BCR sequences essential for transformation by the BCR-ABL oncogene bind to the ABL SH2 regulatory domain in a non-phosphotyrosine-dependent manner.

BCR-ABL is a chimeric oncogene implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. BCR first exon sequences specifically activate the tyrosine kinase and transforming potential of BCR-ABL. We have tested the hypothesis that activation of BCR-ABL may involve direct interaction between BCR sequences and the tyrosine kinase regulatory domains of ABL. Full-length c-BCR as well as BCR sequences retained in BCR-ABL bind specifically to the SH2 domain of ABL. The binding domain has been localized within the first exon of BCR and consists of at least two SH2-binding sites. This domain is essential for BCR-ABL-mediated transformation. Phosphoserine/phosphothreonine but not phosphotyrosine residues on BCR are required for interaction with the ABL SH2 domain. These findings extend the range of potential SH2-protein interactions in growth control pathways and suggest a function for SH2 domains in the activation of the BCR-ABL oncogene as well as a role for BCR in cellular signaling pathways.     

Design and kinetic analysis of hammerhead ribozyme and DNAzyme that specifically cleave TEL-AML1 chimeric mRNA

In order to develop the oligonucleotides to abolish an expression of TEL-AML1 chimeric RNA, which is a genetic aberration that causes the acute lymphoblastic leukemia (ALL), hammerhead ribozymes and deoxyoligoribozymes that can specifically cleave TEL-AML1 fusion RNA were designed. Constructs of the deoxyribozyme with an asymmetric substrate binding arm (Dz26) and the hammerhead ribozyme with a 4 nt-bulged substrate binding arm in the stem III (buRz28) were able to cleave TEL-AML1 chimeric RNA specifically at sites close to the junction in vitro, without cleaving the normal TEL and AML1 RNA. Single-turnover kinetic analysis under enzyme-excess condition revealed that the buRz28 is superior to the Dz26 in terms of substrate binding and RNA-cleavage. In conjunction with current progress in a gene-delivery technology, the designed oligonucleotides that specifically cleave the TEL-AML1 chimeric mRNA are hoped to be applicable for the treatment of ALL in vivo.     

In vitro catalytic activities of DNA/RNA chimeric hammerhead ribozymes against AML1-MTG8 mRNA, a fused gene transcript in acute myeloid leukemia with t(8;21)

In order to design the best construct for therapeutic hammerhead ribozymes against AML1-MTG8, the t(8;21)-associated fusion mRNA of acute myeloid leukemia, we synthesized DNA/RNA chimeric ribozymes directed to the area adjacent to the fusion point between AML1 and MTG8. Catalytic efficiency and fusion gene specificity of ribozymes were examined by kinetic studies of the cleavage reactions of AML1-MTG8, AML1, and MTG8 RNAs transcribed in vitro. Ribozyme 2 (Rz2) specifically cleaved AML1-MTG8 RNA at three nucleotides downstream of the fusion junction with high efficiency. The highest cleavage efficiency was achieved by Rz4.3, which targeted non-contiguous sequences and cleaved at 19 nucleotides downstream of the fusion junction. Rz4.3 also cleaved MTG8 RNA but the cleavage efficiency was three orders of magnitude lower than that for AML1-MTG8 RNA. Therefore, Rz4.3 and Rz2 are the proper ribozymes for in vivo application to modulate gene expression of the AML1-MTG8.     

The effect of structure in a long target RNA on ribozyme cleavage efficiency.

Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif - vpr . The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping. The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme. Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. It is therefore unlikely that steric interference affected hammerhead ribozyme cleavage. Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs.     

Specificity of novel allosterically trans- and cis-activated connected maxizymes that are designed to suppress BCR-ABL expression

Chronic myelogenous leukemia (CML) is associated with the presence of the Philadelphia chromosome, which is generated by the reciprocal translocation of chromosomes 9 and 22. In the case of L6 (b2a2) mRNA, it is difficult to cleave the abnormal mRNA specifically because the mRNA includes no sequences that can be cleaved efficiently by conventional hammerhead ribozymes near the BCR-ABL junction. We recently succeeded in designing a novel maxizyme, which specifically cleaves BCR-ABL fusion mRNA, as a result of the formation of a dimeric structure. As an extension of our molecular engineering of maxizymes, as well as to improve their potential utility, we examined whether an analogous conformational change could be induced within a single molecule when two maxizymes were connected via a linker sequence. An active conformation was achieved by binding of the construct to the BCR-ABL junction in trans, with part of the linker sequence then acting as an antisense modulator in cis (within the complex) to adjust the overall structure. Results of studies in vitro in the presence of cetyltrimethylammonium bromide (CTAB) (but not in its absence) suggested that a certain kind of connected maxizyme (cMzB) might be able to undergo a desired conformational change and, indeed, studies in vivo confirmed this prediction. Therefore, we successfully created a fully functional, connected maxizyme and, moreover, we found that the activity and specificity of catalytic RNAs in vivo might be better estimated if their reactions are monitored in vitro in the presence of CTAB.     

Selective cleavage of bcr-abl chimeric RNAs by a ribozyme targeted to non-contiguous sequences.

Conventionally designed ribozymes may be unable to cleave RNA at sites which are inaccessible due to secondary structure. In addition, it may also be difficult to specifically target a conventionally designed ribozyme to some chimeric RNA molecules. Novel approaches for ribozyme targeting were developed by using the L6 bcr-abl fusion RNA as a model. Using one approach, we successfully directed ribozyme nucleation to a site on the bcr-abl RNA that is distant from the GUA cleavage site. These ribozymes bound to the L6 substrate RNA via an anchor sequence that was complementary to bcr sequences. The anchor was necessary for efficient cleavage as the anchor minus ribozyme, a conventionally designed ribozyme, was inefficient at catalyzing cleavage at this same site. The effect of anchor sequences on catalytic rates was determined for two of these ribozymes. Ribozymes generated by a second approach were designed to cleave at a CUU site in proximity to the bcr-abl junction. Both approaches have led to the development of a series of ribozymes specific for both the L6 and K28 bcr-abl chimeric RNAs, but not normal abl or bcr RNAs. The specificity of the ribozyme correlated in part with the ability of the ribozyme to bind substrate as demonstrated by gel shift analyses. Secondary structure predictions for the RNA substrate support the experimental results and may prove useful as a theoretical basis for the design of ribozymes.     

Suppression of USP7 induces BCR-ABL degradation and chronic myelogenous leukemia cell apoptosis

Chronic myelogenous leukemia (CML) is a clonal malignancy of hematopoietic stem cells featured with the fusion protein kinase BCR-ABL. To elicit the mechanism underlying BCR-ABL stability, we perform a screen against a panel of deubiquitinating enzymes (DUBs) and find that the ubiquitin-specific protease 7 (USP7) drastically stabilizes the BCR-ABL fusion protein. Further studies show that USP7 interacts with BCR-ABL and blocks its polyubiquitination and degradation. Moreover, USP7 knockdown triggers BCR-ABL degradation and suppresses its downstream signaling transduction. In line with this finding, genetic or chemical inhibition of USP7 leads to BCR-ABL protein degradation, suppresses BCR/ABL signaling, and induces CML cell apoptosis. Furthermore, we find the antimalarial artesunate (ART) significantly inhibits USP7/BCR-ABL interaction, thereby promoting BCR-ABL degradation and inducing CML cell death. This study thus identifies USP7 as a putative Dub of BCR-ABL and provides a rationale in targeting USP7/BCR-ABL for the treatment of CML.Subject terms: Deubiquitylating enzymes, Leukaemia     

Chemical and enzymatic probing of effector-mediated changes in the conformation of a maxizyme

The protein encoded by chimeric BCR-ABL mRNA causes chronic myelogenous leukemia (CML). We showed previously that a novel allosterically controllable ribozyme, of the type known as a maxizyme, can cleave this mRNA, with high specificity and high-level activity in vivo. We designed the maxizyme in such a way that it was able to form an active core with which to capture the catalytically indispensable Mg2+ ions only in the presence of the BCR-ABL mRNA junction. In order to probe the putative conformational changes, we used a weakly alkaline solution (pH 9.2) in the presence of 25 mM Mg2+ ions to hydrolyze differentially phosphodiester bonds that were located in different environments. Phosphodiester bonds in single-stranded regions were clearly more susceptible to attack by alkali than those within a double-stranded helix. As indicated by earlier data obtained in vivo, our results demonstrated that the active conformation was achieved only in the presence of the junction within the chimeric BCR-ABL mRNA. Moreover, we demonstrated that the use of mild alkaline solutions to probe RNA structures is very informative.     

Comparative study of DNA enzymes and ribozymes against the same full-length messenger RNA of the vanilloid receptor subtype I.

The efficiencies of 32 antisense oligodeoxynucleotides, 35 DNA enzymes and 6 ribozymes to bind and cleave the full-length messenger RNA of the vanilloid receptor subtype I were analyzed. Systematic screening of the mRNA revealed that good accessibility of a putative cleavage site for antisense oligodeoxynucleotides is a necessary but not a sufficient prerequisite for efficient DNA enzymes. Comparison of DNA enzymes and ribozymes against the same target sites revealed: 1) DNA enzymes were more active with longer recognition arms (9 nucleotides on either side), whereas ribozymes revealed higher activities with shorter recognition arms (7 nucleotides on either side). 2) It does not only depend on the target site but also on the enzyme sequence, whether a DNA enzyme or a ribozyme is more active. 3) The most efficient DNA enzyme found in this study had an approximately 15-fold higher reaction rate, k(react), and a 100-fold higher k(react)/K(m) under single turnover conditions compared with the fastest ribozyme. DNA enzymes as well as ribozymes showed significant activity under multiple turnover conditions, the DNA enzymes again being more active. We therefore conclude that DNA enzymes are an inexpensive, very stable and active alternative to ribozymes for the specific cleavage of long RNA molecules.     

Oligonucleotide facilitators enhance the catalytic activity of RNA-cleaving DNA enzymes.

From in vitro selection studies, DNA structures have been found that cleave target RNA sequence specifically and show a certain similarity to the well-investigated hammerhead ribozymes. Such DNA enzymes are more resistant to nuclease-mediated degradation than RNA enzymes. On the other hand, their cleavage activity is lower than the activity of hammerhead ribozymes. In the present study, we improved the activity of DNA enzymes by adding oligonucleotide facilitators complementary to the 5' and the 3' ends of the substrate to the cleavage reaction. DNA enzyme activity in vitro was monitored under multiple turnover conditions using short RNA model substrates. We have shown that oligonucleotide facilitators strongly enhance the multiple turnover activity of the DNA enzyme reaction. In one of our model systems with a suitable facilitator combination, we were able to observe a more than 200-fold enhancement of the k(cat)/Km value. The comparison of two DNA enzyme-substrate systems showed that the principal effects of the facilitators were independent of the substrate sequence. However, the degree of facilitator effect was noticeably dependent on the basic catalytic efficiency of DNA enzymes. Furthermore, the efficiency of the DNA enzyme reaction with facilitator was compared with the reaction of a DNA enzyme with a stem sequence extended by the sequence of the facilitator. The multiple turnover activity of such a "long DNA enzyme" is higher than the activity of the short DNA enzyme without facilitators. However, when compared with the multiple turnover reactions of the short DNA enzyme with facilitator, the reaction with the long DNA enzyme is considerably slower. The results obtained with our model systems demonstrate that oligonucleotide facilitators enable DNA enzymes to act as effective multiple turnover catalysts by cleavage of RNA substrates.     

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     

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).     

Probing of the secondary structure of maxizymes.

The protein encoded by chimeric BCR-ABL mRNA causes chronic myelogenous leukemia (CML). We showed previously that a novel allosterically controllable ribozyme, of the type known as a maxizyme, can cleave this mRNA, with high specificity and high-level activity in vivo. In order to probe the putative conformational changes, we used a weakly alkaline solution to hydrolyze differentially phosphodiester bonds that were located in different environments. As indicated by earlier data obtained in vivo, our results demonstrated that the active conformation was achieved only in the presence of the junction within the chimeric BCR-ABL mRNA.     

Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.     

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.     

Activity identification of chimeric anti-caspase-3 mRNA hammerhead ribozyme in vitro and in vivo

To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettes in vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ribozyme embedded into the human snRNA U6, and detected the activity of the ribozyme in vitro and in vivo. Meanwhile we compared it with the self-cleaving hammerhead ribozymes that we previously studied, and with the general ribozyme, cloned into RNA polymerase II expression systems. The results showed that the three ribozymes, p1.5RZ107, pRZ107 and pU6RZ107 had the correct structure, and that they could cleave cas-pase-3 mRNA exactly to produce two fragments: 143nt/553nt. p1.5RZ107 has the highest cleavage efficiency in vitro, almost 80%. However, the U6 chimeric ribozyme, pU6RZ107, has the highest cleavage activity in vivo, almost to 65%, though it has lower cleavage activity in vitro. The cleavage results demonstrated that the pU6RZ107, the U6 chimeric ribozyme, could more efficiently expre