FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2014496171> ?p ?o ?g. }

• W2014496171 endingPage "47996" @default.
• W2014496171 startingPage "47987" @default.
• W2014496171 abstract "The intracellular ability of the “10–23” DNAzyme to efficiently inhibit expression of targeted proteins has been evidenced by in vitro and in vivo studies. However, standard conditions for kinetic measurements of the DNAzyme catalytic activity in vitro include 25 mm Mg2+, a concentration that is very unlikely to be achieved intracellularly. To study this discrepancy, we analyzed the folding transitions of the 10–23 DNAzyme induced by Mg2+. For this purpose, spectroscopic analyzes such as fluorescence resonance energy transfer, fluorescence anisotropy, circular dichroism, and surface plasmon resonance measurements were performed. The global geometry of the DNAzyme in the absence of added Mg2+ seems to be essentially extended, has no catalytic activity, and shows a very low binding affinity to its RNA substrate. The folding of the DNAzyme induced by binding of Mg2+ may occur in several distinct stages. The first stage, observed at 0.5 mm Mg2+, corresponds to the formation of a compact structure with limited binding properties and without catalytic activity. Then, at 5 mm Mg2+, flanking arms are projected at right position and angles to bind RNA. In such a state, DNAzyme shows substantial binding to its substrate and significant catalytic activity. Finally, the transition occurring at 15 mm Mg2+ leads to the formation of the catalytic domain, and DNAzyme shows high binding affinity toward substrate and efficient catalytic activity. Under conditions simulating intracellular conditions, the DNAzyme was only partially folded, did not bind to its substrate, and showed only residual catalytic activity, suggesting that it may be inactive in the transfected cells and behave like antisense oligodeoxynucleotide. The intracellular ability of the “10–23” DNAzyme to efficiently inhibit expression of targeted proteins has been evidenced by in vitro and in vivo studies. However, standard conditions for kinetic measurements of the DNAzyme catalytic activity in vitro include 25 mm Mg2+, a concentration that is very unlikely to be achieved intracellularly. To study this discrepancy, we analyzed the folding transitions of the 10–23 DNAzyme induced by Mg2+. For this purpose, spectroscopic analyzes such as fluorescence resonance energy transfer, fluorescence anisotropy, circular dichroism, and surface plasmon resonance measurements were performed. The global geometry of the DNAzyme in the absence of added Mg2+ seems to be essentially extended, has no catalytic activity, and shows a very low binding affinity to its RNA substrate. The folding of the DNAzyme induced by binding of Mg2+ may occur in several distinct stages. The first stage, observed at 0.5 mm Mg2+, corresponds to the formation of a compact structure with limited binding properties and without catalytic activity. Then, at 5 mm Mg2+, flanking arms are projected at right position and angles to bind RNA. In such a state, DNAzyme shows substantial binding to its substrate and significant catalytic activity. Finally, the transition occurring at 15 mm Mg2+ leads to the formation of the catalytic domain, and DNAzyme shows high binding affinity toward substrate and efficient catalytic activity. Under conditions simulating intracellular conditions, the DNAzyme was only partially folded, did not bind to its substrate, and showed only residual catalytic activity, suggesting that it may be inactive in the transfected cells and behave like antisense oligodeoxynucleotide. The typical DNAzyme, 1The abbreviations used are: DNAzymeDNA enzyme suitable for the sequence-specific cleavage of RNAFRETfluorescence resonance energy transferHPLChigh pressure liquid chromatographyHUVEChuman umbilical vein endothelial cell.1The abbreviations used are: DNAzymeDNA enzyme suitable for the sequence-specific cleavage of RNAFRETfluorescence resonance energy transferHPLChigh pressure liquid chromatographyHUVEChuman umbilical vein endothelial cell. known as the “10–23” model, has tremendous potential in gene suppression for both target validation and therapeutic applications (1Santoro S.W. Joyce G.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4262-4266Crossref PubMed Scopus (1244) Google Scholar). It is capable of cleaving single-stranded RNA at specific sites under simulated physiological conditions and can be used to control even complex biological processes such as tumor angiogenesis. For example, DNAzymes to β1 and β3 mRNA reduced expression of targeted integrin subunits in endothelial cells and blocked proliferation, migration, and network formation in a fibrin and Matrigel™ matrix (2Cieslak M. Niewiarowska J. Nawrot M. Koziolkiewicz M. Stec W.J. Cierniewski C.S. J. Biol. Chem. 2002; 277: 6779-6787Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In a cell culture system, a 10–23 deoxyribozyme designed against 12-lipoxygenase mRNA specifically down-regulated expression of this protein and its metabolites, which are known to play a crucial role in tumor angiogenesis (3Liu C. Cheng R. Sun L.Q. Tien P. Biochem. Biophys. Res. Commun. 2001; 284: 1077-1082Crossref PubMed Scopus (26) Google Scholar). Similarly, the DNAzyme to VEGFR2 mRNA cleaved its substrate efficiently and inhibited the proliferation of endothelial cells with a concomitant reduction of VEGFR2 mRNA and blocked tumor growth in vivo (4Zhang L. Gasper W.J. Stass S.A. Ioffe O.B. Davis M.A. Mixson A.J. Cancer Res. 2002; 62: 5463-5469PubMed Google Scholar).The origins of the DNAzyme catalytic activity are not yet fully understood, but the observed rate enhancements probably are generated by a number of factors, including metal ion and nucleobase catalysis and local stereochemical effects. The 10–23 DNAzyme has been developed using an in vitro selection strategy on the basis of its ability to cleave RNA in the presence of Mg2+ (1Santoro S.W. Joyce G.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4262-4266Crossref PubMed Scopus (1244) Google Scholar). It has a catalytic domain of 15 highly conserved deoxyribonucleotides flanked by two substrate-recognition domains and can cleave effectively between any unpaired purine and pyrimidine of mRNA transcripts. Like many other enzymes catalyzing phosphoryl-transfer reactions, it is recognized as a metalloenzyme requiring divalent metal, preferentially Mg2+ ions, for catalytic activity. Divalent cations play a crucial role in these mechanisms, as evidenced by a number of observations. For example, addition of La3+ to the Mg2+-background reaction mixture inhibited the DNAzyme-catalyzed reactions, suggesting the replacement of catalytically and/or structurally important Mg2+ by La3+ (5He Q.C. Zhou J.M. Zhou D.M. Nakamatsu Y. Baba T. Taira K. Biomacromolecules. 2002; 3: 69-83Crossref PubMed Scopus (24) Google Scholar). The function of divalent metal cations in DNAzyme activity is very complex and includes (i) stabilization of the transition state of reaction. The divalent metal cation dependence of the enzyme was described as being the evidence supporting a chemical mechanism involving metal-assisted deprotonation of the 2′-hydroxyl located adjacent to the cleavage site (6Santoro S.W. Joyce G.F. Biochemistry. 1998; 37: 13330-13342Crossref PubMed Scopus (361) Google Scholar). It also includes (ii) neutralization of negative charges of phosphate groups, thus facilitating DNA-RNA interactions. High resolution x-ray crystal structures of Mg2+ and Ca2+ salts of the model B-DNA decamers CCAACGTTGG and CCAGCGCTGG revealed sequence-specific binding of Mg2+ and Ca2+ to the major and minor grooves of DNA, as well as nonspecific binding to backbone phosphate oxygen atoms. This accounts for the neutralization of between 50 and 100% of the negative charges of phosphate groups (7Chiu T.K. Dickerson R.E. J. Mol. Biol. 2000; 25: 915-945Crossref Scopus (183) Google Scholar). (iii) Some of these bound cations may also play a purely structural role by inducing proper folding of the DNAzyme molecule, thus helping to organize the enzyme into its active conformation. There are several reports showing that Mg2+ helps to stabilize different types of double-stranded DNA structures (8Welche J.B. Duckett D.R. Lilley D.M. Nucleic Acids Res. 1993; 21: 4548-4555Crossref PubMed Scopus (58) Google Scholar, 9Soyfer V.N. Potaman V.N. Triple-helical Nucleic Acids. Springer, New York1966: 360Google Scholar) and can induce bending or enhance curvature in DNA (10Brukner I. Susic S. Dlakic M. Savic A. Pongor S. J. Mol. Biol. 1994; 11: 26-32Crossref Scopus (115) Google Scholar). Furthermore, Mg2+ and other divalent cations enhance end-to-end DNA interactions, particularly in the case of fragments with self-complementary ends (11Dahlgreen P.R. Lyubchenko Y.L. Biochemistry. 2002; 41: 11372-11378Crossref PubMed Scopus (28) Google Scholar). These structural effects of cations may be even more profound in a single-stranded and flexible DNAzyme molecule.Mg2+-dependent cleavage has special relevance to biology because it is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo (12Scott W. Klug A. Trends Biochem. Sci. 1996; 21: 220-224Abstract Full Text PDF PubMed Scopus (89) Google Scholar). However, at the present time, it is hard to explain their intracellular catalytic activity, keeping in mind the catalytic dependence upon high concentrations of Mg2+, which is unlikely to be achieved in cytoplasm. To address the question of their intracellular catalytic activity, we attempted to correlate changes in the catalytic activity and configuration of the DNAzyme induced by gradually bound Mg2+. To characterize structural changes in the DNAzyme, we performed fluorescence resonance energy transfer (FRET) analysis, which allowed us to monitor general folding of the molecule based on the measurements of distances between fluorophores linked to 5′ and 3′ side bases, and surface plasmon resonance analysis of the DNAzyme binding to its RNA substrate. Structural changes induced by cations in DNAzymes were also monitored by circular dichroism and fluorescence anisotropy analysis.EXPERIMENTAL PROCEDURESSynthesis of DNAzyme to β3 mRNA—DNAzyme was chemically synthesized on a solid support using an ABI-394 DNA synthesizer, as described previously (13Cierniewski C.S. Babinska A. Swiatkowska M. Wilczynska M. Okruszek A. Stec W. Eur. J. Biochem. 1995; 227: 494-499Crossref PubMed Scopus (19) Google Scholar). This particular DNA sequence (5′-GAGTCCCATAg1g2c3t4a5g6c7t8a9c10a11a12c13g14a15AAGACTTGAG-3′) was used previously to analyze the enzymatic activity, specificity, exonuclease resistance, and ability to inhibit expression of β3 integrins in endothelial cells (2Cieslak M. Niewiarowska J. Nawrot M. Koziolkiewicz M. Stec W.J. Cierniewski C.S. J. Biol. Chem. 2002; 277: 6779-6787Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). For BIAcore experiments, the inactive DNAzyme, β3DE(15)In, with a single substitution (G6 → A) in the reactive loop and antisense oligodeoxynucleotide β3(1245–1265) containing both flanking arms of the β3DE (5′-GAGTCCCATACAAGACTTGAG-3′) were synthesized. Two analogues of β3DE(15)In and β3(1245–1265) were produced as well, which contained the modified oligonucleotides such as phosphorothioates or 2′-O-methyl-substituted residues introduced at both 5′ and 3′ sides. Hence, S-β3DE(15)in and S-β3(1245–1265) have two phosphorothioate substitutions, whereas MeO-β3DE(15)in and MeO-β3(1245–1265) contain two 2′-O-methyl-substituted residues at both their 5′ and 3′ sides, respectively. An additional mutated DNAzyme (MeO-β3DE(11)) with a downsized catalytic loop of a deoxyribozyme from 15-mer to 11-mer (5′-GAGTCCCATAg1g2c3t4a9c10a11a12c13g14a15AAGACTTGAG-3′) was synthesized and used as a control. DNAzymes with the 11-mer catalytic loop were described to be Ca2+-dependent deoxyribozymes and showed significantly reduced binding affinities and catalytic activities in the presence of Mg2+ (14Okumoto Y. Sugimoto N. J. Inorg. Chem. 2000; 82: 189-195Crossref PubMed Scopus (16) Google Scholar). All deoxyoligonucleotides were purified by HPLC and ion exchange chromatography (to 98%), and their purity was checked by PAGE under denaturing conditions.The doubly labeled β3 DNAzymes with the 15-mer and 11-mer catalytic loops were synthesized by the solid-state phosphoramidite approach on an ABI 392 synthesizer, starting from fluorescein CPG support (CPG, Inc.). The 2′-O-Me RNA cyanoethyl phosphoramidites (Glen Research) were used for introduction of 2′-O-Me nucleotide units flanking the sequence from the 3′ and 5′ ends. The synthesis was terminated with the addition of rhodamine cyanoethyl phosphoramidite (CPG, Inc.). Oligonucleotides were cleaved from the solid support and deprotected by brief (4 h at 55 °C) treatment with 30% ammonia. Pure product was isolated by preparative HPLC on a Hamilton PRP1 column. The peak fractions were evaporated to dryness, redissolved in water, and then ethanol-precipitated.The synthetic, biotinylated (at the 3′ end) 21-mer RNA (biotin-CUCAGGGUAUGUUCUGAACUC) used in BIAcore experiments was purchased from Bionovo (Poland). Its sequence corresponded to the 1245–1265 fragment of β3 mRNA. After synthesis, the product was purified by HPLC and its purity was checked by PAGE.Preparation of Target RNA Substrates and Kinetic Analysis—Aliquots of RNA substrates (20 μl, 5 μm) dissolved in a T4 polynucleotide kinase buffer were mixed with [γ-32P]ATP (2 μl, 20 μCi) and T4 polynucleotide kinase (3 units). Reaction was carried out for an h at 37 °C. All reported kinetic values were determined in multiple turnover reactions. Vmax and Km values were determined from the y intercept and slope, respectively, of the best-fit line to a Lineweaver-Burke plot of 1/V versus 1/[S]. Reactions (10 min at 37 °C; total volume = 20 μl) were carried out in 50 mm Tris, pH 8.0, containing 15 mm MgCl2, 0.01% SDS, DNAzyme (0.0125 μm) with the radiolabeled RNA substrate used in a wide range of concentrations. The cleavage reaction was stopped by the addition of 5 μl of 0.5 m EDTA, and the products were separated by electrophoresis in 20% polyacrylamide gels under denaturing conditions. Amounts of the product were evaluated by use of a PhosphorImager (Amersham Biosciences).Cell Culture—Human umbilical vein endothelial cells (HUVEC) were isolated from freshly collected umbilical cords by collagenase treatment (15Jaffe E.A. Minich R. Adelman B. Becker C.G. Nachman R.L. J. Exp. Med. 1976; 144: 209-221Crossref PubMed Scopus (141) Google Scholar, 16Jaffe E.A. Nachman R.L. Becher C.G. Minich C.R. J. Clin. Invest. 1973; 52: 2745-2756Crossref PubMed Scopus (5984) Google Scholar). Cells were grown in gelatin-coated 75-cm2 tissue culture flasks and were maintained at confluence in RPMI 1640 medium supplemented with streptomycin (100 μg/ml), penicillin (100 units/ml), fungizone (2.5 mg/ml), heparin (90 μg/ml), l-glutamine (1 mm), sodium bicarbonate (2 mg/ml), 20% fetal bovine serum, and epidermal growth factor (40 ng/ml) at 37 °C in a humidified 5% CO2 atmosphere. Primary cultures were harvested at confluence with trypsin/EDTA and transferred into gelatin-coated dishes. For the experiments, confluent cultures were used at the second passage.For microscopic examination, cells were plated at a density of 5 × 104 cells/well on Thermanox cover-slips in 8-well tissue culture chamber slides (NUNC) with detachable chambered upper structures. Before performance of assays, the serum-containing medium was changed to a serum-free medium (Opti-MEM). The cultures were gently rinsed three times with the medium and preincubated with fluorophore-labeled MeOβ3DE(15) (0.5 μm) for 6 h in the presence of Lipofectin (5 μg/ml). After that time, the transfection mixture was replaced by normal serum-containing medium, and cells were grown for another 18 h. Attached, treated intact cells were maintained in a CO2 incubator at 37 °C. Two control assays were carried out using either untreated cells or cells exposed to 0.25 μm fluorescein. After incubation, cells were washed three times with phosphate-buffered saline, fixed with freshly prepared 3.5% paraformaldehyde for 15 min at room temperature, washed three times with phosphate-buffered saline, mounted in 2.5% DABCO™ in glycerol, and processed for microscopy.Fluorescence Spectroscopy—Fluorescence emission spectra were measured on an LS-50 spectrofluorometer (PerkinElmer Life Sciences), and spectra were corrected for lamp fluctuations and instrumental variations. Polarization artifacts were avoided by setting excitation and emission polarizers to magic angle conditions (54.74°). All of the fluorescence measurements were performed at the temperature of 23 ± 1 °C. Emission spectra, excitation spectra, and luminescence intensity were recorded with 5-nm band passes for both the excitation and emission monochromators. A cut-off filter in the emission beam was used to eliminate second-order wavelength interference. The excitation wavelengths used were 494 nm and 560 nm for fluorescein- and rhodamine-conjugated constructs, respectively. Emission spectra were corrected for the blank contribution and for the instrument response and normalized to the DNA concentration in a quartz cell with a 1-cm path length. Excitation and emission spectra were automatically corrected for lamp intensity variations. Buffers were degassed by bubbling nitrogen to prevent quenching of fluorescence by dissolved oxygen. The fluorescence emission signals were stable to photobleaching under the experimental conditions of measurement. The apparent interchromophore separation, R, the distance separating the energy donor and acceptor was calculated by the Forster equation: r = R0(1/E – 1)1/6, where E is the efficiency of energy transfer from donor to acceptor. E = 1 – FDA/FD and R0 is the distance for 50% transfer efficiency E. FDA and FD are the fluorescence intensities of the donor in the presence and absence of the acceptor, respectively. FDA and FD were measured at 520 nm, the emission maximum for fluorescein.The fluorescence anisotropy of the fluorescein and rhodamine probes attached to the DNAzyme (Fluo-MeO-β3DE(15)-Rhod, Fluo-MeO-β3DE(11)-Rhod) was monitored in an LS-50 spectrofluorometer (PerkinElmer Life Sciences) equipped with an automatic anisotropy measuring device. The anisotropy r is defined asr=(IVV-G×IVH)/(IVV+2×G×IVH)(Eq. 1) where I is fluorescence intensity. The first and second indices refer to the orientation of excitation and emission polarizers, respectively. G is the correction factor. The cell holder was thermostated at 21 °C.Analysis of Fluorescence Data—Efficiencies of energy transfer were determined from enhancement acceptor fluorescence (17Clegg R.M. Methods Enzymol. 1992; 211: 353-388Crossref PubMed Scopus (649) Google Scholar, 18Clegg R.M. Murchie A.I.H. Zechel A. Lilley D.M.J. (1993).Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2994-2998Crossref PubMed Scopus (267) Google Scholar). The emission at a given wavelength (v1) of a double-labeled sample excited primarily at the donor wavelength (v′) contains emission from the donor, emission from directly excited acceptor, and emission from acceptor excited by energy transfer from the donor, i.e.F(v1,v')=[S]·ϵD(v')·ϕD(v1)·d+·{(1-EFRET)·a++a-+ϵA(v')·ϕA(v1)·a++ϵD(v')·ϕA(v1)·EFRET·d++a+}=FD(v1,v')+FA(v1,v')(Eq. 2) where [S] is the concentration of DNAzyme, d+ and a+ are the molar fraction of DNAzyme molecules labeled with donor and acceptor respectively, and a– is the molar fraction of DNAzyme molecules unlabeled with acceptor. Superscripts D and A refer to donor and acceptor, respectively. ϵD(v′) and ϵA(v′) are the molar absorption coefficients of donor and acceptor, respectively, and φD(v1) and φ (v1) are the fluorescent quantum yields of donor and acceptor, respectively. Thus the spectrum contains the components due to donor emission [FD(v1,v′), i.e. the first term containing φD(v1)] and those due to acceptor emission [FA(v1,v′), i.e. the latter two terms containing φ (v1)]. The first stage of the analysis involves subtraction of the spectrum of DNA labeled only with donor, leaving just the acceptor components, i.e. FA(v1,v′). The pure acceptor spectrum thus derived is normalized to one from the same sample excited at a wavelength (v″) at which only the acceptor is excited, with emission at v2. We then obtain the acceptor ratio(ratio)A=FA(v1,v')/FA(v2,v'')={EFRET·d+·[ϵD(v')/ϵA(v'')+ϵA(v')/ϵA(v'')]}·[ϕA(v1)/ϕA(v2)](Eq. 3) EFRET is directly proportional to (ratio)A and can be easily calculated because ϵD(v′)/ϵA(v″) and ϵA(v′)/ϵA(v″) are measured from absorption spectra, and φA(v1)/φA(v2) is unity when v1 = v2.Analysis of Circular Dichroism—The circular dichroism spectra of MeO-β3DE (1 μm), free or in the complex with the substrate (2 μm), was measured in a solution of 10 mm Tris-HCl, pH 7.5, containing increasing concentrations of MgCl2 at 21 °C. In control experiments, 0.1 m and 1 m NaCl were used. Before measurement, the complex was allowed to form by heating the solution at 95 °C for 2.5 min followed by gradual cooling. Measurements were made in a quartz cuvette (5-mm path length) with a CD spectrometer (model CD6, Jobin Yvon) from 200 to 320 nm in triplicate. The spectra were obtained by smoothing the averaged spectra with a calculator.Surface Plasmon Resonance—The kinetic parameters (association and dissociation rate constants, kon and koff, respectively) and the affinity constant (KD) between DNAzyme and the mRNA substrate were measured by surface plasmon resonance using a BIAcoreX (Amersham Biosciences). Briefly, avidin was covalently attached to carboxymethyl dextran chips (CM5, BIAcore) previously activated with N-hydroxysuccinimide and N-ethyl-N′-dimethylaminopropyl carbodiimide, according to the manufacturer's instructions. Experiments were performed at 37 °C using 50 mm Tris, pH 8.0, containing divalent cations at the indicated concentrations. 15 μl of 5′ end-biotinylated RNA at 100 nm in 50 mm Tris, pH 8.0, was injected at the flow rate of 5 μl/min and consequently immobilized on the bound avidin to give a response of ∼500 resonance units, an arbitrary unit specific for the BIAcore instrument. The levels of immobilized RNA were within the low levels that have to be used to ensure that the observed binding rate will be limited by the reaction kinetics rather than by the mass transport effects of the injected DNAzyme (19Bondeson K. Frostell-Karlsson A. Fagerstam L. Magnusson G. Anal. Biochem. 1993; 214: 245-251Crossref PubMed Scopus (144) Google Scholar). In typical experiments, DNAzyme flowed in two channels of the sensor; the first one contained the RNA substrate attached to avidin, and the second was without the RNA substrate. The latter was used to correct SPR traces and remove the background binding between DNAzyme and the immobilized avidin on the dextran. The concentration of the injected DNAzyme was in the range 20–200 nm, and the flow rate was 5 μl/min. The amount of ligand bound to immobilized RNA substrate was monitored by measuring the variation of the surface plasmon resonance angle as a function of time. Results were expressed in resonance units. In preliminary experiments, the data obtained for at least three different concentrations of DNAzymes were fitted to several models; the best fits (χ2 = 1.4) were obtained by assuming a one-to-one interaction. Then, the association rate constant, kon, and dissociation rate constant, koff, were determined separately from individual association and dissociation phases, respectively. The overall affinity constant, KA, was derived from kon/koff. The sensor chip was regenerated with three 10-μl pulses of 12.5% formamide.RESULTSEnzymatic Characteristics of the DNAzyme to β3 Integrin Subunit—The 10–23 DNAzyme used in these studies was designed to cleave β3 integrin subunit mRNA, and preliminary characterization was done in our recent work (2Cieslak M. Niewiarowska J. Nawrot M. Koziolkiewicz M. Stec W.J. Cierniewski C.S. J. Biol. Chem. 2002; 277: 6779-6787Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Standard conditions for kinetic measurements of the catalytic activity of DNAzyme in vitro include 25 mm MgCl2 (20Kumar P.K.R. Zhou D.-M. Yoshinari K. Taira K. Eckstein F. Lilley D.M.J. Catalytic RNA, Nucleic Acids and Molecular Biology. Vol. 10. Springer-Verlag, Berlin1996: 217-230Google Scholar). Under such conditions, the 10–23 DNAzyme shows optimal enzymatic activity, whereas its mutant MeO-β3DE(11) with the shortened catalytic loop is inactive (Fig. 1A). In this experiment, enzymatic reactions were performed in 50 mm Tris-HCl, pH 8.0, containing 0–25 mm MgCl2, under multiple turnover conditions. The 32P-labeled mRNA substrate was mixed with the DNAzyme β3DE in the molar ratio of 80:1 and incubated at 37 °C; aliquots were withdrawn after 10 min. The cleavage reaction was stopped by the addition of 5 μl of 0.5 m EDTA, and products were separated by electrophoresis in 20% polyacrylamide gels under denaturing conditions. Amounts of the product were evaluated by use of a PhosphorImager. Fig. 1B shows a composition of cleavage mixtures obtained after a 60-min incubation of 32P-labeled mRNA substrates with 0.025 μm β3DE added at a molar ratio ranging from 5:1 to 80:1. Each of the DNAzymes, unmodified (β3DE) and modified (MeO-β3DE), cleaved the substrate at the predicted site. Interestingly, DNAzyme with the 2′-MeO residues showed a significantly higher enzymatic activity than β3DE, as evidenced by the catalytic efficiency kcat/Km of 3.62 × 106 and 1.09 × 106m–1 min–1, respectively.Study of Ion-induced Folding of the DNAzyme by Fluorescence Resonance Energy Transfer—To follow the folding transitions of the DNAzyme induced upon binding of Mg2+ ions, FRET was utilized. For this purpose, β3DE with the 15-mer and 11-mer catalytic loops was modified by attachment of the donor and acceptor fluorophores, rhodamine and fluorescein, to the 5′ and 3′ ends of the flanking arms, respectively. Next, a series of experiments were designed to analyze whether the fluorophores attached to the terminal bases affect the ability of the oligodeoxynucleotide construct to function as an active DNAzyme species. As seen in Fig. 2A, incubation of the Fluo-MeO-β3DE(15)-Rhod with the 32P-labeled substrate in the presence of 25 mm Mg2+ under multiple-turnover conditions at 37 °C leads to cleavage at the correct site. When such a construct was incubated with endothelial cells, it remained resistant to intracellular nucleases and, even after 24 h, was located exclusively within the cytoplasm, particularly in the perinuclear organelles. The cellular uptake, intracellular distribution, and stability of the Fluo-MeO-β3DE(15)-Rhod were the same as those recently reported for another 10–23 DNAzyme (21Dass C.R. Saravolac E.G. Li Y. Sun L.Q. Antisense Nucleic Acid Drug Dev. 2002; 12: 289-299Crossref PubMed Scopus (116) Google Scholar). Cellular transport of the Fluo-MeO-β3DE(15)-Rhod as a function of the external oligonucleotide concentration was non-linear, being more efficient at concentrations below 2 μm. The punctate fluorescence distribution observed even after 24 h of exposure to the DNAzyme seems to suggest that endosomal vesicles are the primary targets of the probes under study (Fig. 2B). Fluo-MeO-β3DE(15)-Rhod could be detected intracellularly, both when emission of either fluorescein or rhodamine was measured and both fluorophores showed full colocalization. Thus, the attached fluorophores did not influence the enzymatic activity and biological properties of the DNAzyme, including the ability to interact with cellular components responsible for its transport. Transfection of endothelial cells with the DNAzyme (5 μm) efficiently reduced expression of β3 integrin subunit measured at the level of β3 mRNA by reverse transcriptase-PCR (Fig. 2C) and at the cellular surface by flow cytometry (not shown). Although MeO-β3DE(11)-Rhod had the same cellular distribution as MeO-β3DE(15)-Rhod, it did not show any biological activity detectable at the level of β3 mRNA or β3 expression at the cell surface. These data provide the evidence that the 10–23 DNAzyme has an intracellular catalytic or antisense activity, even at the much lower cation concentrations than those normally used in in vitro analysis.Fig. 2Biological activity of Fluo-MeO-β3DE-Rhod.A, DNAzyme activity of the fluorophore-labeled construct identical to that used for the FRET analysis. The cleavage activity was examined after incubation of Fluo-MeO-β3DE-Rhod with a 20-fold excess of 5′-32P-labeled RNA substrate in the presence of 15 mm MgCl2 at 37 °C. A sample was removed at different time points (lanes 1-5). B, the fluorescence image of endothelial cells treated with Fluo-MeO-β3DE-Rhod. Endothelial cells, exposed to 0.5 μm of the fluorophore-labeled construct for 24 h at 37 °C and processed as described under “Experimental Procedures,” were analyzed by confocal fluorescence microscopy. The punctate fluorescence distribution within the cytoplasm was detected by monitoring both fluorescein and rhodamine attached to DNAzyme. C, the reduced expression of β3 mRNA in HUVECs treated with MeO-β3DE when compared with unchanged expression in untreated cells. β3 mRNA was evaluated by relative quantitative reverse transcriptase-PCR using glyceraldehyde-3-phosphate dehydrogenase mRNA as an intrinsic control.View Large Image Figure ViewerDownload (PPT)Both Fluo-MeO-β3DE(15)-Rhod and Fluo-MeO-β3DE(11)-Rhod were next used to measure energy transfer resulting from a dipolar coupling between the transition moments of the two fluorophores, fluorescein as the energy donor and rhodamine as the energy acceptor. When the fluorescence spectrum of one fluorophore (the donor) overlaps with the excitation spectrum of another fluorophore (the acceptor), the excitation of the donor" @default.
• W2014496171 created "2016-06-24" @default.
• W2014496171 creator A5008525064 @default.
• W2014496171 creator A5065679567 @default.
• W2014496171 creator A5066355029 @default.
• W2014496171 creator A5078328470 @default.
• W2014496171 date "2003-11-01" @default.
• W2014496171 modified "2023-10-06" @default.
• W2014496171 title "Structural Rearrangements of the 10–23 DNAzyme to β3 Integrin Subunit mRNA Induced by Cations and Their Relations to the Catalytic Activity" @default.
• W2014496171 cites W1769395946 @default.
• W2014496171 cites W1975105337 @default.
• W2014496171 cites W1977709085 @default.
• W2014496171 cites W1978903221 @default.
• W2014496171 cites W1979871091 @default.
• W2014496171 cites W1981949351 @default.
• W2014496171 cites W1987208051 @default.
• W2014496171 cites W1992541367 @default.
• W2014496171 cites W1997694257 @default.
• W2014496171 cites W2002048308 @default.
• W2014496171 cites W2009461154 @default.
• W2014496171 cites W2026256712 @default.
• W2014496171 cites W2030973999 @default.
• W2014496171 cites W2063865555 @default.
• W2014496171 cites W2067947663 @default.
• W2014496171 cites W2078224461 @default.
• W2014496171 cites W2081125994 @default.
• W2014496171 cites W2083929510 @default.
• W2014496171 cites W2086654802 @default.
• W2014496171 cites W2087644304 @default.
• W2014496171 cites W2089163172 @default.
• W2014496171 cites W2090909297 @default.
• W2014496171 cites W2093115020 @default.
• W2014496171 cites W2094165999 @default.
• W2014496171 cites W2101669631 @default.
• W2014496171 cites W2102683170 @default.
• W2014496171 cites W2123498957 @default.
• W2014496171 cites W2141157874 @default.
• W2014496171 cites W2157603347 @default.
• W2014496171 cites W2173835561 @default.
• W2014496171 cites W2234783182 @default.
• W2014496171 doi "https://doi.org/10.1074/jbc.m300504200" @default.
• W2014496171 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12952967" @default.
• W2014496171 hasPublicationYear "2003" @default.
• W2014496171 type Work @default.
• W2014496171 sameAs 2014496171 @default.
• W2014496171 citedByCount "67" @default.
• W2014496171 countsByYear W20144961712012 @default.
• W2014496171 countsByYear W20144961712013 @default.
• W2014496171 countsByYear W20144961712015 @default.
• W2014496171 countsByYear W20144961712016 @default.
• W2014496171 countsByYear W20144961712017 @default.
• W2014496171 countsByYear W20144961712018 @default.
• W2014496171 countsByYear W20144961712019 @default.
• W2014496171 countsByYear W20144961712020 @default.
• W2014496171 countsByYear W20144961712021 @default.
• W2014496171 countsByYear W20144961712022 @default.
• W2014496171 countsByYear W20144961712023 @default.
• W2014496171 crossrefType "journal-article" @default.
• W2014496171 hasAuthorship W2014496171A5008525064 @default.
• W2014496171 hasAuthorship W2014496171A5065679567 @default.
• W2014496171 hasAuthorship W2014496171A5066355029 @default.
• W2014496171 hasAuthorship W2014496171A5078328470 @default.
• W2014496171 hasBestOaLocation W20144961711 @default.
• W2014496171 hasConcept C104292427 @default.
• W2014496171 hasConcept C104317684 @default.
• W2014496171 hasConcept C105580179 @default.
• W2014496171 hasConcept C161790260 @default.
• W2014496171 hasConcept C185592680 @default.
• W2014496171 hasConcept C552990157 @default.
• W2014496171 hasConcept C55493867 @default.
• W2014496171 hasConcept C84211826 @default.
• W2014496171 hasConcept C86803240 @default.
• W2014496171 hasConcept C95444343 @default.
• W2014496171 hasConceptScore W2014496171C104292427 @default.
• W2014496171 hasConceptScore W2014496171C104317684 @default.
• W2014496171 hasConceptScore W2014496171C105580179 @default.
• W2014496171 hasConceptScore W2014496171C161790260 @default.
• W2014496171 hasConceptScore W2014496171C185592680 @default.
• W2014496171 hasConceptScore W2014496171C552990157 @default.
• W2014496171 hasConceptScore W2014496171C55493867 @default.
• W2014496171 hasConceptScore W2014496171C84211826 @default.
• W2014496171 hasConceptScore W2014496171C86803240 @default.
• W2014496171 hasConceptScore W2014496171C95444343 @default.
• W2014496171 hasIssue "48" @default.
• W2014496171 hasLocation W20144961711 @default.
• W2014496171 hasOpenAccess W2014496171 @default.
• W2014496171 hasPrimaryLocation W20144961711 @default.
• W2014496171 hasRelatedWork W1988567260 @default.
• W2014496171 hasRelatedWork W2014970772 @default.
• W2014496171 hasRelatedWork W2340510833 @default.
• W2014496171 hasRelatedWork W2496039254 @default.
• W2014496171 hasRelatedWork W2948105872 @default.
• W2014496171 hasRelatedWork W3047657684 @default.
• W2014496171 hasRelatedWork W4232144512 @default.
• W2014496171 hasRelatedWork W4253008687 @default.
• W2014496171 hasRelatedWork W4378802211 @default.
• W2014496171 hasRelatedWork W4380203878 @default.
• W2014496171 hasVolume "278" @default.

• next