Trans-cleaving activity of hepatitis delta virus genomic ribozymes

Objective: To study whether 2 hepatitis delta virus genomic ribozymes(g. Rz) have trans-cleaving activity. Methods: g. Rz74 and g. Rz55 were transcribed from their templates which were synthesized and cloned into plasmid pGEM-4Z; Homologous substrate RNA was transcribed for pRz277B and labelled withα-32 P-UTP. The trans-cleaving activity was studied by mixing the ribozymes and substrate in appropriate who under certain conditions. Results: The trans-cleaved products were generated after mixing the ribozyme with the substrate for 30 min, and accumulated more for 90 min. Conclusion: Both g. Rz74 and g. Rz55 possess trans-cleaving activity.

人工设计的Ribozymes体外切割HPV16 E6和E7 mRNA的研究

人乳头状瘤病毒(human papillomavirus,HPV)是一类感染人体的皮肤粘膜并引起乳头状瘤病变的病毒。目前发现的HPV已有63型,不同型的HPV引起人体不同部位的病变。HPV 16主要与生殖系统的肿瘤尤其是子宫颈癌有密切关系。

Reversal of HCC Drug Resistance by Using Hammerhead Ribozymes against Multidrug Resistance 1 Gene

To reverse multidrug resistance（MDR） of HepG2 by anti-MDR1 hammerhead ribozyme, an anti-MDR1 hammerhead ribozyme was developed and delivered to P-gp-overproducing human hepatocarcinoma cell line HepG2 by a retroviral vector containing RNA polymerase Ⅲ promoter. The expression of mdrl/Pgp and Rz was detected in HepG2, HepG2 muhidrug-resistant cell line and HepG2 Rz-transfected cells by semi-quantitative RT-PCR and Western blot methods. Moreover, MTT assay was employed to detect the sensitivity of these ribozyme-transfected cells, and Rhodamine123 （Rh123） was used to test the function of Pgp. The Rz- transfected HepG2 cells became doxorubicin-sensitive, which was concomitant with the decreased MDR1 expression. The study showed that the retrovirus vector encoding the anti-MDR1 ribozyme may be applicable to the treatment of MDR cells.

GENE THERAPY USING RETROVIRAL VECTOR OF bcr-abl SPECIFIC MULTI-UNIT RIBOZYMES COULD INHIBIT CML CELL GROWTH AND INDUCE APOPTOSIS

Objective: To investigate the in vitro cleavage ability and effects on apoptosis and cell growth of the bcr-abl fusion gene specific multi-unit ribozymes. Methods: Three fusion point specific ribozymes were designed and the multi-unit ribozymes?in vitro transcription vector and retroviral vector were constructed. The in vitro cleavage ability was tested. The retroviral vector was transfected into K562 cell and the effects on proliferation, apoptosis, cell cycle and cell structure were observed. Results: Multi-unit ribozymes had in vitro cleavage efficiency of 70.8%, which was more efficient than single-unit and double-unit ribozymes. Transfection of the retroviral vector of the ribozyme into K562 cells, induced inhibition of cell growth and apoptosis. The incorporation rate of DNA in ribozymes transfected K562 cells was greatly decreased along with time passed, with an inhibition rate of more than 50% after 96 h of transfection. Under FCM, 18.4% of the cells underwent apoptosis 72 h after transfection and more cells were blocked in G phase, with the ratio in S phase greatly decreased (41.9%). Under electron microscope, compaction of nuclear chromatin and apoptosis bodies were observed. Conclusion: Multi-unit ribozymes specific to bcr-abl fusion gene can be used to treat CML and to purge bone marrow for self-grafting.

Inhibition of Hepatitis B Virus Replication and Expression in Vitro and in Vivo by the Hammerhead Ribozymes Targeted Different Sites

Objective To develop an effective and specific medicine targeting hepatitis B virus(HBV) pregenome. Based on the identified accessible target sites for hammerhead ribozyme in our previous researches, a recombinant hepatitis delta virus(HDV) ribozyme was chosen and used to demonstrate the effective cleavage in vitro and in vivo. Methods Three hammerhead ribozymes for potential target sites(S, X and C genes) and co-expression plasmid(pTr-dB, pTdδ-dB, pTrX-dB and pTrC-dB) as well as four HDV-ribozyme chimera constructs with HBV(pTdXX, pTdXC, pTdSX and pTdSC) were severally chosen to validate the inhibition of the replication and expression of HBV. The co-expression plasmids(pTdδ and pTr-Db) in physiological saline were hydrodynamically injected to mice by tail vein. Results Compared with the group injected with pTr-dB in Huh-7 cell, hepatitis B surface antigen(HBsAg) was reduced by 31% in the group injected with pTdδ-dB, by 54%, 26%, 72% and 97% in the group injected with recombinant-ribozymes pTdSX, pTdSC, pTdXC and pTdXX, respectively. The inhibiting effects of endogenous ribozymes RzX and RzC on the HBsAg expression were 66% and 57%, respectively. Compared with the positive control, the amount of HBsAg was decreased in mice injected with pTdXX through tail vein by 88% and 96% on the second day and the third day, respectively. HBsAg was undetectable on the 6th day and could not primitively be detected on the 9th day in the sera from all mice. HBV DNA was not detected in the sera of BALB/c mice injected with pTdXX-dB, pTrX-dB or replicating-defective plasmid pHBV, while HBV DNA replication in control group could be detected on the 6th day. While HBcAg could not be detected in liver tissues of mice injected with plasmid pTdXX-dB on the 3rd day. Conclusions Encoding regions of HBV S, C and X gene were the effective cleavage sites for hammerhead ribozyme in vitro and in vivo, which provides basis for further construction of therapeutic recombinant HDV and the development of targeting antiviral gene therapy.

Ribozymes:an anti-viral agent

The discovery that RNA can act as an enzyme led Thomas Cech to win the Nobel Prize in Chemistry and led immediately to the next wave of attempts to find an effective RNA-based therapy.The tantalizing idea that RNA enzymes called trans-cleaving ribozymes enables them to act as potential antiviral and powerful tool for functional genomic studies.The efficacy of ribozyme function in a complex intracellular environment is dependent on the intracellular fate of the RNA that is being targeted.Recently,ribozymes have been used successfully to inhibit gene expression in a variety of biological systems in vitro and in vivo.Ribozyme has also been used successfully to combat many cases of viral infection,as clinical trial.Despite it needs to be investigated and explored as far as its structural and functional aspects are concern.In view of the significance of ribozyme in modern medicine,we reviewed the recent literature on general approach to control viral infection.

Comparison of cleavage activity in vitro between two ribozymes targeted against different sites of PDGF receptor β subunit

Objective: To compare the cleavage activity of ribozymes directed against 2 sites of PDGF receptorP subunit cDNA gene in vitro. Methods: The 608 bp fragment of PDGF receptor β subunit cDNA was clonedinto T-vector under the control of T7 promoter, named pPDGFR-β. Two ribozymes were designed to cleavethe CUU sequence at codon 45 and codon 252 of PDGF receptor β subunit mRNA respectively. These 2 ham-merhead ribozyme genes were cloned into vector PI. 5 between 5' -cis ribozyme and 3' -cis ribozyme to gener-ate the plasmids of pRZ1 and pRZ2. The pPDGFR-β, pRZl and pRZ2 were linearized and then transcribedwith T7 promoter in vitro. Results: The RZ1 showed high cleavage activity in vitro, but the RZ2 showed nocleavage activity under the same condition. Conclusion: The cleavage site selection is an important factor in-fluencing the cleavage activities of ribozymes.

Evaluating Performance of Different RNA Secondary Structure Prediction Programs Using Self-cleaving Ribozymes

Accurate identification of the correct,biologically relevant RNA structures is critical to understanding various aspects of RNA biology since proper folding represents the key to the functionality of all types of RNA molecules and plays pivotal roles in many essential biological processes.Thus,a plethora of approaches have been developed to predict,identify,or solve RNA structures based on various computational,molecular,genetic,chemical,or physicochemical strategies.Purely computational approaches hold distinct advantages over all other strategies in terms of the ease of implementation,time,speed,cost,and throughput,but they strongly underperform in terms of accuracy that significantly limits their broader application.Nonetheless,the advantages of these methods led to a steady development of multiple in silico RNA secondary structure prediction approaches including recent deep learning-based programs.Here,we compared the accuracy of predictions of biologically relevant secondary structures of dozens of self-cleaving ribozyme sequences using seven in silico RNA folding prediction tools with tasks of varying complexity.We found that while many programs performed well in relatively simple tasks,their performance varied significantly in more complex RNA folding problems.However,in general,a modern deep learning method outperformed the other programs in the complex tasks in predicting the RNA secondary structures,at least based on the specific class of sequences tested,suggesting that it may represent the future of RNA structure prediction algorithms.

Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing

Development of tools for targeted modifications of specific DNA sequences in plants is of great importance to basic plant biology research as well as crop improvement.The ability to cut DNA at specific locations in the genome to generate doublestrand breaks（DSBs）in vivo is a prerequisite for any genome editing efforts.

CLEAVAGES OF TRANSCRIPTS OF TOBACCO MOSAIC VIRUS BY SYNTHETIC RIBOZYMES in vitro

Based upon computer-assisted predictions on the secondary structures of tobacco mosaicvirus (TMV) genomic RNA (both polarities), hammerhead type ribozymes were synthesizedin vitro, which all shared a conserved domain adapted from satellite tobacco ringspot virus(sTobRV)RNA. Ribozymes RZ1, RZ2 and RZ3 were designed to cleave the phosphodiester bondsimmediate to the 3'--end of GUC between the residues 5384-5385 and 6312--6313 on the plusstrand and 1214-1215 on the minus strand, respectively. The in vitro data indicated that RZ1 wasable to cleave completely its substrates BT1(+ ) and BT2(+ ), representing partial sequencesof the plus strand of the TMV MP region at 50, 37 and 30℃ with a molar ratio of ribozymeto the target as low as 1:1. Its two iso-ribozymes RZ1A and RZ1B which were respectivelymodified to contain a CUUCGG sequence in the conserved region and in an additional 3'-ter-minal stem-loop of UUUUUCUUCGGAAAAA were able to cleave BT1(+) and BT2(+) asefficiently as RZ1. Ribozyme RZ3 cleaved, with less efficiency, its substrate BT2(-), repre-senting the minus strand of the TMV MP gene at 50, 37 or 30℃ even with a molar ratio ofribozyme to the target as high as 4:1. Ribozyme RZ2, however, had not any visible activityto its substrate BT3(+ ), representing the sequence was involved in the 3'--terminal tRNA--likesequence even though it was incubated for 5 min at 65℃ before being exposed to the ribozyme.

Blocking Translation of Oncogenic mRNA

Double-stranded RNA-mediated interference (RNAi), antisense oligonucleotides (ASO), and ribozymes have excellent specificity to their target oncogenic mRNA. They also seem to show great promise when it comes to treating cancer. The problem is that RNAi, ASO, and ribozymes have poor stability and are constantly being degraded by nucleases. Researchers have made some efforts to increase antisense oligonucleotides’ stability by creating phospharimidate and Phosphorothioate. Currently, ribozymes, antisense oligonucleotides, and (RNAi) are the three main methods used to target RNA. These methods are currently undergoing clinical trials for the purpose of focusing on specific RNAs involved in disorders like cancer and neurodegeneration. In fact, ASOs that target amyotrophic lateral sclerosis and spinal muscular atrophy have produced promising results in clinical trials. The formation of chemical alterations that boost affinity and selectivity while reducing noxiousness owing to off-target impacts are two benefits of ASOs. Another benefit is increased affinity. With a focus on RNAi and ASOs, this review illustrated the main therapeutic strategies of RNA therapy now in use.

The Possibility of Nucleic Acids to Act as Anti-Viral Therapeutic Agents—A Review

For just about 30 years, researchers have considered the likelihood to utilize nucleic acids as antiviral therapeutics. In principle, small single-stranded nucleotide sequence (oligonucleotide) could hybridize to a particular gene or messenger RNA and diminish transcription or translation, respectively, in this manner decreasing the amount of protein that is synthesized. Until now, an incredible number of antisense oligonucleotides, double-stranded oligonucleotides, aptamers, ribozymes, deoxyribozymes, interfering RNAs, chimeric RNA-DNA molecules, antibody genes has been created artificially and applied effectively for comprehension and manipulating biological processes and in clinical preliminaries to treat a variety of diseases. Their versatility and potency make them similarly fit candidates for fighting viral infections. However, troubles with their efficiency, off-target effects, toxicity, delivery, and stability halted the development of nucleic acid-based therapeutics that can be utilized in the clinic. The potential for nucleic acid therapeutic agents is significant and is quite recently beginning to be realized. In this review, we have summarized some of the recent advancements made in the area of nucleic acid based therapeutics and focused on the methods of their delivery and associated challenges.

抗c-erbB-2 ribozyme的计算机设计

目的:设计能特异性切割人癌基因c-erbB-2 mRNA的核酶(ribozyme).方法:根据Symons的“锤头结构”,以人癌基因c-erbB-2 mRNA为靶RNA,用计算机对其可能为核酶切割的位点进行分析以寻找最佳切点;对底物及核酶的二级结构进行预测.结果:c-erbB-2mRNA第2428～2776位和第2820～3004位之间的二级结构相对稳定,是重要的生物学功能区,因而也是核酶理想的攻击区域.该区第2438,2477,2751位是核酶的最佳切割位点.针对这三个位点设计了三个核酶并分别命名为RZ1,RZ2,RZ3.计算机模拟显示,它们均能与底物切点两翼碱基结合而形成锤头状结构.通过计算机基因库检索,在已知人基因中,其它基因的mRNA均不含有上述三个核酶切点两翼碱基的同源序列.结论:并非所有的GUX或CUX均可作为核酶的理想切割位点,c-erbB-2mRNA上第2438,2477,2751位可能是核酶的最佳切割位点.

Ribozyme和靶RNA的相互作用对于切割反应的影响

锤头结构Ｒｉｂｏｚｙｍｅ（Ｒｚ）通过与靶ＲＮＡ上切卢、二狈帕５核普酸序列的碱基配对识别和切割靶ＲＮＡ。通过改变Ｒｉｂｏｚｙｍｅ与靶ＲＮＡ之间碱基配对的长度以及性质，研究了Ｒｉｂｏｚｙｍｅ与靶ＲＮＡ相互作用对于它的切割反应的影响。结果表明：（１）缩短碱基配对长度和改变碱基配对的性质，使Ｒｉｔｉｏｚｙｍｅ与底物的熔卢、（Ｔｍ）降低，同时反应最适温度（Ｔｏｐｔ）也降低。（２）动力学分析表明，反应的Ｋｍ值随温度升高而增加，当温度接近或高于Ｒｉｂｏｚｙｍｅ与底物的熔点时，Ｋｍ值增加大大加快。（３）反应的Ｋｃａｔ也随温度升高而增加，但Ｋｃａｔ增加的速度继而逐渐变慢，最后Ｋｃａｔ下降。

目的Ribozyme两侧长片段附加序列（LAS）的去除及其转录载体构建

选择小鼠腺苷脱氨酶ｍＲＮＡ特异Ｒｉｂｏｚｙｍｅ做为目的Ｒｉｂｏｚｙｍｅ（Ｒｚ），在目的Ｒｉｂｏｚｙｍｅ两侧分别设计５＇－顺式Ｒｚ和３＇－顺式Ｒｚ，在目的Ｒｉｂｏｚｙｍｅ上设计ＢａｍＨＩ酶切位点，并构建了上述Ｒｉｂｏｚｙｍｅ转录载体ｐＳＣＲｚ２６２，采用该质粒进行体外转录，并对转录靶ＲＮＡ分子进行了切割反应。结果显示ｐＳＣＲｚ２６２在转录过程中发生了自身切割，并释放出目的Ｒｉｂｏｚｙｍｅ－Ｒｚ２６２，该目的Ｒｉｂｏｚｙｍｅ不但去除了两侧长片段附加序列，而且保持了正确的生物学活性，通过ＢａｍＨＩ切点的设计简化了该质粒的筛选。

抗HPV16 E7-ribozyme在CV-1细胞中的稳定表达

构建了由RSV-LTR启动子带动并能在细胞内稳定复制的Ribozyme的自身修剪表达质粒pRSV-Rz523、Ribozyme反义对照质粒pRSV-AE7及人增殖细胞核抗原基因(PCNA)启动子带动的HPV16 E7片段(+554～+686)的真核表达质粒pPCNA-E7。经G418抗性筛选获得了稳定表达Ribozyme的CV-1细胞克隆,其表达水平约为9.0pmol/10~6个细胞,其中活性Ribozyme的量大于50fmol/10~6个细胞,分离得到的Ribozyme可在体外特异切割E7靶RNA片段。通过共转染Ribozyme(或反义对照)和底物表达质粒并筛选出细胞克隆,研究了Ribozyme在细胞中对底物表达水平的影响。初步结果显示,Ribozyme的导入可使细胞内底物E7的RNA表达水平降低了90％(反义对照使E7 RNA表达降低20％)。上述结果提示:在CV-1细胞中表达的Ribozyme不仅在体外,同时在细胞内具有一定的生物学活性,有可能应用于逆转宫颈癌细胞的恶性表型。

抗猪瘟病毒Ribozyme基因表达载体的构建

根据猪瘟病毒（ＨＣＶ）基因组ＲＮＡ的序列和Ｒｉｂｏｚｙｍｅ的结构特点，设计并合成了针对ＨＣＶ基因组Ｐ（８０）编码区长为５６碱基对（ｂｐ）的榔头状ＨＣＶＲｉｂｏｚｙｍｅ（ＨＲ）基因。将此基因插入质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ中，转化ＸＬ１－ｂｌｕｅ细胞，经限制性内切酶鉴定，获得６个阳性克隆ｐＢＨＲ。可用于ＨＣＶ抗性的体外表达研究。又将羊金属巯因启动子（ｏＭＴ－ｐｒｏｍｏｔｅｒ）插入ｐＢＨＲ，构建重组质粒ｐｏＭＨＲ，经序列分析插入的ＨＲ基因序列和方向与设计相符。再将ｏＭＴＨＲ基因插入质粒ｐｍＭＴ中，最终构成ｐＭＨＲ表达载体，经酶切分析和聚合酶链式反应（ＰＣＲ）鉴定表为此表达载体中确含ｏＭＴ启动子和ＨＣＶ－Ｒｉｂｏｚｙｍｅ基因，可用于ＨＣＶ抗性的体内表达研究。

抗肿瘤多药抗性核酶转录质粒的构建及其体外切割研究

目的 探讨体外核酶对肿瘤多药抗性相关基因mdr1靶RNA分子的切割作用及其影响因素。方法 利用计算机软件设计能特异切割mdr1 mRNA的锤头状核酶（ribozyme），并构建抗mdr1-ribozyme和靶分子的体外转录质粒，进行体外切割实验。结果 发现抗mdr1-ribozyme能较好地切割mdr1 mRNA。 结论 抗肿瘤多药抗性核酶的体外研究为其体内应用提供了参考，ribozyme有可能成为逆转肿瘤多药抗性的新药物。

RNA研究进展之一:Ribozyme

Cech和Altman因发现酶活性RNA(Ribozyme)而获得1989年诺贝尔化学奖。本文重点介绍Ribozyme的应用研究。现在已知的Ribozyme绝大部分参与RNA的加工和成熟。它们可以分为四类。(1).

反义技术在感染性疾病防治研究中的应用

反义技术在感染性疾病防治研究中的应用张德礼，高步先，高显明，邓柏林，朱关福（北京军区后勤部卫生防疫队，北京军区兽医防治中心病毒基因工程研究室，北京１０００７１）Ａｂｓｔｒａｃｔ：Ｔｈｅｕｐｄａｔｅａｄｖａｎｃｅｓ，ｅｘｉｓｔｉｎｇｑｕｅｓｔｉｏｎｓ，...