tailieunhanh - Báo cáo Y học: Site-specific modification of functional groups in genomic hepatitis delta virus (HDV) ribozyme

Human hepatitis delta (HDV) ribozyme is one of small ribozymes, such as hammerhead and hairpin ribozymes, etc. Its secondary structure shows pseudoknot structure com-posed of four stems (I to IV) and three single-stranded regions (SSrA, -B and -C). The 3D structure of 3¢-cleaved product of genomic HDV ribozyme provided extensive information about tertiary hydrogen bonding interactions between nucleotide bases, phosphate oxygens and 2¢OHs including new stem structure . | Eur. J. Biochem. 269 5792-5803 2002 FEBS 2002 doi j Site-specific modification of functional groups in genomic hepatitis delta virus HDV ribozyme Fumiko Nishikawa Miho Shirai and Satoshi Nishikawa Functional Nucleic Acids Group Institute for Biological Resources and Functions National Institute of Advanced Industrial Science and Technology AIST Ibaraki Japan Human hepatitis delta HDV ribozyme is one of small ribozymes such as hammerhead and hairpin ribozymes etc. Its secondary structure shows pseudoknot structure composed of four stems I to IV and three single-stranded regions SSrA -B and -C . The 3D structure of 3 -cleaved product of genomic HDV ribozyme provided extensive information about tertiary hydrogen bonding interactions between nucleotide bases phosphate oxygens and 2 OHs including new stem structure . To analyze the role of these hydrogen bond networks in the catalytic reaction sitespecific atomic-level modifications such as deoxynucleotides deoxyribosyl-2-aminopurine deoxyribosylpurine 7-deaza-ribonucleotide and inosine were incorporated in the smallest trans-acting HDV ribozyme 47-mer . Kinetic analysis of these ribozyme variants demonstrated the importance of the two W-C base pairs of for cleavage in addition the results suggest that all hydrogen bond interactions detected in the crystal structure involving 2 -OH andN7atoms arepresent in most of the variants the relative reduction in kobs caused by substitution of the 2 -OH group correlated with the number of hydrogen bonds affected by the substitution. However G74 and C75 may have more than one hydrogen bond involving the 2 -OH in both the trans- and cis-acting HDV ribozyme. Moreover in variants in which N7 was deleted kobs was reduced 5- to -5-fold it may suggest that N7 assists in coordinating Mg2 ions or water molecules which bind with weak affinity in the active structure. Keywords HDV ribozyme RNA structure and function .

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