The DNAzymes Rs6, Dz13, and DzF have potent biologic effects independent of catalytic activity

DNAzymes are catalytic DNA molecules capable of cleaving RNA substrates and therefore constitute a possible gene-suppression technology. We examined whether the previously reported potency of a DNAzyme targeting c-jun (Dz13) could be improved with judicious use of sequence and chemical modifications. Catalytic activity was measured to establish correlations between catalytic activity and biological potency. Surprisingly, Dz13 had significant cytotoxic activity against cells of rodent origin (IC(50) = 20-50 nM) despite having greatly reduced catalytic activity against a rodent target substrate (<25%), the latter being the result of a mismatch to the rodent c-jun sequence. In contrast, a modified Dz13 matching the rodent c-jun sequence (DT1501b) had no activity at similar concentrations against human or rodent cells despite being able to efficiently cleave the rodent c-jun sequence. Overall, catalytic activity against synthetic substrates did not correlate with cytotoxic activity and catalytically inactive mutants had in some cases equal or superior potency in cell cytotoxicity assays. Further examination of other previously published DNAzymes (Rs6 and DzF) revealed other occurrences of this anomalous behaviour. The active sequences all have G-rich 5 termini, suggesting that G-quadruplex formation might be involved. Consistent with this, deaza-guanosine substitutions abrogated cytotoxicity of Dz13. However, Dz13 did not show evidence of quadruplex formation as determined by circular dichroism studies and native electrophoresis. These data reveal that the biologic activity of several published DNAzymes is not mediated through the catalytic degradation of target mRNA.     

Introduction of guanidinium-modified deoxyuridine into the substrate binding regions of DNAzyme 10–23 to enhance target affinity: Implications for DNAzyme design

Deoxyribozymes (DNAzymes) are important catalysts for potential therapeutic RNA destruction and no DNAzyme has received as much notoriety in terms of therapeutic use as the Mg²⁺-dependent RNA-cleaving DNAzyme 10–23 (Dz10–23). As such, we have investigated the synthetic modification of Dz10–23 with a guanidinium group, a functionality that reduces the anionic nature and can potentially enhance the membrane permeability of oligonucleotides. To accomplish this, we synthesized a heretofore unknown phosphoramidite, 5-(N,N′-biscyanoethoxycarbonyl)-guanidinoallyl-2′-deoxyuridine and then incorporated it into oligonucleotides via solid phase synthesis to study duplex stability and its effect on Dz10–23. This particular modification was chosen as it had been used in the selection of Mg²⁺-free self-cleaving DNAzymes; as such this will enable the eventual comparison of modified DNAzymes that do or do not depend on Mg²⁺ for catalysis. Consistent with antecedent studies that have incorporated guanidinium groups into DNA oligonucleotides, this guanidinium-modified deoxyuridine enhanced the thermal stability of resulting duplexes. Surprisingly however, Dz10–23, when synthesized with modified residues in the substrate binding regions, was found to be somewhat less active than its non-modified counterpart. This work suggests that this particular system exhibits uniform binding with respect to ground state and transition state and provides insight into the challenge of re-engineering a Mg²⁺-dependent DNAzyme with enhanced catalytic activity.     

DNAzymes to beta 1 and beta 3 mRNA down-regulate expression of the targeted integrins and inhibit endothelial cell capillary tube formation in fibrin and matrigel.

A novel approach based on DNA-cleaving deoxyribozymes (DNAzymes) was developed to control expression of beta(1) and beta(3) integrins in endothelial cells. To engineer a specific cleavage site in mRNA, the flanking domains of DNAzymes were derived from oligodeoxynucleotides complementary to sequences corresponding to 1053-1070 and 1243-1267 in beta(1) and beta(3) mRNA, respectively. Phosphorothioate analogues of these antisense oligodeoxynucleotides, designated beta1-1053 and beta3-1243, significantly inhibited expression of beta(1) and beta(3) integrin subunits in endothelial and K562 cells at the level of mRNA and protein synthesis. They also specifically decreased the cell surface expression of corresponding subunits in endothelial cells and K562 cells, as measured by flow cytometry. In functional tests, beta1-1053 and beta3-1243 markedly reduced adhesion of cells to fibronectin and vitronectin, respectively. We designed DNAzymes to beta(1) and beta(3) mRNAs containing a 15-deoxynucleotide catalytic domain that was flanked by two substrate recognition segments of 8 and 10 deoxynucleotides for beta(1) and beta(3) DNAzymes, respectively. Both DNAzymes in the presence of Mg(2+) specifically cleaved their substrates, synthetic beta(1) and beta(3) mRNA fragments. Although DNAzymes were partially modified with phosphorothioate and with 2'-O-methyl groups at both the 5' and 3' ends indicated similar kinetic parameters, they were significantly more potent than the unmodified DNAzymes because of their much higher resistance to nuclease degradation. Similar to the antisense oligonucleotides, DNAzymes abolished microvascular endothelial cell capillary tube formation in fibrin and Matrigel. In conclusion, DNAzymes to beta(1) and beta(3) mRNAs with 2'-O-methyl modifications are potentially useful as gene-inactivating agents and may ultimately provide a therapeutic means to inhibit angiogenesis in vivo.     

bla_(TEM-116)型超广谱β-内酰胺酶的表达、纯化及其应用

为大量获取低成本的TEM-116超广谱β-内酰胺酶,并分析其降解环境中β-内酰胺类抗生素残留物的可行性,本研究在Escherichia coli BL21(DE3)菌株中表达了重组TEM-116超广谱β-内酰胺酶,经亲和层析纯化、柱复性与分子筛层析纯化,得到了高纯度的目的蛋白,对其理化性质进行了分析。结果表明,重组TEM-116超广谱β-内酰胺酶的分子量、比活性分别为30kDa和476IU/mg,与天然酶性质相近。重组酶在体内外对多种青霉素、头孢菌素类药物均具有较高降解效率:10IU酶可清除1L发酵液中7000mg的青霉素G;320IU酶可清除1L尿液中各200mg的青霉素G、氨苄青霉素和头孢唑林混合抗生素;1.0～2.5IU的酶可在4℃～37℃温度范围内清除1L牛奶中80U的青霉素G;2.0×104～2.3×104IU/(kg·bw)的酶能够清除小鼠体内8.0×104～9.1×104μg/(kg·bw)的青霉素G。     

Substitution of serine for arginine in position 162 of TEM-type beta-lactamases extends the substrate profile of mutant enzymes, TEM-7 and TEM-101, to ceftazidime and aztreonam

TEM-7 is a novel broad-spectrum beta-lactamase (Bla), selected in vivo, with a resistance profile similar to that of TEM-1 and TEM-2, but extended to ceftazidime (Caz) and aztreonam. Nucleotide sequencing revealed that the TEM-7 gene is almost identical with that of TEM-2. There was 1 bp change which would result in the substitution of Ser (TEM-7) for Arg (TEM-2) in amino acid (aa) position 162 (i.e., aa position 139 of the mature enzyme). This substitution, also found in TEM-101, a spontaneous in vitro derivative of TEM-1 selected on Caz, was assumed to be responsible for the extension of the substrate profile. The assumption was verified by exchange of a DNA fragment, carrying the mutation of the TEM-7-coding gene, with the homologous fragment of the TEM-1-coding gene in pBR322. In the three-dimensional model of class-A Bla [Joris et al., Biochem. J. 250 (1988) 313-324], aa 139 is located at the rim of the groove which contains the active center and adjacent to the evolutionarily conserved BoxV. It is speculated that extra free hydroxyl groups in this area may participate in the stabilization of otherwise non-substrate compounds.     

Brief communication: stability and catalytic activity of novel circular DNAzymes

DNAzymes represent a new generation of catalytic nucleic acids for specific RNA targeting in order to inhibit protein translation from the specifically cleaved mRNA. The 10-23 DNAzyme was found to hydrolyze RNA in a sequence-specific manner both in vitro and in vivo. Although single-stranded DNAzymes may represent the most effective nucleic acid drug to date, they are nevertheless sensitive to nuclease degradation and require modifications for in vivo application. However, previously used stabilization of DNAzymes by site-specific phosphorothioate (PT) modifications reduces the catalytic activity, and the PTO displays toxic side effects when applied in vivo. Thus, improving the stability of DNAzymes without reducing their catalytic activity is essential if the potential of these compounds should be realized in vivo. RESULTS: The Circozyme was tested targeting the mRNA of the most common genetic rearrangement in pediatric acute lymphoblastic leukemia TEL/AML1 (ETV6/RUNX1). The Circozyme exhibits a stability comparable to PTO-modified DNAzymes without reduction of catalytic activity and specificity and may represent a promising tool for DNAzyme in vivo applications. CONCLUSION: The inclusion of the catalytic site and the specific mRNA binding sequence of the DNAzyme into a circular loop-stem-loop structure (Circozyme) of approximately 70 bases presented here represents a new effective possibility of DNAzyme stabilization.     

Nucleic Acid-Mediated Cleavage of M1 Gene of Influenza A Virus Is Significantly Augmented by Antisense Molecules Targeted to Hybridize Close to the Cleavage Site

Influenza A virus genome segment 7 encodes protein M1, which is the matrix protein playing crucial role in the virus life cycle. Any antiviral strategy that aims at reducing, in particular, the expression of this genome segment should, in principle, reduce the infectivity of the virus. We developed a specific antiviral approach at the molecular level and designed several novel 10–23 DNAzymes (Dz) and hammerhead ribozymes (Rz), specifically targeted to cleave at the conserved domains of the influenza virus M1 RNA. We sought to use antisense molecules with the hope that it will facilitate the ribozyme-mediated cleavage. We observed that the Mg²⁺-dependent sequence-specific cleavage of M1 RNA was achieved by both the Dz and Rz in a dose-dependent manner. This combination of catalytic Dz and Rz with antisense molecules, in principle, resulted in more effective gene suppression, inhibited the whole virus replication in host cell, and thus could be exploited for therapeutic purposes.     

Targeting insulin-like growth factor I with 10-23 DNAzymes: 2'-O-methyl modifications in the catalytic core enhance mRNA cleavage

Insulin-like growth factor I (IGF-I) and its cognate receptor (IGF-1R) contribute to normal cell function and to tumorigenesis. The role of IGF-I signaling in tumor growth has been demonstrated in vivo using nucleic acid-based strategies. Here, we designed the first 10-23 DNAzymes directed against IGF-I mRNA. Unlike antisense approaches and RNA interference that require protein catalysis, DNAzymes catalyze protein-free RNA cleavage. We identified target sequences and measured catalytic properties of differently designed DNAzymes on short synthetic RNA targets and on in vitro transcribed IGF-I mRNA. The most efficient cleavers were then transfected into cells, and their inhibitory effect was analyzed using reporter gene assays. We found that increasing the size of DNAzyme flanking sequences and modifications of the termini with 2'-O-methyl residues improved cleavage rates of target RNAs. Modification of the catalytic loop with six 2'-O-methyl ribonucleotides at nonessential positions increased or decreased catalytic efficiency depending on the mRNA target site. In cells, DNAzymes with 2'-O-methyl-modified catalytic cores and flanking sequences were able to inhibit reporter gene activity because of specific recognition and cleavage of IGF-I mRNA sequences. Mutant DNAzymes with inactive catalytic cores were unable to block reporter gene expression, demonstrating that the RNA cleaving ability of 10-23 DNAzymes contributed to inhibitory mechanisms. Our results show that nuclease-resistant 2'-O-methyl-modified DNAzymes with high catalytic efficiencies are useful for inhibiting IGF-I gene function in cells.     

Characterization of non-8-17 sequences uncovers structurally diverse RNA-cleaving deoxyribozymes

RNA-cleaving deoxyribozymes (DNAzymes) can be isolated from random-sequence DNA pools via the process of in vitro selection. However, small and simple catalytic motifs, such as the 8-17 DNAzyme, are commonly observed in sequence space, presenting a challenge in discovering large and complex DNAzymes. In an effort to investigate underrepresented molecular species derived from in vitro selection, in this study we sought to characterize non-8-17 sequences obtained from a previous in vitro selection experiment wherein the 8-17 deoxyribozyme was the dominant motif. We examined 9 sequence families from 21 motifs by characterizing their structural and functional features. We discovered 9 novel deoxyribozyme classes with large catalytic domains (>40 nucleotides) utilizing three-way or four-way junction structural frameworks. Kinetic studies revealed that these deoxyribozymes exhibit moderate to excellent catalytic rates (k(obs) from 0.003 to 1 min(-1)), compared to other known RNA-cleaving DNAzymes. Although chemical probing experiments, site-directed mutational analyses, and metal cofactor dependency tests suggest unique catalytic cores for each deoxyribozyme, common dinucleotide junction selectivity was observed between DNAzymes with similar secondary structural features. Together, our findings indicate that larger, structurally more complex, and diverse catalytic motifs are able to survive the process of in vitro selection despite a sequence space dominated by smaller and structurally simpler catalysts.     

10-23型DNA酶作为鉴定mRNA靶点有效性的新工具

10-23DNA酶是能主动切割mRNA的一类反义寡核苷酸.利用10-23DNA酶的直接切割作用验证mRNA结构靶点的有效性.对筛选的绿色荧光蛋白(GFP)基因mRNA的4个靶点平行设计了4条反义寡核苷酸和4条10-23DNA酶,对照组反义寡核苷酸将最佳靶点——靶点2的反义寡核苷酸突变2个碱基,对照组10-23DNA酶将靶点2的10-23DNA酶结合臂中央突变2个碱基.体外4条10-23DNA酶切割mRNA的结果和相应的4条反义寡核苷酸依赖的RNaseH降解结果完全相似,细胞内4条10-23DNA酶对绿色荧光蛋白的表达抑制作用与相应的4条反义寡核苷酸相似,表明10-23DNA酶显示的最佳作用靶点同样是最佳作用效果的反义寡核苷酸结合靶.10-23DNA酶可以作为评价mRNA结构靶点有效性的新工具.     

Hairpin DNAzymes: a new tool for efficient cellular gene silencing

BACKGROUND: RNA-based gene silencing is potentially a powerful therapeutic strategy. Catalytic 10-23 DNAzymes bind to target RNA by complimentary sequence arms on a Watson-Crick basis and thus can be targeted to effectively cleave specific mRNA species. However, for in vivo applications it is necessary to stabilise DNAzymes against nucleolytic attack. Chemical modifications can be introduced into the binding arms to increase stability but these may alter catalytic activity and in some cases increase cell toxicity. METHODS: We designed novel 10-23 DNAzyme structures that incorporate stem-loop hairpins at either end on the DNAzyme binding arms. The catalytic activity of hairpin DNAzymes (hpDNAzyme) were tested in vitro against 32P-labelled cRNA encoding the muscle acetylcholine receptor (AChR) alpha-subunit. Resistance of hpDNAzymes to nucleolytic degradation was tested by incubation of the hpDNAzymes with Bal-31, DNase1 or HeLa cell extract. Gene silencing by hpDNAzymes was assessed by measuring reduced fluorescence from DsRed2 and EGFP reporters in cell culture systems, and reduced 125I-alpha-bungarotoxin binding in cells transfected with cDNA encoding the AChR. RESULTS: We show that hpDNAzymes show remarkable resistance to nucleolytic degradation, and demonstrate that in cell culture systems the hpDNAzymes are far more effective than standard 10-23 DNAzymes in down-regulating protein expression from target mRNA species. CONCLUSION: hpDNAzymes provide new molecular tools that, without chemical modification, give highly efficient gene silencing in cells, and may have potential therapeutic applications.     

Suppression of platelet-type 12-lipoxygenase activity in human erythroleukemia cells by an RNA-cleaving DNAzyme.

Human platelet-type 12-lipoxygenase (12-LOX) and its metabolites play a crucial role in tumor angiogenesis. A "10-23" deoxyribozyme (DNAzyme) and its phosphorothioate-modified version were designed and synthesized against the 12-LOX mRNA. Both DNAzymes were able to cleave their substrate efficiently in a time- and concentration-dependent manner in vitro. Under a multiple turnover condition, both performed well at 37 degrees C, showing the k(cat) of 1 and 0.26 min(-1), respectively. The phosphorothioate modification of the DNAzyme significantly increased its stability in cells without a substantial loss of kinetic efficiency in vitro. In a cell culture system, transfection of the DNAzymes into HEL cells resulted in a significant down-regulation of the 12-LOX mRNA. Furthermore, the cell extracts from the DNAzyme-transfected cells exhibited a marked reduction in the 12-LOX enzyme activity. The present results indicated the potential use of DNAzyme technology for gene function study and cancer therapy.     

Immunomodulating effects of tofizopam (Grandaxin) and diazepam in vitro

Benzodiazepines (BDZs) are known to act not only in the central nervous system, but on peripheral cells and tissues binding to the peripheral-type benzodiazepine receptors. In the present study, the influence of two different BDZs (diazepam (Dz) and tofizopam (Tof) on several immune functions has been examined in vitro. Some differences between Dz and Tof in their effects on human lymphocyte proliferative response, changes in glucocorticoid-induced suppression of cell proliferation and influence on cytokine production (tumor necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2)) have been determined. Dz suppressed mitogen-induced peripheral blood mononuclear cell (PBMC) proliferation, enhanced dexamethasone-induced inhibition of PBMC proliferative response, and suppressed lymphocyte production of TNF-alpha and IL-2. Tof usually enhanced PBMC proliferation and IL-2 production in low and moderate doses, but in high doses it suppressed both. Tof in all investigated doses enhanced dexamethasone-induced suppression of lymphocyte proliferation and depressed TNF-alpha production. Thus, both Dz and Tof are shown to have immunomodulating effects in vitro. Tof, opposite to Dz even in the therapeutic doses, is able to enhance in vitro mitogen-induced lymphocyte proliferation and IL-2 production.     

Fas基因mRNA反义靶点筛选和体外验证

应用PARASS(poly-A anchored RNA accessible sites screening) 技术筛选Fas基因mRNA 获得3个潜在反义作用靶点,靶点1、2、3分别位于Fas基因297nt-317nt、619nt-639nt和662nt-682nt。设计了对应靶点的反义寡核苷酸A1、A2、A3,和10-23型DNAzyme D1、D2和D3。将反义寡核苷酸和Fas基因RNA结合再加入RNase H进行反应,10-23型DNAzyme则直接与Fas基因RNA作用,结果表明：3个靶点的反义寡核苷酸组及DNAzyme均能降解Fas基因RNA,为有效靶点,其靶点反应优势次序为靶点3>靶点1>靶点2;而非靶点对照组和有效靶点突变了2个碱基的对照组均没有反应。靶点2和靶点3与ISIS公司经过多次实验筛选到的Fas反义作用靶点位置基本相同,表明PARASS技术的有效性和可靠性。获得的有效反义寡核苷酸和DNAzyme为后续研究打下基础。     

6-(1-Hydroxyalkyl))penam sulfone derivatives as inhibitors of class A and class C beta-lactamases II

Two stereoselective processes for the synthesis of novel 3,6-disubstituted penam sulfone derivatives were developed. One 6beta-(1-hydroxyethyl) and four 6beta-hydroxymethyl penam sulfone derivatives were synthesized. All four 6beta-(hydroxymethyl)penam sulfone derivatives demonstrated good IC50 against both TEM-1 and AmpC beta-lactamases. Of these, 6beta-hydroxymethyl penam sulfone derivative 25 was the most active inhibitor which was able to restore the activity of piperacillin in vitro and in vivo against both TEM-1 and AmpC beta-lactamases producing organisms.     

Backbone dynamics of TEM-1 determined by NMR: evidence for a highly ordered protein

Backbone dynamics of TEM-1 beta-lactamase (263 amino acids, 28.9 kDa) were studied by 15N nuclear magnetic resonance relaxation at 11.7, 14.1, and 18.8 T. The high quality of the spectra allowed us to measure the longitudinal relaxation rate (R1), the transverse relaxation rate (R2), and the {1H}-15N NOE for up to 227 of the 250 potentially observable backbone amide groups. The model-free formalism was used to determine internal motional parameters using an axially anisotropic model. TEM-1 exhibits a small prolate axial anisotropy (D(parallel)/D(perpendicular) = 1.23 +/- 0.01) and a global correlation time (tau(m)) of 12.41 +/- 0.01 ns. The unusually high average generalized order parameter (S2) of 0.90 +/- 0.02 indicates that TEM-1 is one of the most ordered proteins studied by liquid-state NMR to date. Although the omega-loop has a high degree of order in the picosecond-to-nanosecond time scale (mean S2 value of 0.90 +/- 0.02), we observed the presence of microsecond-to-millisecond time scale motions for this loop, as for the vicinity of the active site. These motions could be relevant for the catalytic function of TEM-1. Amide exchange experiments were also performed, and several amide groups were not exchanged after 12 days, an indication that global motions in TEM-1 are also very limited. Although detailed dynamics characterization by NMR cannot be readily applied to TEM-1 in the presence of relevant substrates, the unusual picosecond-to-nanosecond dynamics behavior of TEM-1 presented here will be essential to the validation and improvement of future molecular dynamics simulations of TEM-1 in the presence of functionally relevant substrates.