Comparative analysis of cleavage rates after systematic permutation of the NUX consensus target motif for hammerhead ribozymes.

A trans-cleaving asymmetric hammerhead ribozyme directed against an AUC decreases target motif within an RNA specific for human immunodeficiency virus type 1 (HIV-1) was generated. The AUC decreases motif of the target RNA was permutated in order to generate all 12 variants of an NUX decreases consensus target motif, wherein N = A, C, G or U and X = A, C or U. Four asymmetric hammerhead ribozymes differing in the nucleotide that is complementary to N were generated, of which each was specific for three of the 12 target motifs. The residual sequence context within helices I and III remained unchanged. All 12 combinations resulted in cleavage of the target RNA. Using single-turnover conditions, the detectable cleavage rate constants at 37 degrees C were determined, which varied considerably depending on the NUX decreases motif. The NUC decreases motifs were cleaved more efficiently, with AUC decreases being cleaved best. Comparison with previous studies indicates that the sequence context of the NUX decreases motif plays a major role for the detectable cleavage activity.     

稳定的RNA发夹结构及其生物学功能

以UNCG、GNRA、CUUG(N=A、U、C或G,R=G或A)为端环能够形成稳定的、保守的发夹结构。高分辨率的溶液结构、晶体结构和计算机模拟等方法从原子水平上解析了这些发夹特殊的结构特征。在体内,它们发挥着重要的生物学功能:在折叠过程中作为折叠的起始位置帮助组织RNA分子正确折叠;与核酸受体结合参与三级相互作用;与蛋白质发生相互作用;阻止逆转录酶的延伸等等。另外,由于C(UUCG)G发夹极其稳定的特征,在体外RNA分子的实验测定中它还是稳定核酸结构的理想工具。这些稳定的发夹广泛分布于体内rRNA、催化RNA和非编码mRNA中。但在对人类编码区mRNA结构特征的研究当中,却未发现C(UUCG)G发夹。     

Mapping of psoralen cross-linked nucleotides in RNA

A method is described for using the cross-linking reagent 4'-(hydroxy-methyl)-4,5',8-trimethylpsoralen (HMT) to map base paired regions and higher-order structure within RNA molecules. Applying this method to yeast tRNAPhe, we have specifically identified cross-links within the acceptor stem between U6 X U68, in the D-stem between C11 X C25, and in the T psi-stem between U50 X C63 and U52 X C63. We have also identified a unique cross-link between U8 X C48 which are trans pyrimidines in the core region due to tertiary interactions between U8:A14 and C48:G15. The precise point of cross-linking was deduced in every case by using purine-specific U2 ribonuclease along with cytidine-specific CL3 ribonuclease which will anomalously cleave after photoreversed pyrimidines. The ability to map the precise point of cross-linking should prove invaluable in identifying nucleotides in close proximity within the tertiary structure of other RNA molecules.     

Design of hairpin ribozyme variants with improved activity for poorly processed substrates

Application of ribozymes for knockdown of RNA targets requires the identification of suitable target sites according to the consensus sequence. For the hairpin ribozyme, this was originally defined as Y?2 N?1 *G+1 U+2 Y+3 B+?, with Y = U or C, and B = U, C or G, and C being the preferred nucleobase at positions -2 and +4. In the context of development of ribozymes for destruction of an oncogenic mRNA, we have designed ribozyme variants that efficiently process RNA substrates at U?2 G?1 *G+1 U+2 A+3 A+? sites. Substrates with G?1 *G+1 U+2 A+3 sites were previously shown to be processed by the wild-type hairpin ribozyme. However, our study demonstrates that, in the specific sequence context of the substrate studied herein, compensatory base changes in the ribozyme improve activity for cleavage (eight-fold) and ligation (100-fold). In particular, we show that A+3 and A+? are well tolerated if compensatory mutations are made at positions 6 and 7 of the ribozyme strand. Adenine at position +4 is neutralized by G? →U, owing to restoration of a Watson-Crick base pair in helix 1. In this ribozyme-substrate complex, adenine at position +3 is also tolerated, with a slightly decreased cleavage rate. Additional substitution of A? with uracil doubled the cleavage rate and restored ligation, which was lost in variants with A?, C? and G?. The ability to cleave, in conjunction with the inability to ligate RNA, makes these ribozyme variants particularly suitable candidates for RNA destruction.     

Ribozyme mediated destruction of RNA in vivo.

Previous studies have demonstrated that high ribozyme to substrate ratios are required for ribozyme inhibitory function in nuclear extracts. To obtain high intracellular levels of ribozymes, tRNA genes, known to be highly expressed in most tissues, have been modified for use as ribozyme expression cassettes. Ribozyme coding sequences were placed between the A and the B box, internal promoter sequences of a Xenopus tRNAMet gene. When injected into the nucleus of frog oocytes, the ribozyme tRNA gene (ribtDNA) produces 'hammerhead' ribozymes which cleave the 5' sequences of U7snRNA, its target substrate, with high efficiency in vitro. Oocytes were coinjected with ribtDNA, U7snRNA and control substrate RNA devoid of a cleavage sequence. It was found that the ribtRNA remained localized mainly in the nucleus, whereas the substrate and the control RNA exited rapidly into the cytoplasm. However, sufficient ribtRNA migrated into the cytoplasm to cleave, and destroy, the U7snRNA. Thus, the action of targeted 'hammerhead' ribozymes in vivo is demonstrated.     

In vitro construction of effective M1GS ribozymes targeting HCMV UL54 RNA segments

Seven sequence-specific ribozymes (M1GS RNAs) derived in vitro from the catalytic RNA subunit of Escherichia coli RNase P and targeting the mRNAs transcribed by the UL54 gene encoding the DNA polymerase of human cytomegalovirus were screened from 11 ribozymes that were designed based on four rules: (1) the NCCA-3′ terminal must be unpaired with the substrate; (2) the guide sequence (GS) must be at least 12 nt in length; (3) the eighth nucleotide must be U, counting from the site-1; and (4) around the cleavage site, the sites -1/ 1/ 2 must be U/G/C or C/G/C. Further investigation of the factors affecting the cleavage effect and the optimal ratio for M1GS/substrate was carried out. It was determined that the optimal ratio for M1GS/substrate was 2:1 and too much M1GS led to substrate degrading. As indicated above, several M1GS that cleaved HCMV UL54 RNA segments in vitro were successfully designed and constructed.Our studies support the use of ribozyme M1GS as antisense molecules to silence HCMV mRNA in vitro, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.     

A method for prediction of accessible sites on an mRNA sequence for target selection of hammerhead ribozymes.

Hammerhead ribozymes are short RNA molecules endowed with endoribonucleolytic activity. Their use as molecular tools for specific inhibition of gene translation is affected by many factors including the target accessibility. A method for the prediction of accessible target sites for hammerhead ribozymes within a given RNA sequence is described. This method maps all putative NUH cleavage sites (N = A, C, G, U and H = A, C, U) and picks out short flanking regions as the binding domain for the corresponding ribozyme. The probabilistic level of unfolding, accessibility score (AS), is then calculated for each target region on the basis of a comparison of all folding structures obtained for the target RNA and arranged according to the Boltzmann's distribution. At the end, a series of imposed limits gives the best target sequences endowed with highly probable accessibility and with a potentially active catalytic structure of the hammerhead sequence. A successive experimental approach to verify the effective accessibility of selected targets was used. For that, antisense oligonucleotides addressed to the coding region of bcl2 mRNA were synthesized and administered to the MCF7 human cell line. The inhibition of gene expression, as measured by western analysis of the BCL2 protein, demonstrated that all target sites selected on the basis of their putative accessibility were actually sensitive to antisense treatments while the inaccessible ones were not. The application of this target discovery method to ribozyme design is proposed in order to satisfy a crucial condition.     

Hydrogen and hydration of DNA and RNA oligonucleotides

In this paper, hydrogen bonding interaction and hydration in crystal structures of both DNA and RNA oligonucleotides are discussed. Their roles in the formation and stabilization of oligonucleotides have been covered. Details of the Watson-Crick base pairs G.C and A.U in DNA and RNA are illustrated. The geometry of the wobble (mismatched) G.U base pairs and the cis and almost trans conformations of the mismatched U.U base pairs in RNA is described. The difference in hydration of the Watson-Crick base pairs G.C, A.U and the wobble G.U in different sequences of codon-anticodon interaction in double helical molecules are indicative of the effect of hydration. The hydration patterns of the phosphate, the 2'-hydroxyl groups, the water bridges linking the phosphate group, N7 (purine) and N4 of Cs or O4 of Us in the major groove, the water bridges between the 2'-hydroxyl group and N3 (purine) and O2 (pyrimidine) in the minor groove are discussed.     

A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.     

Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages.

Hammerhead ribozymes are considered to be potential therapeutic agents for HIV virus because of their site-specific RNA cleavage activities. In order to elucidate structure--function relationship and also to hopefully endow ribozymes with resistance to ribonucleases, we firstly synthesized chimeric DNA/RNA ribozymes in which deoxyribonucleotides were substituted for ribonucleotides at noncatalytic residues (stems I, II, and III). Kinetic analysis revealed that (i) DNA in the hybridizing arms (stems I and III) enhanced the chemical cleavage step. (ii) stem II and its loop do not affect its enzymatic activity. Secondly, we introduced deoxyribonucleotides with phosphorothioate linkages to the same regions (stems I, II, and III) in order to test whether such thio-linkages further improve their resistance to nucleases. Kinetic measurements revealed that this chimeric thio-DNA/RNA ribozyme had seven-fold higher cleavage activity (kcat = 27 min-1) than that of the all-RNA ribozyme. In terms of stability in serum, DNA-armed ribozymes gained about 10-fold higher stability in human serum but no increase in stability was recognized in bovine serum, probably because the latter serum mainly contained endoribonucleases that attacked unmodified catalytic-loop regions of these ribozymes. Thirdly, in order to protect them from endoribonucleases, three additional modifications were made at positions U7, U4 and C3 within the internal catalytic-loop region, that succeeded in gaining more than a hundred times greater resistance to nucleases in both serums. More importantly, these catalytic-loop modified ribozymes had the comparable cleavage activity (kcat) to the wild-type ribozyme. Since these chimeric thio-DNA/RNA ribozymes are more resistant to attack by both exonucleases and endoribonucleases than the wild-type all-RNA ribozymes in vivo and since their cleavage activities are not sacrificed, they appear to be better candidates than the wild type for antiviral therapeutic agents.     

Conserved unpaired adenine residues are important for ordered structures of 5S ribosomal RNA. An infrared study of the secondary and tertiary structure of Thermus thermophilus 5S rRNA

An improved set of infrared calibration spectra for the determination of G X C and A X U base pairs leads to 32 +/- 3 G X C (+ G X U) and 4 +/- 1 A X U base pairs for Thermus thermophilus 5S RNA in the presence and absence of Mg2+. These results give further support for the consensus secondary structure of 5S RNA recently proposed by several groups. T. thermophilus 5S RNA shows, in the presence of Mg2+, a distinct two-step thermal melting of its ordered structure. Based on new data about the stacking dependence of infrared intensities of unpaired ribonucleotides the spectral changes of the low-temperature transition should be explained by melting of stacked arrangements of unpaired bases and/or non-standard base pairs. Striking is the reduction in A stacking, which is not related to the melting of A X U base pairs, indicating the importance of the mostly conserved unpaired adenines for the Mg2+ stabilized higher-order structures especially within internal loops of 5S RNA.     

A ribonuclease from human seminal plasma active on double-stranded RNA

A ribonuclease, active on single- and double-stranded RNAs, has been isolated from human seminal plasma 3-5 micrograms of enzyme were recovered per ml of seminal plasma, equivalent to 71% of total activity and a 2500-fold purification (measured with poly(A) X poly(U) as substrate) from the initial dialyzed material. Similar amounts of RNAase were found per g (wet weight) of human prostate, where the enzyme appears to be produced. Human seminal RNAase degrades poly(U) 3-times faster than poly(A) X poly(U), and poly(C) or viral single-stranded RNA about 10-times faster than poly(U). Degradation of poly(A) X poly(U), viral double-stranded RNA, and poly(A) by human seminal RNAase is 500-, 380- and 140-times more efficient, respectively, than by bovine RNAase A. The enzyme, a basic protein with maximum absorbance at 276 nm, occurs in two almost equivalent forms, one of which is glycosylated. Mr values of the glycosylated and non-glycosylated form are 21000 and 16000, respectively. The amino-acid composition of the RNAase is very similar to that of human pancreatic RNAase. The same is true for the carbohydrate content of its glycosylated form.     

Base pairing between Escherichia coli RNase P RNA and its substrate.

Base pairing between the substrate and the ribozyme has previously been shown to be essential for catalytic activity of most ribozymes, but not for RNase P RNA. By using compensatory mutations we have demonstrated the importance of Watson-Crick complementarity between two well-conserved residues in Escherichia coli RNase P RNA (M1 RNA), G292 and G293, and two residues in the substrate, +74C and +75C (the first and second C residues in CCA). We suggest that these nucleotides base pair (G292/+75C and G293/+74C) in the ribozyme-substrate complex and as a consequence the amino acid acceptor stem of the precursor is partly unfolded. Thus, a function of M1 RNA is to anchor the substrate through this base pairing, thereby exposing the cleavage site such that cleavage is accomplished at the correct position. Our data also suggest possible base pairing between U294 in M1 RNA and the discriminator base at position +73 of the precursor. Our findings are also discussed in terms of evolution.     

RNA priming of DNA replication by bacteriophage T4 proteins

Bacteriophage T4 DNA replication proteins have been shown previously to require ribonucleoside triphosphates to initiator new DNA chains on unprimed single-stranded DNA templates in vitro. This DNA synthesis requires a protein controlled by T4 gene 61, as well as the T4 gene 41, 43 (DNA polymerase), 44, 45, and 62 proteins, and is stimulated by the gene 32 (helix-destabilizing) protein. In this paper, the nature of the RNA primers involved in DNA synthesis by the T4 proteins has been determined, using phi X174 and f1 DNA as model templates. The T4 41 and "61" proteins synthesize pentanucleotides with the sequence pppA-C(N)3 where N in positions 3 and 4 can be G, U, C, or A. The same group of sequences is found in the RNA at the 5' terminus of the phi X174 DNA product made by the seven T4 proteins. The DNA product chains begin at multiple discrete positions on the phi X174 DNA template. The characteristics of the T4 41 and "61" protein priming reaction are thus appropriate for a reaction required to initiate the synthesis of discontinuous "Okazaki" pieces on the lagging strand during the replication of duplex DNA.     

A thermodynamic study of unusually stable RNA and DNA hairpins.

About 70% of the RNA tetra-loop sequences identified in ribosomal RNAs from different organisms fall into either (UNCG) or (GNRA) families (where N = A, C, G, or U; and R = A or G). RNA hairpins with these loop sequences form unusually stable tetra-loop structures. We have studied the RNA hairpin GGAC(UUCG)GUCC and several sequence variants to determine the effect of changing the loop sequence and the loop-closing base pair on the thermodynamic stability of (UNCG) tetra-loops. The hairpin GGAG(CUUG)CUCC with the conserved loop G(CUUG)C was also unusually stable. We have determined melting temperatures (Tm), and obtained thermodynamic parameters for DNA hairpins with sequences analogous to stable RNA hairpins with (UNCG), C(GNRA)G, C(GAUA)G, and G(CUUG)C loops. DNA hairpins with (TTCG), (dUdUCG), and related sequences in the loop, unlike their RNA counterparts, did not form unusually stable hairpins. However, DNA hairpins with the consensus loop sequence C(GNRA)G were very stable compared to hairpins with C(TTTT)G or C(AAAA)G loops. The C(GATA)G and G(CTTG)C loops were also extra stable. The relative stabilities of the unusually stable DNA hairpins are similar to those observed for their RNA analogs.     

A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability.

Hammerhead ribozymes cleave RNA substrates containing the UX sequence, where X = U, C or A, embedded within sequences which are complementary to the hybridising 'arms' of the ribozyme. In this study we have replaced the RNA in the hybridising arms of the ribozyme with DNA, and the resulting ribozyme is many times more active than its precursor. In turnover-kinetics experiments with a 13-mer RNA substrate, the kcat/Km ratios are 10 and 150 microM-1min-1 for the RNA- and DNA-armed ribozymes, respectively. The effect is due mainly to differences in kcat. In independent experiments where the cleavage step is rate-limiting, the DNA-armed ribozyme cleaves the substrate with a rate constant more than 3 times greater than the all-RNA ribozyme. DNA substrates containing a ribocytidine at the cleavage site have been shown to be cleaved less efficiently than their all-RNA analogues; again however, the DNA-armed ribozyme is more effective than the all-RNA ribozyme against such DNA substrates. These results demonstrate that there are no 2'-hydroxyl groups in the arms of the ribozyme that are required for cleavage; and that the structure of the complex formed by the DNA-armed ribozyme with its substrate is more favourable for cleavage than that formed by the all-RNA ribozyme and its substrate.