Folding of the hammerhead ribozyme: pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme-substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair.     

Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing

Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites.     

A helical twist-induced conformational switch activates cleavage in the hammerhead ribozyme

We have captured the structure of a pre-catalytic conformational intermediate of the hammerhead ribozyme using a phosphodiester tether formed between I and Stem II. This phosphodiester tether appears to mimic interactions in the wild-type hammerhead RNA that enable switching between nuclease and ligase activities, both of which are required in the replicative cycles of the satellite RNA viruses from which the hammerhead ribozyme is derived. The structure of this conformational intermediate reveals how the attacking nucleophile is positioned prior to cleavage, and demonstrates how restricting the ability of Stem I to rotate about its helical axis, via interactions with Stem II, can inhibit cleavage. Analogous covalent crosslinking experiments have demonstrated that imposing such restrictions on interhelical movement can change the hammerhead ribozyme from a nuclease to a ligase. Taken together, these results permit us to suggest that switching between ligase and nuclease activity is determined by the helical orientation of Stem I relative to Stem II.     

Threshold Occupancy and Specific Cation Binding Modes in the Hammerhead Ribozyme Active Site are Required for Active Conformation

The relationship between formation of active in-line attack conformations and monovalent (Na⁺) and divalent (Mg²⁺) metal ion binding in hammerhead ribozyme (HHR) has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of the threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence and in the absence of a Mg²⁺ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the HHR folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of HHR catalysis and support the supposition that Mg²⁺, in addition to stabilizing active conformations, plays a specific chemical role in catalysis.     

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.     

Chrysanthemum chlorotic mottle viroid RNA: dissection of the pathogenicity determinant and comparative fitness of symptomatic and non-symptomatic variants

Chrysanthemum chlorotic mottle viroid (CChMVd) is a small RNA (398-401nt) with hammerhead ribozymes in both polarity strands that mediate self-cleavage of the oligomeric RNA intermediates generated in a rolling-circle mechanism of replication. Within the in vivo branched RNA conformation of CChMVd, a tetraloop has been identified as a major determinant of pathogenicity. Here we present a detailed study of this tetraloop by site-directed mutagenesis, bioassay of the CChMV-cDNA clones and analysis of the resulting progenies. None of the changes introduced in the tetraloop, including its substitution by a triloop or a pentaloop, abolished infectivity. In contrast to observations for other RNAs, the thermodynamically stable GAAA tetraloop characteristic of non-symptomatic CChMVd-NS strains was not functionally interchangeable for other stable tetraloops of the UNCG family, suggesting that the sequence, rather than the structure, is the major factor governing conservation of this motif. In most cases, the changes introduced initially led to symptomless infections, which eventually evolved to be symptomatic concurrently with the prevalence in the progeny of the UUUC tetraloop characteristic of symptomatic CChMVd-S strains. Only in one case did the GAAA tetraloop emerge and eventually dominate the progeny in infected plants that were non-symptomatic. These results revealed two major fitness peaks in the tetraloop (UUUC and GAAA), whose adjacent stem was also under strong selection pressure. Co-inoculations with CChMVd-S and -NS variants showed that only when the latter was in a 100- or 1000-fold excess did the infected plants remain symptomless, confirming the higher biological fitness of the S variant and explaining the lack of symptom expression previously observed in cross-protection experiments.     

A pH-dependent conformational change, rather than the chemical step, appears to be rate-limiting in the hammerhead ribozyme cleavage reaction.

We have investigated the chemical basis for a previously observed 7.8 A conformational change in the hammerhead ribozyme that positions the substrate for in-line attack. We have found that the conformational change can only be observed at or above pH 8.5 (in the presence of Co(2+)) and requires the presence of an ionizable 2'-OH at the cleavage site, and note that this observed apparent pK(a) of 8.5 for the conformational change is within experimental error (+/-0.5) of the previously reported apparent kinetic pK(a) of 8.5 for the hammerhead ribozyme in the presence of Co(2+). We have solved two crystal structures of hammerhead ribozymes having 2'-OCH(3) or 2'-F substitutions at the cleavage site and have found that these will not undergo a conformational change equivalent to that observed for the hammerhead ribozyme having an unmodified attacking nucleophile under otherwise identical conditions. We have also characterized the kinetics of cleavage in the crystal. In addition to verifying that the particular sequence of RNA that we crystallized cleaves faster in the crystal than in solution, we also find that the extent of cleavage in the crystal is complete, unlike in solution where this and most other hammerhead ribozyme substrates are cleaved only to about 70 % completion. The initial cleavage rate in the crystal obeys the expected log-linear relation between cleavage-rate and pH with a slope of 0.7, as has been observed for other hammerhead ribozyme sequences in solution, indicating that in both the crystal and in solution the pH-dependent step is rate-limiting. However, the cleavage rate in the crystal is biphasic, with the most dramatic distinction between initial (slower) and final (faster) phases appearing at pH 6.0. The initial phase corresponds to the pH-dependent cleavage rate observed in solution, but the second, faster phase is roughly pH-independent and closely parallels the cleavage rate observed at pH 8 (0.4/minute). This result is particularly remarkable because it entails that the rapidly cleaving phase at pH 6 is comparable to the cleavage rate for the fastest cleaving hammerhead ribozymes at pH 6. Based upon these observations, we conclude that the pH-dependent conformational change is the rate-determining step under standard conditions for the hammerhead ribozyme self-cleavage reaction, and that an ionizable 2'-proton at cleavage site is required for this conformational change. We further hypothesize that deprotonation of the cleavage-site 2'-oxygen drives this conformational change.     

Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme

The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma hammerhead ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this ribozyme. The divalent metals ions Ca²⁺, Mg²⁺, Mn²⁺, and Sr²⁺ have a relatively small variation (less than sixfold) in their ability to globally fold the hammerhead ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma hammerhead ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma hammerhead ribozyme are discussed.     

Deprotonation stimulates productive folding in allosteric TRAP hammerhead ribozymes

Hammerhead ribozymes in crystals change conformation in response to deprotonation of the nucleophilic 2' OH, thereby aligning the hydroxyl for in-line displacement at the scissile phosphate. Published data do not address whether deprotonation affects folding in solution. Allosteric hammerhead "TRAPs," when activated by the appropriate oligonucleotide, show the expected log-linear relation between initial cleavage rate and pH. In contrast, attenuated TRAPs shows biphasic kinetics in which a rapid burst is followed by slow cleavage that is nearly independent of pH. Attenuated ribozymes are stimulated by urea at both low and high pH, confirming that rearrangement of secondary structure is rate-limiting for the attenuated ribozymes once they have folded. Plots of burst magnitude versus pH in the absence of urea show a sharp transition around pH 8.3, which is near the kinetic pKa for the cleavage reaction in Mg2+. Raising the pH after folding at pH 7.5 did not activate attenuated ribozymes even when the RNA was incubated at the elevated pH for extended periods prior to addition of Mg2+. In contrast, lowering the pH after folding at pH 9.5 rapidly re-established attenuation. Deprotonation of the ribozyme-substrate complex thus appears to alter the folding landscape such that a metastable "pre-activated" complex forms before the thermodynamically more stable attenuated state can be attained. From the initial partition into active and inactive conformers, we estimate that this deprotonation contributes approximately 1.2 kcal/mol toward stabilization of the active fold at a crucial step during folding of the TRAP. Assuming that the nucleophilic 2' OH is the relevant acid, its deprotonation would thus serve a dual role of favoring productive fold and enhancing the nucleophilicity of this oxygen.     

一个双价锤头型核酶在体外对两种不同植物病毒RNA的专一切割作用

根据锤头核酶模型,设计合成了一个以黄瓜花叶病毒（ＣＭＶ） 外壳蛋白（ＣＰ） 亚基因组ＲＮＡ 为底物的锤头型核酶（ＲＺＣ） 。在证明它能有效切割该底物后,再将这个核酶与一个能专一性切割烟草花叶病毒（ＴＭＶ） 移动蛋白（ ＭＰ） 亚基因组ＲＮＡ 的锤头型核酶（ＲＺ１） 相互串联构成了一个双价核酶（ＲＺ１Ｃ） 。体外结果表明,这个双价核酶能与相应的单价核酶ＲＺ１ 和ＲＺＣ 一样专一而有效地切割ＣＭＶＣＰ和ＴＭＶ ＭＰＲＮＡ。     

Expanded hammerhead ribozymes containing addressable three-way junctions

Recently, hammerhead ribozyme (HHR) motifs have been utilized as powerful tools for gene regulation. Here we present a novel design of expanded full-length HHRs that allows attaching additional functionalities to the ribozyme. These features allowed us to construct a very efficient artificial riboswitch in bacteria. Following the design of naturally occurring three-way junctions we attached an additional helix (IV) to stem I of the HHR while maintaining very fast cleavage rates. We found that the cleavage activity strongly depends on the exact design of the junction site. Incorporation of the novel ribozyme scaffold into a bacterial mRNA allowed the control of gene expression mediated by autocatalytic cleavage of the ribozyme. Appending an aptamer to the newly introduced stem enabled the identification of very powerful theophylline-inducible RNA switches by in vivo screening. Further investigations revealed a cascading system operating beyond the ribozyme-dependent mechanism. In conclusion, we extended the hammerhead toolbox for synthetic biology applications by providing an additional position for the attachment of regulatory modules for in vivo control of gene expression.     

锤头状核酶在HepG2细胞中抑制adr亚型的乙型肝炎病毒

针对HBV基因组设计了三种锤头状核酶－－RS3、RC2和RC1。将裸露的和用tRNA包埋的核酶（RtS3、RtC2和RtC1）基因插入RNA修剪质粒pRG523中,然后转变为真核表达载体,使用同样克隆技术,构建由不同长度（2、4、8、12个）RS3或12个RtS3连成的鸟枪型真核表达载体,将它们分别与带有HBV基因组的质粒共转染人肝癌细胞系HepG2,用G418筛选抗性细胞。分析不同种类的核酶在G418抗性细胞中的HBV抑制活性,包括子代HBV－DNA、HBV－RNA和抗原（HBsAg或HBcAg/HBeAg)表达的减少。结果表明,所有设计的核酶对HBV有＞70％的抑制活性,而tRNA包埋核酶的活性略高于裸露核酶。特别值得提出的是,由8个或12个相同核酶连接的鸟枪型质粒（8RS3、12RS3和12RsS3）具有很高的抑制HBV活性,达到＞9－％的抑制。     

Applications of nucleic acid technology in the CNS

This review examines applications of nucleic acid technology in the form of catalytic nucleic acids (ribozymes and DNAzymes) and RNA interference (RNAi) in the CNS. The basic mechanism of catalytic nucleic acids and RNAi is reviewed, and potentials and problems highlighted. Recent advances in chemical modifications and delivery techniques are summarized. Applications in the CNS, including their use in primary neuronal cells, organotypic slice culture and the brain in vivo are further discussed.     

锤头型核酶作用机理的研究进展

概述了锤头型核酶的二级结构特征,动力学反应的特点以及核酶切割反应的催化机制,提出了锤头型核酶作用机理有待于深入研究的问题.     

丙型肝炎病毒RNA特异性核酶的切割活性研究

针对丙型肝炎病毒RNA(HCV-RNA)的5′非编码区和部分C区的二级结构,设计并合成了四个不同的锤头型核酶(ribozyme A, ribozyme B, ribozyme C₁, ribozyme C₂).首先应用体外切割实验筛选出作用于HCV-RNA起始密码子上游GTA↓位点的核酶RzA有较好的活性.为初步验证核酶RzA在细胞内的切割活性,经脂质体介导,将RzA-RNA与另一携带该核酶靶基因的质粒表达载体pCl-neo-luciferase(载体中荧光素酶基因受核酶靶基因的调控)共转染HepG₂细胞.通过测定荧光素酶基因的表达证实了核酶在细胞内有较好的切割活性.在此实验基础上,把RzA基因克隆至pCl-neo质粒表达载体中,再次经脂质体介导,将重组的表达载体pCl-neo-RzA与携带该核酶靶基因的质粒表达载体pCl-neo-luciferase共转染HepG₂细胞,获得了更好的切割效果.