Intracellular inhibition of the replication of hepatitis B virus by hammerhead ribozymes.

BACKGROUND: Chronic hepatitis B virus (HBV) infection is frequently associated with cirrhosis and hepatocellular carcinoma, and so has become a major worldwide health problem. Hammerhead ribozymes have recently gained some attention as potential tools to inhibit viral infection, for which there are no general effective therapies available. METHODS: A hammerhead ribozyme, RzC, was designed to target the sequence encoding the tail region of the HBV core protein. The activities of the ribozyme were analyzed in vitro and in human hepatoma (HepG2) cells. RESULTS: In vitro, RzC cleaves HBV-RNA at its target site up to 30%, while the disabled ribozyme, dRzC, which has a one-base mutation in the catalytic site, did not cleave the target RNA at all. When the ribozymes were cotransfected into HepG2 cells with the HBV genome-containing plasmid, p3.6II, the inhibition of HBV replication by RzC was greater than that by dRzC, indicating that the active catalytic domain of the hammerhead ribozyme could increase the extent of antisense-mediated inhibition. In addition, there was a gradient of effectiveness in which the greater the amount of released ribozyme, the greater the reduction in HBV progeny DNA. CONCLUSIONS: These results suggest the possibility of hammerhead ribozyme-mediated gene therapy for the treatment of HBV infections.

Inhibition of hepatitis B virus by hammerhead ribozyme targeted to the poly(A) signal sequence in cultured cells.

The deviant poly(A) signal of hepatitis B virus (HBV) not only controls the formation of the 3' end of all the viral RNA, but is also crucial for HBV replication. Hence, a cis-releasing hammerhead ribozyme (RzA) targeted to the poly(A) signal region of HBV subtype adr was investigated for its antiviral effects. In vitro, RzA cleaved HBV RNA at its target site up to 70%, while the disabled ribozyme (dRzA), which had a one-base mutation in the catalytic site, did not cleave the target RNA at all. When the ribozymes were cotransfected into HepG2 cells with the HBV genome-containing plasmid p3.6II, the wild-type ribozyme RzA could effectively decrease HBV RNA levels and inhibit HBV replication, whereas its disabled form, dRzA, had much weaker effects, indicating that the active catalytic domain of the hammerhead ribozyme could markedly increase the extent of antisense-mediated inhibition. In addition, there was a gradient of effectiveness: the higher the amount of released ribozyme, the more the reduction in target HBV RNA in cells as well as progeny DNA reduction. These results suggest the possibility of the hammerhead ribozyme RzA to be used for the gene therapy of HBV infection.

[Effect of inhibition of expression PCNA with ribozyme on the proliferation of HeLa cells].

The Proliferating Cell Nuclear Antigen (PCNA), which is an auxiliary protein for DNA polymerase delta, is found to be essential for cellular DNA replication. A designed hammerhead ribozyme, with high efficiency to cleave the PCNA mRNA site-specificly in vitro, was constructed into a self-trimming expression plasmid, and then was introduced into HeLa cells by lipofectin reagent. Small molecular RNAs, isolated from total cellular RNA with the same length as active ribozyme, can cleave the target RNA in vitro, which suggested that this expression plasmid can yield active ribozyme molecules in cells. In comparison with the vector control, the entrance of S phase of the HeLa cells transfected by the ribozyme expression plasmid was delayed 8 hours after serum stimulation. Mean-while, those cells transfected by mutant inactive ribozyme as antisense RNA control was delayed only 3 hours. These results demonstrated that this ribozyme can inhibit the DNA replication in HeLa cells effectively and could be used as a potential tool to study the function of PCNA in cellular DNA replication and cell cycle progression.

In vitro cleavage of HPV16 E6 and E7 RNA fragments by synthetic ribozymes and transcribed ribozymes from RNA-trimming plasmids.

A RNA-trimming plasmid pRG523 is constructed, in which three Rz genes, GR5(5'-cis-Rz gene), HR2G(trans-Rz gene) and GR3(3'-cis-Rz gene), are arranged in the order from 5' to 3' downstream from the T7 promoter. In vitro transcription of this plasmid shows that the trans-Rz can be trimmed to definite lengths by the cis-Rz on both sides of the trans-Rz. In vitro cleavage of HPV16 E6 and E7 RNA fragments of different lengths by synthetic Rz and that of E7 RNA with a length of 171 nt by synthetic Rz and transcribed Rzs with different lengths of flanking sequences is studied. The results show that the non-base-pairing flanking sequences on both Rz and target RNA can affect the cleavage reaction.

The conformation of single-stranded nucleic acids tDNA versus tRNA.

Conformational analyses using the single-strand-specific nuclease from mung bean and restriction endonucleases have been performed on a series of DNA fragments related to the sequence of the yeast initiator tRNA(Met). Mung bean nuclease cleaves DNA fragments exclusively in some, but not all, single-stranded regions as predicted by RNA secondary structural rules. Comparison of cleavage patterns of yeast initiator tRNA(Met), tDNA(Met) (a DNA oligomer having the sequence of tRNA(Met] and the anti-tDNA(Met) (the complement of tDNA(Met] suggests that the conformation of the three molecules is very similar. Furthermore, both tDNA and anti-tDNA are cleaved by HhaI and CfoI restriction endonucleases at two GCG/C sites which would be in double-stranded regions (the acceptor and dihydrouridine stem), if the two molecules adopt the tRNA cloverleaf structure. On the other hand, minor cleavage products show that the core region, i.e. the extra loop area, is slightly more exposed in tDNA and in anti-tDNA than in tRNA. Therefore, we submit that the global conformation of nucleic acids is primarily dictated by the interaction of purine and pyrimidine bases with atoms and functional groups common to both RNA and DNA. In this view the 2'-hydroxyl group, in tRNA at least, is an auxiliary structural feature whose role is limited to fostering local interactions, which increase the stability of a given conformation.

Studies on the sites expressing evolutionary changes in the structure of eukaryotic 5S ribosomal RNA.

We have determined the complete sequences of 5S rRNAs from a lamprey (Lampetra reissneri), a lancelet (Branchiostoma belcheri), silkworms (Philosamia cynthia ricini, Bombyx mori, Antheraea pernyi), and a silkworm hybrid (artificially fertilized hybrid species of Philosamia cynthia ricini male x Bombyx mori female), as well as those of cotton seeds (Gossypium hirsutum L.). Having compared more than 170 eukaryotic 5S rRNAs of which seven sequences have been determined by our group as mentioned above, we have found that the "evolutionary sites" that exist at special locations in these structures are closely related to the evolution of eukaryotes. The changes proceed step by step in an orderly way, i.e., the change in nucleotide residues of the "evolutionary sites" depends on the order of the evolution of the species and shows group-specific patterns.

Restriction of single-stranded M13 DNA using synthetic oligonucleotides: the structural requirement of restriction enzymes.

A targeted ss (single stranded) DNA cleavage technique is reported which involves the use of synthetic oligomers complementary to the ss M13 DNA polylinker. BamHI, SmaI, and KpnI restriction enzymes were tested with a partial duplex DNA formed from ss M13 DNA and a nested series of fragments derived from a synthetic 21-mer which were complementary to the polylinker region. These enzymes require up to two flanking nucleotides in addition to the hexameric recognition site for efficient cleavage. This technique could be useful for effecting unique cleavages of DNA with enzymes which generally give a large number of fragments and for strategies of ss DNA manipulation.

Synthesis of the 5'-half molecule of yeast alanine tRNA.

In this paper, we report the synthesis of the 5'-half molecule of yeast alanine tRNA (tRNAAlay) by ligating three oligonucleotide fragments corresponding to the nucleotide sequences 1-13, 14-22 and 23-35 respectively under the catalysis of T4 RNA ligase (Fig. 1). Because of the high purity of the oligonucleotide fragments and the excellent quality of T4RNA ligase and polynucleotide kinase we prepared, the isolation steps were simplified and the overall yields were much higher. The ligating yield of the docosamer (IV) was 75%, that of the pentatriacontamer (V), 90%, and the isolated yield of the final product was 21% calculated on the basis of the tridecamer (III) used in the first reaction. Under the action of T4 RNA ligase the synthetic 5'-half molecule was joined with the natural 3'-half molecule forming a semi-synthetic tRNAAlay, which possessed the biological activities of both accepting (3H)-alanine and incorporating it into proteins. The correctness of the structure of the synthetic 5'-half molecule was verified by both chemical analysis and biological activity assay.