Inhibition of fibroblast growth factors

Summary The potential roles of members of the fibroblast growth factor family in tumor angiogenesis and metastasis and their mechanisms of release from cells are discussed. Furthermore, we review methods of therapeutic targeting of these polypeptides. In particular, we focus on the possibility to inhibit fibroblast growth factors with drugs that mimic heparin-like cellular binding sites and thus can interfere with growth factor receptor recognition. In addition, we discuss antibodies, antisense oligodeoxynucleotides, and ribozymes as approaches to inhibit production and activity of these growth factors.List of abbreviations aFGF acidic fibroblast growth factor (=FGF-1) - bFGF basic FGF (=FGF-2) - HBGF heparin-binding growth factor - HGF hepatocyte growth factor - HSPG heparansulfate proteoglycan - PTN pleiotrophin - TGF transforming growth factor - VEGF vascular endothelial cell growth factor Presented at the symposium "New Approaches in the Therapy of Breast Cancer", Georgetown University Medical Center, Washington DC, October 1994, generously supported by an education grant from Bristol-Myers Squibb.     

Use of RNA in drug design

This is a review of RNA as a target for small molecules (ribosomes, riboswitches, regulatory RNAs) and RNA-derived oligonucleotides as tools (antisense/small interfering RNA, ribozymes, aptamers/decoy RNA and microRNA). This review highlights the present state of research using RNA as a drug target or as a potential drug candidate and explains at which stage and to what extent rational design could eventually be involved. Special attention has been paid to the recent potential clinical applications of RNA either as drugs or drug targets. The review deals mainly with mechanistic approaches rather than with physicochemical or computational aspects of RNA-based drug design.     

Protein-free small nuclear RNAs catalyze a two-step splicing reaction

Pre-mRNA splicing is a crucial step in eukaryotic gene expression and is carried out by a highly complex ribonucleoprotein assembly, the spliceosome. Many fundamental aspects of spliceosomal function, including the identity of catalytic domains, remain unknown. We show that a base-paired complex of U6 and U2 small nuclear RNAs, in the absence of the ≈200 other spliceosomal components, performs a two-step reaction with two short RNA oligonucleotides as substrates that results in the formation of a linear RNA product containing portions of both oligonucleotides. This reaction, which is chemically identical to splicing, is dependent on and occurs in proximity of sequences known to be critical for splicing in vivo. These results prove that the complex formed by U6 and U2 RNAs is a ribozyme and can potentially carry out RNA-based catalysis in the spliceosome.     

“Hairpin” and “hammerhead” ribozymes directed towards the Mumps virus nucleocapsid RNA: Specific cleavage of a small synthetic RNA substrate and full-length mRNA

In an attempt to develop efficient antiviral agents against Mumps virus, we designed ribozymes targeting the nucleocapsid (NP) mRNA. Transacting catalytic RNAs of the hammerhead and hairpin types were synthesized; they contained specific motifs, shared similar flanking regions and were directed against a 5GUC3 target immediately downstream to the initiation codon of NP mRNA. Both ribozymes were first assayed on a synthetic 16 bases target RNA and found to catalytically and efficiently cleave the substrate in a sequence specific way. No cleavage, however, occurred when mutated forms of the ribozymes were used. In addition, both ribozyme types, when tested on the full length NP mRNA, were also able to cleave the substrate although turnover could not be demonstrated. As a rule, the hammerhead ribozyme proved more efficient than its hairpin counterpart, as well on the synthetic RNA substrate as on the full length NP mRNA target.     

不同长度丁型肝炎病毒核酶的体外自剪切活性及其意义

目的分析不同长度丁型肝炎病毒核酶的体外自剪切活性.方法用Xba Ⅰ,EcoRⅠ或BamHI将含有HDV.Rz序列的pRz277正向质粒(pRz277A)和pRz277反向质粒(pRz277B)线性化,以α-32p-UTP标志和T7噬菌体RNA聚合酶转录出不同长度的基因组核酶(g.Rz)和抗基因组(即复制中间体)核酶(ag.Rz),在一定条件下进行自剪切反应,然后作聚丙烯酰胺凝胶电泳(PAGE)和放射自显影.结果g.Rz24/100(自剪切位点5和3端分别含24nt和100nt,下同)、ag.Rz38/119在适当条件下绝大部分发生自剪切.g.Rz24/253和ag.Rz38/239也具有一定自剪切活性,但显著低于前两者,即使温度升至55℃或加入去离子甲酰胺也是如此.结论适宜长度的HDV.Rz在体外具有较高的自剪切活性.这些发现有助于我们下一步研究HDV.Rz的反式剪切作用.     

抗丙型肝炎病毒锤头结构核酶的计算机设计

目的设计可位点特异切割丙型肝炎病毒（ＨＣＶ）５’非编码区（５’ＮＣＲ）和Ｃ区的多位点核酶（ＲＺ）．方法根据“锤头结构”ＲＺ的设计原理，以ＨＣＶＨ（１ａ）株５’ＮＣＲ和Ｃ区ＲＮＡ为靶序列，计算机预测其二级结构，运用能量最低化程序，选择理论上理想的ＲＺ切割位点，并比较５株ＨＣＶ序列ＲＺ切点两翼的同性．结果在ＨＣＶ５’ＮＣＲ和Ｃ区１２４个自然切割位点（ＣＵＸ和ＧＵＸ）中选出２１３（ＣＵＣ），２６０（ＧＵＡ），４０７（ＧＵＣ）和４９８（ＣＵＵ）４个切割位点，不同株之间切点两翼ＲＺ结合序列同源性为１００％．结论计算机可作为抗病毒ＲＺ设计的辅助工具；ＨＣＶ上述４个位点可能是最理想的切割位点．     

Nonenzymatic assembly of active chimeric ribozymes from aminoacylated RNA oligonucleotides

Aminoacylated transfer RNAs, which harbor a covalent linkage between amino acids and RNA, are a universally conserved feature of life. Because they are essential substrates for ribosomal translation, aminoacylated oligonucleotides must have been present in the RNA world prior to the evolution of the ribosome. One possibility we are exploring is that the aminoacyl ester linkage served another function before being recruited for ribosomal protein synthesis. The nonenzymatic assembly of ribozymes from short RNA oligomers under realistic conditions remains a key challenge in demonstrating a plausible pathway from prebiotic chemistry to the RNA world. Here, we show that aminoacylated RNAs can undergo template-directed assembly into chimeric amino acid–RNA polymers that are active ribozymes. We demonstrate that such chimeric polymers can retain the enzymatic function of their all-RNA counterparts by generating chimeric hammerhead, RNA ligase, and aminoacyl transferase ribozymes. Amino acids with diverse side chains form linkages that are well tolerated within the RNA backbone and, in the case of an aminoacyl transferase, even in its catalytic center, potentially bringing novel functionalities to ribozyme catalysis. Our work suggests that aminoacylation chemistry may have played a role in primordial ribozyme assembly. Increasing the efficiency of this process provides an evolutionary rationale for the emergence of sequence and amino acid–specific aminoacyl-RNA synthetase ribozymes, which could then have generated the substrates for ribosomal protein synthesis.

The evolution of ribosomal protein synthesis would have required the presence of 2′,3′-aminoacylated RNAs, which are the universal substrates for protein synthesis. Without specialized aminoacyl-RNA synthetase enzymes, the initial aminoacylation of the 2′,3′-diol of RNA must have been mediated by spontaneous chemical processes. However, current chemical pathways leading to RNA aminoacylation are inefficient or rely on unstable, activated amino acids. For example, nonenzymatic aminoacylation of the 2′,3′-diol of RNA oligonucleotides with imidazole-activated amino acids affords low levels of aminoacylation (1, 2), primarily due to the pronounced hydrolytic instability of protonated aminoacyl esters (half-life of ca. 50 to 180 min at 22 °C, pH 8, depending on the amino acid residue) (3–5). Alternatively, interstrand transfer of an amino acid from a 5′-phosphate mixed anhydride to the 2′,3′-diol generates aminoacylated oligonucleotides but depends on the efficient synthesis of phospho-carboxy anhydrides, which are prone to hydrolysis and CO₂-mediated degradation (6, 7). Given these limitations, spontaneous synthesis alone may not have generated sufficient aminoacylated RNA substrates to facilitate the emergence of a primitive ribosome. Furthermore, spontaneous synthesis of aminoacylated RNAs is unlikely to have been highly sequence and amino acid specific; this lack of specificity would have made the evolution of coded peptide synthesis problematic. Here, we consider a scenario in which the initial inefficient and minimally specific aminoacylation of RNA played an alternative role that was nonetheless advantageous to primordial protocells. Such a role might have provided a selective pressure favoring the evolution of ribozymes that generated and maintained high levels of RNA aminoacylation. Accordingly, we have sought to identify a role for aminoacylated RNAs that could have been beneficial to primitive protocells, independent of ribosomal protein synthesis.Early life is thought to have used ribozymes to catalyze primordial metabolic reactions as well as RNA replication prior to the evolution of protein enzymes (i.e., the RNA world) (8, 9). In this scenario, the first ribozymes were generated through the nonenzymatic ligation of short RNA oligonucleotides and/or the template-directed polymerization of activated nucleotide monomers. Imidazole and its derivatives have traditionally been used to activate RNA building blocks (10, 11), and recent work has expanded on the prebiotic plausibility of imidazole-activated RNAs (12–15). While the activation of ribonucleotides with imidazoles has facilitated the assembly and templated copying of short RNAs (10, 16–19), oligomers long enough to exhibit enzymatic properties have remained out of reach. A major obstacle to efficient ribozyme assembly is the relatively poor nucleophilicity of the terminal 2′,3′-diol of RNA, mandating high Mg²⁺ concentrations that are not compatible with fatty acid-based vesicle models of protocells (20). Carbodiimide activation of phosphate (21, 22) or replacement of the 3′-hydroxyl with an amino group allow for efficient ligation of RNA oligonucleotides (23–28), but the prebiotic relevance of these approaches remains unclear.In the 1970s, Shim and Orgel demonstrated that glycylated nucleotides can take part in template-directed polymerization with imidazole-activated nucleotides, forming phosphoramidate linkages between the Gly amine and the 5′-phosphate of an adjacent nucleotide (29). Building on this discovery, recent investigations have focused on the possibility of an RNA–amino acid copolymer world (30) and the conditions that permit conjugation of peptido RNAs (31). The Richert group has recently expanded our understanding of peptidoyl (peptide-bridged) RNAs and how they may have played a role in template-directed peptide condensation (31, 32). However, to our knowledge, chimeric, amino acid–bridged ribozymes created with the building blocks of both proteins and RNA have not yet been reported. In our previous work, short aminoacylated RNAs were shown to form amino acid bridges in nonenzymatic ligation reactions with imidazole-activated oligonucleotides at rates much higher than observed with unmodified RNA (5). Here, we extend that approach to show that multiple ligation reactions can generate chimeric amino acid–bridged RNAs long enough to constitute catalytic RNAs. Importantly, we show that these amino acid–bridged RNAs function as catalytically proficient ribozymes. We demonstrate the assembly of three chimeric ribozymes that perform RNA cleavage, RNA ligation, and RNA aminoacylation, all thought to have been important enzymatic functions in the RNA world. We generate the chimeric ribozymes at 2.5 mM Mg²⁺, conditions that are compatible with fatty acid–based vesicles (20) and limit RNA and activated RNA degradation (33) but generally yield no RNA ligation. We further show that aminoacylated RNA assembly on splint templates can generate a chimeric ribozyme that functions in the same pot without purification, thereby highlighting the potential prebiotic plausibility of our chimeric ribozyme assembly method. Our work reveals a link between aminoacylation chemistry and the nonenzymatic assembly of RNA oligonucleotides into functional ribozymes. This functional link provides a potential rationale for the evolution of ribozyme-catalyzed RNA aminoacylation prior to the evolution of ribosomal protein synthesis.     

抗慢性粒细胞性白血病bcr/abl mRNA真核表达载体的构建

目的：构建一个含M1RNA、具有特异性抗慢性粒细胞性白血病（CML）细胞融合基因bcr/ablmRNA的真核表达载体，以用于CML的分子靶向治疗。方法：以pTK117为模板，通过PCR方法合成一个带有导引序列（GS）的M1RNA，再将PCR产物克隆到真核表达载体pNAV-1上，得到重组质粒pAVGS4，转化大肠埃希菌JM109，以碱裂解法小量抽提，酶切电泳鉴定，并测序鉴定。结果：以EcoRI和SalI酶切、1％的琼脂糖凝胶电泳显示在500和6500bp附近各有一条明亮的条带。应用PCR方法测序的结果与模板序列一致。结论：构建的pNAV-1经酶切和测序鉴定，与目标序列一致，含有核酶、具有抗bcr/abl mRNA的真核表达载体构建成功，预期可用于CML细胞株和原代细胞的实验研究。     

Trans-cleaving activity of hepatitis delta virus genomic ribozymes

It is ~rted lhat hepatitis delta virus (HDV)genondc HNA can self-cleave intramolecularly in the sitebetween Ugh, and G@, ~rted by Makino et .ill]. ThisPIDCess is catalyZed by HDV genondc RNA itself whichbelongs to the fthely of catalytic RNAs named riboZymes.The intramolecular self-cleaVage is also called ets-cleavage, and the self-cleaving HDV genondc riboZyme(g. Rz)is defined as ets-g. Rz. SOme reseaxches have shown lhatfoe genondc riboZyme which possesses the shortest natoalseq…