Formation of spatial structure of RNA molecules

In this review we consider several experimental and theoretical approaches for investigation of RNA folding and determination of nucleotides that play an important role upon folding of such molecules as tRNA and several classes of ribozymes. It has been shown that nucleotides in the D- and T-loop regions are the last to be involved in tRNA structure or they are not included in the folding nucleus of tRNA. Using the specially elaborated method SHAPE it has been demonstrated that the model of hierarchical folding which was recognized for a long time is not correct for tRNA folding. In the second part of the given review the algorithms and programs used for the prediction of secondary structures of RNA as well as for modeling of RNA folding are considered.     

Formation of RNA spatial structures

The review considers different experimental and theoretical approaches to the investigation of RNA folding and identification of nucleotides that critically affect the folding of molecules, such as tRNA, and several classes of ribozymes. For instance, it has been shown that nucleotides of the D- and T-loop regions are the last to be involved in the tRNA structure, or, rather, they are not included in the tRNA folding nucleus. A specially developed SHAPE method was used to show that the long-recognized hierarchical folding model does not hold true for tRNA folding. In the second part of the review, algorithms and programs used for the prediction of RNA secondary structures, as well as for modeling RNA folding, are considered.     

A long-range pseudoknot is required for activity of the Neurospora VS ribozyme.

Four small RNA self-cleaving domains, the hammerhead, hairpin, hepatitis delta virus and Neurospora VS ribozymes, have been identified previously in naturally occurring RNAs. The secondary structures of these ribozymes are reasonably well understood, but little is known about long-range interactions that form the catalytically active tertiary conformations. Our previous work, which identified several secondary structure elements of the VS ribozyme, also showed that many additional bases were protected by magnesium-dependent interactions, implying that several tertiary contacts remained to be identified. Here we have used site-directed mutagenesis and chemical modification to characterize the first long-range interaction identified in VS RNA. This interaction contains a 3 bp pseudoknot helix that is required for tertiary folding and self-cleavage activity of the VS ribozyme.     

NMR spectroscopic characterization of a model RNA duplex reflecting the core sequence of hammerhead ribozymes

ABSTRACT

Hammerhead ribozymes are a model system for studying molecular mechanism of RNA catalysis. Physicochemical data-driven mechanistic studies are an indispensable step towards understanding the catalysis of hammerhead ribozymes. Here we characterized a model RNA duplex with catalytically important sheared-type G12-A9 base pair and A9-G10.1 metal ion-binding motif in hammerhead ribozymes. By using high magnetic field NMR, all base proton signals, including catalytic residues, were unambiguously assigned. We further characterized structural features of this RNA molecule and found that it reflects the structural features of the A9-G10.1 motif of hammerhead ribozymes. Therefore, this RNA molecule is suitable for extracting an intrinsic physicochemical properties of catalytically important residues.     

Effects of variations in length of hammerhead ribozyme antisense arms upon the cleavage of longer RNA substrates.

The efficacy of intracellular binding of hammerhead ribozyme to its cleavage site in target RNA is a major requirement for its use as a therapeutic agent. Such efficacy can be influenced by several factors, such as the length of the ribozyme antisense arms and mRNA secondary structures. Analysis of various IL-2 hammerhead ribozymes having different antisense arms but directed to the same site predicts that the hammerhead ribozyme target site is present within a double-stranded region that is flanked by single-stranded loops. Extension of the low cleaving hammerhead ribozyme antisense arms by nucleotides that base pair with the single-stranded regions facilitated the hammerhead ribozyme binding to longer RNA substrates (e.g. mRNA). In addition, a correlation between the in vitro and intracellular results was also found. Thus, the present study would facilitate the design of hammerhead ribozymes directed against higher order structured sites. Further, it emphasises the importance of detailed structural investigations of hammerhead ribozyme full-length target RNAs.     

Combinatorial properties of RNA secondary structures.

The secondary structure of an RNA molecule is of great importance and possesses influence, e.g., on the interaction of tRNA molecules with proteins or on the stabilization of mRNA molecules. The classification of secondary structures by means of their order proved useful with respect to numerous applications. In 1978, Waterman, who gave the first precise formal framework for the topic, suggested to determine the number a(n,p) of secondary structures of size n and given order p. Since then, no satisfactory result has been found. Based on an observation due to Viennot et al., we will derive generating functions for the secondary structures of order p from generating functions for binary tree structures with Horton-Strahler number p. These generating functions enable us to compute a precise asymptotic equivalent for a(n,p). Furthermore, we will determine the related number of structures when the number of unpaired bases shows up as an additional parameter. Our approach proves to be general enough to compute the average order of a secondary structure together with all the r-th moments and to enumerate substructures such as hairpins or bulges in dependence on the order of the secondary structures considered.     

作用于HBV（adr亚型）RNA的tRNA—包埋锤头状核酶的研究

设计了针对ＨＢＶ（ａｄｒ亚型）ＲＮＡ的二个锤头状核酶（ＲＳ３和ＲＣ２）,并将其插入ｔＲＮＡ反密码环中（ＲｔＳ３和ＲｔＣ２）,以增加其稳定性。实验表明,虽然插入ｔＲＮＡ中的核酶与裸露核酶相比,催化活性有所下降,但在胎牛血清和ＨｅｐＧ２细胞抽提液中的稳定性却明显提高。因此,ｔＲＮＡ－包埋核酶有可能提高在体内的抗病毒能力。     

Structural RNA has lower folding energy than random RNA of the same dinucleotide frequency

We present results of computer experiments that indicate that several RNAs for which the native state (minimum free energy secondary structure) is functionally important (type III hammerhead ribozymes, signal recognition particle RNAs, U2 small nucleolar spliceosomal RNAs, certain riboswitches, etc.) all have lower folding energy than random RNAs of the same length and dinucleotide frequency. Additionally, we find that whole mRNA as well as 5'-UTR, 3'-UTR, and cds regions of mRNA have folding energies comparable to that of random RNA, although there may be a statistically insignificant trace signal in 3'-UTR and cds regions. Various authors have used nucleotide (approximate) pattern matching and the computation of minimum free energy as filters to detect potential RNAs in ESTs and genomes. We introduce a new concept of the asymptotic Z-score and describe a fast, whole-genome scanning algorithm to compute asymptotic minimum free energy Z-scores of moving-window contents. Asymptotic Z-score computations offer another filter, to be used along with nucleotide pattern matching and minimum free energy computations, to detect potential functional RNAs in ESTs and genomic regions.     

On realizing shapes in the theory of RNA neutral networks

It is known (Reidys et al., 1997b. Bull. Math. Biol. 59(2), 339-397) that for any two secondary structures S,S' there exists an RNA sequence compatible with both, and that this result does not extend to more than two secondary structures. Indeed, a simple formula for the number of RNA sequences compatible with secondary structures S,S' plays a role in the algorithms of Flamm et al. (2001. RNA 7, 254-265) and of Abfalter et al. (2003. Proceedings of the German Conference on Bioinformatics, ) to design an RNA switch. Here we show that a natural extension of this problem is NP-complete. Unless P=NP, there is no polynomial time algorithm, which when given secondary structures S1,...,S(k), for k4, determines the least number of positions, such that after removal of all base pairs incident to these positions there exists an RNA nucleotide sequence compatible with the given secondary structures. We also consider a restricted version of this problem with a "fixed maximum" number of possible stars and show that it has a simple polynomial time solution.     

Secondary structure prediction of interacting RNA molecules

Computational tools for prediction of the secondary structure of two or more interacting nucleic acid molecules are useful for understanding mechanisms for ribozyme function, determining the affinity of an oligonucleotide primer to its target, and designing good antisense oligonucleotides, novel ribozymes, DNA code words, or nanostructures. Here, we introduce new algorithms for prediction of the minimum free energy pseudoknot-free secondary structure of two or more nucleic acid molecules, and for prediction of alternative low-energy (sub-optimal) secondary structures for two nucleic acid molecules. We provide a comprehensive analysis of our predictions against secondary structures of interacting RNA molecules drawn from the literature. Analysis of our tools on 17 sequences of up to 200 nucleotides that do not form pseudoknots shows that they have 79% accuracy, on average, for the minimum free energy predictions. When the best of 100 sub-optimal foldings is taken, the average accuracy increases to 91%. The accuracy decreases as the sequences increase in length and as the number of pseudoknots and tertiary interactions increases. Our algorithms extend the free energy minimization algorithm of Zuker and Stiegler for secondary structure prediction, and the sub-optimal folding algorithm by Wuchty et al. Implementations of our algorithms are freely available in the package MultiRNAFold.     

Eggplant latent viroid,the candidate type species for a new genus within the family Avsunviroidae (hammerhead viroids)

Viroids, small circular RNAs that replicate independently and in most cases incite diseases in plants, are classified into the families Pospiviroidae, composed of species with a central conserved region (CCR) and without hammerhead ribozymes, and Avsunviroidae, composed of three members lacking CCR but able to self-cleave in both polarity strands through hammerhead ribozymes. Here we report the biological and molecular properties of Eggplant latent viroid (ELVd). Purified circular ELVd induces symptomless infections when inoculated into eggplant seedlings. ELVd can be transmitted horizontally and through seed. Sequencing 10 complete cDNA clones showed that ELVd is a circular RNA of 332 to 335 nucleotides with high variability. This RNA can adopt a quasi-rod-like secondary structure of minimal free energy and alternative foldings that permit formation of stable hammerhead structures in plus and minus strands. The ribozymes are active in vitro and, most likely, in vivo. Considering the ELVd properties to be intermediate between those of the two genera of family Avsunviroidae, we propose ELVd as the type species of a third genus with the name ELAVIROID:     

Comparison of the specificities and catalytic activities of hammerhead ribozymes and DNA enzymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA.

With the eventual goal of developing a treatment for chronic myelogenous leukemia (CML), attempts have been made to design hammerhead ribozymes that can specifically cleave BCR-ABL fusion mRNA. In the case of L6 BCR-ABL fusion mRNA (b2a2 type; BCR exon 2 is fused to ABL exon 2), which has no effective cleavage sites for conventional hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to cleave the chimeric mRNA specifically. Several hammerhead ribozymes with relatively long junction-recognition sequences have poor substrate-specificity. Therefore, we explored the possibility of using newly selected DNA enzymes that can cleave RNA molecules with high activity to cleave L6 BCR-ABL fusion (b2a2) mRNA. In contrast to the results with the conventional ribozymes, the newly designed DNA enzymes, having higher flexibility for selection of cleavage sites, were able to cleave this chimeric RNA molecule specifically at sites close to the junction. Cleavage occurred only within the abnormal BCR-ABL mRNA, without any cleavage of the normal ABL or BCR mRNA. Thus, these chemically synthesized DNA enzymes seem to be potentially useful for application in vivo , especially for the treatment of CML, if we can develop exogenous delivery strategies.     

Nuclease resistant ribozymes with high catalytic activity.

Hammerhead ribozymes are efficient RNA enzymes characterized by a typical hammerhead secondary structure and a number of conserved bases. Little is known about the role of the ribose-phosphate backbone, although it is obviously important since a DNA molecule with the same base sequence is not a catalyst. Here we describe the synthesis of artificial ribozymes where modified (2'-O-allyl- and 2'-O-methyl-) ribonucleotides substitute for the corresponding ribonucleotides. A systematic analysis of partially substituted polymers identified a minimum set of six non-contiguous positions where insertion of modified ribonucleotides strongly affects catalytic activity. Surprisingly, ribozymes completely substituted except for these six ribonucleotides are still very active. These molecules efficiently cleave in trans target RNAs in a sequence-specific way, but, unlike RNA ribozymes, are very resistant to nuclease degradation and are very stable in serum. These properties make such synthetic polymers potentially useful for in vivo gene expression studies and therapeutic applications.     

Alternative tertiary structure attenuates self-cleavage of the ribozyme in the satellite RNA of barley yellow dwarf virus.

A self-cleaving satellite RNA associated with barley yellow dwarf virus (sBYDV) contains a sequence predicted to form a secondary structure similar to catalytic RNA molecules (ribozymes) of the 'hammerhead' class (Miller et al., 1991, Virology 183, 711-720). However, this RNA differs from other naturally occurring hammerheads both in its very slow cleavage rate, and in some aspects of its structure. One striking structural difference is that an additional helix is predicted that may be part of an unusual pseudoknot containing three stacked helices. Nucleotide substitutions that prevent formation of the additional helix and favor the hammerhead increased the self-cleavage rate up to 400-fold. Compensatory substitutions, predicted to restore the additional helix, reduced the self-cleavage rate by an extent proportional to the calculated stability of the helix. Partial digestion of the RNA with structure-sensitive nucleases supported the existence of the proposed alternative structure in the wildtype sequence, and formation of the hammerhead in the rapidly-cleaving mutants. This tertiary interaction may serve as a molecular switch that controls the rate of self-cleavage and possibly other functions of the satellite RNA.     

Computing the partition function and sampling for saturated secondary structures of RNA, with respect to the Turner energy model.

An RNA secondary structure is saturated if no base pairs can be added without violating the definition of secondary structure. Here we describe a new algorithm, RNAsat, which for a given RNA sequence a, an integral temperature 0 相似文献   

Selection and characterization of active hammerhead ribozymes targeted against cyclin E and E2F1 full-length mRNA.

Proliferation of vascular smooth muscle cells is generally accepted as a key event in the development of restenosis following percutaneous transluminal angioplasty. To prevent human restenosis, we have designed a molecular strategy based on hammerhead ribozymes targeted against the mRNA of cyclin E and E2F1, two proteins relevant in cell cycle progression whose regulation is interconnected by a positive feedback loop. Following the identification of accessible ribozyme target sites by RNase H mapping, several hammerhead ribozymes were generated that cleave with comparable efficiency two different splice forms of cyclin E mRNA and the full-length and a truncated form of E2F1 RNA, respectively. The most active ribozymes were tested in vitro under single-turnover conditions yielding k(react)/K(m) ratios between 36 and 73 x 10(4) M(-1) min(-1), which places them in the top range ribozymes targeted against long and structured substrates. In addition, we show that the most active ribozyme selected in vitro reduces specifically and significantly (p < 0.0028) proliferation of cultured human vascular smooth muscle cells (VSMC).     

Inverse folding of RNA pseudoknot structures

Background  

RNA exhibits a variety of structural configurations. Here we consider a structure to be tantamount to the noncrossing Watson-Crick and G-U-base pairings (secondary structure) and additional cross-serial base pairs. These interactions are called pseudoknots and are observed across the whole spectrum of RNA functionalities. In the context of studying natural RNA structures, searching for new ribozymes and designing artificial RNA, it is of interest to find RNA sequences folding into a specific structure and to analyze their induced neutral networks. Since the established inverse folding algorithms, RNAinverse, RNA-SSD as well as INFO-RNA are limited to RNA secondary structures, we present in this paper the inverse folding algorithm Inv which can deal with 3-noncrossing, canonical pseudoknot structures.     

Design, characterization and testing of tRNA3Lys-based hammerhead ribozymes

A hammerhead ribozyme targeted against the HIV-1 env coding region was expressed as part of the anticodon loop of human tRNA3Lys without sacrificing tRNA stability or ribozyme catalytic activity. These tRNA-ribozymes were isolated from a library which was designed to contain linkers (sequences connecting the ribozyme to the anticodon loop) of random sequence and variable length. The ribozyme target site was provided in cis during selection and in trans during subsequent characterization. tRNA-ribozymes that possessed ideal combinations of linkers were expected to recognize the cis target site more freely and undergo cleavage. The cleaved molecules were isolated, cloned and characterized. Active tRNA-ribozymes were identified and the structural features conducive to cleavage were defined. The selected tRNA-ribozymes were stable, possessed cleavage rates lower or similar to the linear hammerhead ribozyme, and could be transcribed by an extract containing RNA polymerase III. Retroviral vectors expressing tRNA-ribozymes were tested in a human CD4+ T cell line and were shown to inhibit HIV-1 replication. These tRNA3Lys-based hammerhead ribozymes should therefore prove to be valuable for both basic and applied research. Special application is sought in HIV-1 or HIV-2 gene therapy.     

抗HPV11 E2核酶的计算机设计

 借助计算机软件分析 ,设计出能特异性切割HPV11型 6 4 4ntE2mRNA的核酶 (ribozyme) .遵循Symon′s锤头状核酶结构和GUX剪切位点原则 ,靶序列存在 32个这样的剪切位点 .通过计算机软件分析出核酶的最佳剪切位点 ,并对底物及核酶的二级结构进行预测及进行相应基因生物学功能和基因同源性分析 ,筛选出 2个锤头结构核酶 .针对这两位点设计的核酶分别命名为RZ2 777和RZ32 81.计算机分析显示 ,两核酶与底物切点两翼碱基形成锤头状结构 ,切点所在基因序列具有相对松弛的二级结构 ,位于该基因重要生物功能区内 ,是核酶的理想攻击区域 .通过基因库检索 ,在已知人类基因排除了与上述两核酶切点两翼碱基有基因同源性序列的可能性 .将两核酶用于体外剪切实验取得了良好的实验结果 ,认为借助计算机分析可帮助尽快从多个剪切位点选择出最适核酶