepatitis Delta Virus - genetics; Magnesium - pharmacology; Molecular Sequence Data; Nucleic Acid Conformation; Nucleic Acid Denaturation; Nucleic acids; RNA, Catalytic - chemistry; Rna, ribonucleoproteins; RNA, Viral - chemistry; Sulfuric Acid Esters - pharmacology; Virus; Delta agent; Ribozyme; Pseudoknot structure; Models; Secondary structure; Intramolecular interaction; Structural analysis
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00168a025

We have investigated in detail the higher order structure of the genomic hepatitis delta virus (HDV) ribozyme using various base-specific chemical probes under native, semi-denaturing, and denaturing conditions. The bases of the HDV ribozyme were probed by treatment with dimethyl sulfate [which reacts with A (at N1) and C (at N3)] and a carbodiimide [which reacts with U (at N3) and G (at N1)]. In addition, for probing G residues (at N7), RNA samples were treated with NaBH4 and aniline after modification by treatment with dimethyl sulfate. The sites of modified positions were identified by primer extension analysis with reverse transcriptase. In general, our results are consistent with the proposed pseudoknot model of secondary structure, a model that is based on data from ribonucleolytic cleavage experiments. Our results provide clues to the identification of interacting bases in the HDV ribozyme. Furthermore, using this method we identified local conformational changes in several stem variants.