Site-specific modification of functional groups in genomic hepatitis delta virus (HDV) ribozyme

The human hepatitis delta virus (HDV) ribozyme is a small ribozyme, similar to hammerhead and hairpin ribozymes. Its secondary structure forms a pseudoknot consisting of four stems (I to IV) and three single-stranded regions (SSrA, SSrB, and SSrC). The three-dimensional structure of the 3′-cleaved product of the genomic HDV ribozyme has provided detailed insights into tertiary hydrogen bonding interactions among nucleotide bases, phosphate oxygens, and 2′-hydroxyl groups, including the identification of a new stem structure, P1.1.

To investigate the role of these hydrogen bond networks in catalysis, site-specific atomic-level modifications—including deoxynucleotides, deoxyribosyl-2-aminopurine, deoxyribosylpurine, 7-deaza-ribonucleotide, and inosine—were introduced into the smallest trans-acting HDV ribozyme (47-mer). Kinetic analyses of these ribozyme variants revealed that the two Watson-Crick base pairs within P1.1 are critical for cleavage activity. Additionally, the findings indicate that all hydrogen bond interactions identified in the crystal structure involving 2′-OH groups and N7 atoms are preserved in the active ribozyme conformation.

In most variants, the relative decrease in the observed rate constant (k_obs) resulting from 2′-OH group substitutions correlated with the number of hydrogen bonds disrupted by the modification. However, nucleotides G74 and C75 appear to form multiple hydrogen bonds involving the 2′-OH group in both trans-acting and cis-acting HDV ribozymes. Furthermore, in variants where the N7 position was removed, k_obs was reduced by 5- to 15-fold, suggesting that the N7 atom may facilitate the coordination of Mg^2+ ions or C75 trans water molecules that interact with weak affinity within the active site structure.