Modified binary hammerhead ribozymes with high catalytic activity

A series of binary hammerhead ribozymes was designed and assessed in terms of cleavage activity and nuclease resistance. Enhanced nuclease resistance of binary ribozymes was achieved by incorporation of Z-modified nucleotides at the selective positions along with addition of 3'-3-linked thymidine cap. These modified binary ribozymes efficiently cleave 190-nucleotides long MDR1 mRNA fragment and display catalytic activity much higher then respective full-length analogs.     

Binary hammerhead ribozymes with improved catalytic activity

A new design of binary hammerhead ribozymes displaying high catalytic activity and nucleolytic stability is described. These catalytic structures consist of two partially complementary oligoribonucleotides, capable of assembling into the hammerhead-like structure without tetraloop II on binding to the RNA target. A series of these binary ribozymes targeting the translation initiation region of multiple drug resistance gene mdr1 mRNA was synthesized and assessed in terms of catalytic activity under single and multiple reaction turnover conditions. Enhanced nuclease resistance of the binary ribozymes was achieved by incorporation of 2'-modified nucleotides at selected positions, along with addition of a 3'-3'-linked thymidine cap. The new binary ribozymes exhibit higher RNA cleavage activity than their full-length analogs because of faster dissociation of cleavage products. Furthermore, an excess of one of the ribozyme strands provides the possibility to unfold structured regions of the target RNA and facilitate productive complex formation.     

2′-SUBSTITUTED PHOSPHOROTHIOATE CONTAINING OLIGORIBONUCLEOTIDES: AN APPLICATION TO THE SYNTHESIS AND PURIFICATION OF HAMMERHEAD RIBOZYMES

Abstract

A systematic study of the catalytic activity and nuclease stability of selectively modified hammerhead ribozymes has resulted in the identification of a generic motif containing 5 ribose residues and 31 2′- modified sugars (1). This substructure has been further elaborated to include phosphorothioate linkages. Although oligodeoxyribonucleotides containing phosphorothioate linkages have been studied extensively, similarly substituted RNA molecules or ribozymes have not been explored at-length. The synthesis and purification of these ribozymes is discussed (2).     

2′-O-Methyl Thiomethyl Modifications in Hammerhead Ribozymes

Abstract

The synthesis of all four phosphoramidites of 2′-O-methylthiomethyl ribonucleosides and their incorporation into hammerhead ribozymes and influence on nuclease stability and catalytic activity is described.     

A circular trans-acting hepatitis delta virus ribozyme.

A circular trans-acting ribozyme designed to adopt the motif of the hepatitis delta virus (HDV) trans-acting ribozyme was produced. The circular form was generated in vitro by splicing a modified group I intron precursor RNA in which the relative order of the 5' and 3' splice sites, flanking the single HDV-like ribozyme sequence-containing exon, is reversed. Trans-cleavage activity of the circular HDV-like ribozyme was comparable to linear permutations of HDV ribozymes containing the same core sequence, and was shown not to be due to linear contaminants in the circular ribozyme preparation. In nuclear and cytoplasmic extracts from HeLa cells, the circular ribozyme had enhanced resistance to nuclease degradation relative to a linear form of the ribozyme, suggesting that circularization may be a viable alternative to chemical modification as a means of stabilizing ribozymes against nuclease degradation.     

丁型肝炎病毒核酶的结构特点与催化作用机制

丁型肝炎病毒(HDV)核酶是小核酶的一种,在分子结构和作用机制等方面都有许多不同于其它核酶的特性。以其晶体结构的揭示为基础,近几年对其立体构型及催化机制方面的研究取得了很大进展,尤其是发现HDV核酶的胞嘧啶侧链在生理条件下能发挥一般酸碱催化作用(generalacidbasecatalysis),引起了极大关注。对HDV核酶结构和催化机制的研究,将使核酶被有目的地改造,并极大地推动它在应用方面的研究。     

Nucleic acid molecular switches

Natural and artificial ribozymes can catalyse a diverse range of chemical reactions. Through recent efforts in enzyme engineering, it has become possible to tailor the activity of ribozymes to respond allosterically to specific effector compounds. These allosteric ribozymes function as effector-dependent molecular switches that could find application as novel genetic-control elements, biosensor components or precision switches for use in nanotechnology.     

Small, efficient hammerhead ribozymes

The hammerhead ribozyme is able to cleave RNA in a sequence-specific manner. These ribozymes are usually designed with four basepairs in helix II, and with equal numbers of nucleotides in the 5′ and 3′ hybridizing arms that bind the RNA substrate on either side of the cleavage site. Here guidelines are given for redesigning the ribozyme so that it is small, but retains efficient cleavage activity. First, the ribozyme may be reduced in size by shortening the 5′ arm of the ribozyme to five or six nucleotides; for these ribozymes, cleavage of short substrates is maximal. Second, the internal double-helix of the ribozyme (helix II) may be shortened to one or no basepairs, forming a miniribozyme or minizyme, respectively. The sequence of the shortened helix+loop II greatly affects cleavage rates. With eight or more nucleotides in both the 5′ and the 3′ arms of a miniribozyme containing an optimized sequence for helix+loop II, cleavage rates of short substrates are greater than for analogous ribozymes possessing a longer helix II. Cleavage of genelength RNA substrates may be best achieved by miniribozymes.     

Cathepsin B promotes both motility and invasiveness of oral carcinoma cells

We previously demonstrated that overexpression of cathepsin B (CB) protease in oral squamous cell carcinomas correlated positively with advanced tumor stage and poor histologic malignancy grade. Here we examined whether CB contributes to the invasiveness of oral carcinoma cells. For RNA-mediated inhibition, two ribozymes that target CB mRNA were designed and stably expressed in the oral squamous cell carcinoma cell line 1386Tu. Both ribozymes diminished expression of CB mRNA, protein, and activity, without affecting cathepsin D or beta-actin, as determined by quantitative real-time PCR, Western blots, and protease activity assays. Matrigel invasion assays showed that the invasiveness of the cells was significantly reduced by the expressed ribozymes and, surprisingly, the motilities of the ribozyme-transfected cells were also diminished. Our results document a direct role for CB in promoting oral cancer spread and invasion, and open the possibility of controlling oral carcinoma malignancy and metastasis by targeting CB with RNA inhibitor strategies.     

Transient transfection of a synthetic hammerhead ribozyme targeted against human MGMT gene to cells in culture potentiates the genotoxicity of the alkylation damage induced by mitozolomide.

Unmodified and chemically modified forms of a synthetic hammerhead ribozyme with the mRNA of methylguanine-DNA methyltransferase (MGMT) gene as substrate were characterized for their in vitro and in vivo activities. The unmodified ribozyme efficiently cleaved in vitro a short synthetic substrate, and it was rapidly degraded in fetal bovine serum (FBS). The introduction of phosphorothioates and the substitution of uridine with thymidine at probable nuclease-sensitive sites slightly increased the nuclease resistance of the ribozyme. Conversely, pyrimidine nucleoside substitution with 2'NH2 and 2'F nucleosides strongly enhanced nuclease resistance. The in vivo activity was determined by measuring the genotoxicity induced by the alkylating drug mitozolomide, the damage of which is repaired by MGMT enzyme. CHO/47 cells, temporarily depleted of the MGMT protein, were first transfected with the various synthetic ribozymes and subsequently treated with mitozolomide. At equivalent concentration of the drug, the induction of sister chromatid exchanges was higher in ribozyme-transfected than in untransfected cells, indicating that the synthetic ribozymes potentiated the genotoxicity of mitozolomide. Moreover, the concomitant occurrence of messenger RNA reduction in ribozyme-transfected cells indicated that the inhibition of MGMT resynthesis was the basis of the enhanced genotoxicity.     

Synthesis and characterization of small interfering RNAs with haloalkyl groups at their 3′-dangling ends

With the aim to create a small interfering RNA (siRNA) with enhanced activity and resistance to nuclease degradation, we synthesized and evaluated the properties of the following siRNAs containing haloalkyl β-d-ribofuranosides at their 3′-dangling ends: 2,2,2-trifluoroethyl β-d-ribofuranoside, 2,2,2-trichloroethyl β-d-ribofuranoside and 2,2,2-tribromoethyl β-d-ribofuranoside. The gene silencing activities of the modified siRNAs were investigated through a dual luciferase reporter assay using HeLa cells. The highest silencing activity was observed for the trichloroethyl analog modified siRNA, which was closely followed by the trifluoroethyl and tribromoethyl analogs. The modified siRNAs were found to show increased binding affinity towards the Piwi-Argonaute-Zwille (PAZ) domain protein based on computational analysis and an experimental study. Furthermore, the RNAs modified with the analogs at their 3′-ends exhibited improved resistance to hydrolysis by a 3′-exonuclease.     

Ribozymes: recent advances in the development of RNA tools

The discovery 20 years ago that some RNA molecules, called ribozymes, are able to catalyze chemical reactions was a breakthrough in biology. Over the last two decades numerous natural RNA motifs endowed with catalytic activity have been described. They all fit within a few well-defined types that respond to a specific RNA structure. The prototype catalytic domain of each one has been engineered to generate trans-acting ribozymes that catalyze the site-specific cleavage of other RNA molecules. On the 20th anniversary of ribozyme discovery we briefly summarize the main features of the different natural catalytic RNAs. We also describe progress towards developing strategies to ensure an efficient ribozyme-based technology, dedicating special attention to the ones aimed to achieve a new generation of therapeutic agents.     

含四个串联核酶和GFP基因的转基因质粒的构建

以含绿色荧光蛋白（GFP）基因的质粒pSK100-DS、含切割对虾杆状病毒基因的核酶Rz1的质粒pRGRzl、含核酶Rz2的质粒pRGRz2和转基因空质粒pcDNA3为基础,把绿色荧光蛋白GFP基因克隆于pcDNA3的SV40启动了下面,由SV40启动子控制,含四个两种核酸基因的四联体克隆于pcDNA3的多克隆位点区,由T7启动子控制,构建成含两个Rz1、两个Rz2和GFP基因的转基因质粒pGTR,以用于转基因抗病毒对虾的研究。     

Design and Anti-HIV-1 Activity of Hammerhead and Hairpin Ribozymes Containing a Stable Loop#

Abstract

Three ribozymes, a hairpin ribozyme (HR112) and two hammerhead ribozymes (RZ115 and RZ119) containing a 5′C(UUCG)G3′ loop were designed to cleave the U5 region in the long terminal repeat (LTR) of HIV-1 RNA. The t_½ values of chemically synthesized substrates mediated by three ribozymes were measured. The transformed CEM cells possessing these three ribozyme-encoding genes were challenged with a HIV-1_IIIB strain, and two of these three ribozymes, HR112 and RZ119, were shown to possess strong anti-HIV-1 activity.

     

Synthesis of 6-Aza- & 6-Methyl-pyrimidine Ribonucleoside Phosphoramidites and Their Incorporation in Hammerhead Ribozymes

Abstract

The synthesis of phosphoramidites of 6-modified pyrimidine ribonucleosides and their incorporation into hammerhead ribozymes and influence on nuclease stability and catalytic activity is described.     

2′-DMAOE RNA: Emerging Oligonucleotides with Promising Antisense Properties

Abstract

Oligonucleotides with modifications at the carbohydrate 2′-position offer potential second-generation drug candidates¹. ISIS 13312, a chimeric compound targeting CMV retinitis, has 2′-O-methoxyethyl² (2′-MOE) modifications at the ends to offer enhanced binding affinity and nuclease resistance is an example of this trend. 2′-MOE modification offers high binding affinity and nuclease resistance presumably due to conformational constraints placed on the linkage by the oxygen-oxygen gauche effect³. On the other hand, 2′-O-aminopropyl modification (2′-AP) exhibits the highest nuclease resistance⁴, due to the presence of a cationic charge at the physiological pH. However, it lacks the binding affinity advantage of MOE due to the lack of oxygen-oxygen gauche effect. To optimize the antisense properties of both 2′-MOE and 2′-AP modifications, we have designed and constructed 2′-O-(aminooxyethyl) modification (2′-AOE)⁵ and 2′-O-(dimethylaminooxy ethyl) modification (2′-DMAOE) and synthesized oligomers having these modifications. 2′-DMAOE oligomers demonstrate higher binding affinity and nuclease resistance than 2′-MOE oligomers and stand out as promising candidates for future antisense oligonucleotide drug development.     

Continuous In Vitro Evolution of Ribozymes That Operate Under Conditions of Extreme pH

Continuous in vitro evolution methods were used to study the behavior of an evolving population of RNA ligase ribozymes in response to selection pressures involving conditions of extreme pH. The starting population consisted of randomized variants of a ribozyme that had been optimized for activity at pH 8.5. The ribozymes were subjected to repeated rounds of selective amplification under progressively more acidic or more alkaline conditions. The two final evolved populations of ribozymes were able to operate at either pH 5.8 or pH 9.8, respectively. Representative individuals from the two final populations were isolated and characterized. The low-pH ribozyme exhibited a 10-fold increase in catalytic rate at pH 5.8 compared to the starting molecule. The high-pH ribozyme retained its structural integrity and activity at pH 9.8, whereas the starting molecule was denatured under this condition. These findings demonstrate that a population of functional macromolecules can adapt to stringent environmental conditions through the acquisition of relatively few mutations. The results establish continuous in vitro evolution as a useful model system for exploring the evolution of enzymatic function in extreme environments. Present address (Henriette Kühne): Cardinal Health, 2950 Trade Place, San Diego, CA 92126, USA     

Properties of Antigenomic Hepatitis Delta Virus Ribozyme Cis‐ and Trans‐ Analogs

A series of permuted variants of antigenomic HDV ribozyme and trans‐acting variants were constructed. The catalytic activity study of the ribozymes has shown that all the variants were capable of self‐cleaving with equally biphasic kinetics. Ribonuclease and Fe(II)‐EDTA cleavage have provided evidence that all designed ribozymes fold according to the pseudoknot model and the conformations of the initial and cleaved ribozyme are different. A scheme of HDV ribozyme self‐cleavage reaction was suggested. The role of hydrogen bonds in the reaction was evaluated by substitution of ribose in the ribozyme for deoxyribose. It was found that the 2′‐OH group of U23 and C27 is critical for the reaction to occur; the 2′‐OH group of U32 and U39 is important, while 2′‐OH groups of other nucleotides of loop 3, stem 4 and stem 1 are unimportant for the cleavage activity.