THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 48, Issue of November 28, pp. 47987–47996, 2003 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A. Structural Rearrangements of the 10 –23 DNAzyme to 3 Integrin
Subunit mRNA Induced by Cations and Their Relations to the
Catalytic Activity*

Received for publication, January 16, 2003, and in revised form, July 22, 2003 Published, JBC Papers in Press, September 2, 2003, DOI 10.1074/jbc.M300504200 Marcin Cieslak‡, Jacek Szymanski§, Ryszard W. Adamiak¶, and Czeslaw S. Cierniewski§**
From the Centers for Molecular and Macromolecular Studies and Medical Biology and Microbiology, Polish Academy ofSciences, §Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz, Poland andInstitute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland The intracellular ability of the "10 –23" DNAzyme to
biological processes such as tumor angiogenesis. For example, efficiently inhibit expression of targeted proteins has
DNAzymes to ␤1 and ␤3 mRNA reduced expression of targeted been evidenced by in vitro and in vivo studies. However,
integrin subunits in endothelial cells and blocked proliferation, standard conditions for kinetic measurements of the
migration, and network formation in a fibrin and Matrigel™ DNAzyme catalytic activity in vitro include 25 mM Mg2,
matrix (2). In a cell culture system, a 10 –23 deoxyribozyme a concentration that is very unlikely to be achieved
designed against 12-lipoxygenase mRNA specifically down-reg- intracellularly. To study this discrepancy, we analyzed
ulated expression of this protein and its metabolites, which are the folding transitions of the 10 –23 DNAzyme induced
known to play a crucial role in tumor angiogenesis (3). Simi- by Mg2. For this purpose, spectroscopic analyzes such
larly, the DNAzyme to VEGFR2 mRNA cleaved its substrate as fluorescence resonance energy transfer, fluorescence
efficiently and inhibited the proliferation of endothelial cells anisotropy, circular dichroism, and surface plasmon
with a concomitant reduction of VEGFR2 mRNA and blocked resonance measurements were performed. The global
geometry of the DNAzyme in the absence of added Mg2

tumor growth in vivo (4).
seems to be essentially extended, has no catalytic activ-
The origins of the DNAzyme catalytic activity are not yet ity, and shows a very low binding affinity to its RNA
fully understood, but the observed rate enhancements probably substrate. The folding of the DNAzyme induced by bind-
are generated by a number of factors, including metal ion and ing of Mg2may occur in several distinct stages. The
nucleobase catalysis and local stereochemical effects. The first stage, observed at 0.5 mM Mg2, corresponds to the
10 –23 DNAzyme has been developed using an in vitro selection formation of a compact structure with limited binding
strategy on the basis of its ability to cleave RNA in the presence properties and without catalytic activity. Then, at 5 mM
of Mg2⫹ (1). It has a catalytic domain of 15 highly conserved Mg2, flanking arms are projected at right position and
deoxyribonucleotides flanked by two substrate-recognition do- angles to bind RNA. In such a state, DNAzyme shows
mains and can cleave effectively between any unpaired purine substantial binding to its substrate and significant cat-
and pyrimidine of mRNA transcripts. Like many other en- alytic activity. Finally, the transition occurring at 15 mM
zymes catalyzing phosphoryl-transfer reactions, it is recog- Mg2leads to the formation of the catalytic domain, and
nized as a metalloenzyme requiring divalent metal, preferen- DNAzyme shows high binding affinity toward substrate
tially Mg2⫹ ions, for catalytic activity. Divalent cations play a and efficient catalytic activity. Under conditions simu-
crucial role in these mechanisms, as evidenced by a number of lating intracellular conditions, the DNAzyme was only
partially folded, did not bind to its substrate, and

observations. For example, addition of La3⫹ to the Mg2⫹-back- showed only residual catalytic activity, suggesting that
ground reaction mixture inhibited the DNAzyme-catalyzed re- it may be inactive in the transfected cells and behave
actions, suggesting the replacement of catalytically and/or like antisense oligodeoxynucleotide.
structurally important Mg2⫹ by La3⫹ (5). The function of diva-lent metal cations in DNAzyme activity is very complex andincludes (i) stabilization of the transition state of reaction. The The typical DNAzyme,1 known as the "10 –23" model, has divalent metal cation dependence of the enzyme was described tremendous potential in gene suppression for both target vali- as being the evidence supporting a chemical mechanism involv- dation and therapeutic applications (1). It is capable of cleaving ing metal-assisted deprotonation of the 2⬘-hydroxyl located ad- single-stranded RNA at specific sites under simulated physio- jacent to the cleavage site (6). It also includes (ii) neutralization logical conditions and can be used to control even complex of negative charges of phosphate groups, thus facilitating DNA-RNA interactions. High resolution x-ray crystal structuresof Mg2⫹ and Ca2⫹ salts of the model B-DNA decamers * This work was supported by Grant Z-KBN 004/PO4/98 from the CCAACGTTGG and CCAGCGCTGG revealed sequence-spe- Polish Committee for Scientific Research. The costs of publication ofthis article were defrayed in part by the payment of page charges. This cific binding of Mg2⫹ and Ca2⫹ to the major and minor grooves article must therefore be hereby marked "advertisement" in accordance of DNA, as well as nonspecific binding to backbone phosphate with 18 U.S.C. Section 1734 solely to indicate this fact.
oxygen atoms. This accounts for the neutralization of between ** To whom correspondence should be addressed: Dept. of Molecular 50 and 100% of the negative charges of phosphate groups (7).
and Medical Biophysics, Medical University of Lodz, 6/8 MazowieckaStreet, 92–215 Lodz, Poland. Tel.: 48-42-6783393; Fax: 48-42-6789433; (iii) Some of these bound cations may also play a purely struc- tural role by inducing proper folding of the DNAzyme molecule, 1 The abbreviations used are: DNAzyme, DNA enzyme suitable for thus helping to organize the enzyme into its active conforma- the sequence-specific cleavage of RNA; FRET, fluorescence resonance tion. There are several reports showing that Mg2⫹ helps to energy transfer; HPLC, high pressure liquid chromatography; HUVEC,human umbilical vein endothelial cell.
stabilize different types of double-stranded DNA structures (8, This paper is available on line at http://www.jbc.org

Structural Transitions Induced in the DNAzyme by Mg2⫹ FIG. 1. Cleavage of 3 integrin subunit mRNA substrate by DNAzymes in vitro. A, the effect of Mg2⫹ concentration on enzymatic activity
of DNAzymes. MeO-␤3DE (–) and its mutant MeO-␤3DE (f–f) were incubated with the RNA substrate (molar ratio, 1:80) for 10 min in the presence of increasing concentrations of Mg2⫹ ranging from 0 to 25 mM. The cleavage reaction was stopped by the addition of 0.5 M EDTA, andthe products were separated by electrophoresis in 20% polyacrylamide gels under denaturing conditions. Relative amounts of cleavage products(% of cleavage) are plotted versus Mg2⫹ concentration (MgCl [m M]). Inset, autoradiogram of the gel showing the cleavage product obtained at different Mg2⫹ concentrations (0 –25 mM). B, catalytic activity of ␤3DE and MeO-␤3DE. In these experiments, aliquots of the 32P-labeled mRNAsubstrate were incubated with DNAzymes. ␤3DE and MeO-␤3DE were used at a molar ratio (substrate:enzyme) ranging from 5:1 to 80:1 for upto 60 min at 37 °C. The cleavage products obtained after a 10-min incubation of 32P-labeled mRNA substrate are shown alone (lane 1) or withDNAzymes (␤3DE, MeO-␤3DE) mixed at the ratio 5:1 (lanes 2 and 7), 10:1 (lanes 3 and 8), 20:1 (lanes 4 and 9), 40:1 (lanes 5 and 10), and 80:1 (lanes6 and 11). DNAzymes were used at the concentration of 0.025 ␮M. Reactions were carried out in 50 mM Tris, pH 8.0, containing 15 mM MgCl , 0.01% SDS. Amounts of the product were evaluated by a PhosphorImager (Amersham Biosciences) and used to calculate kinetic parameters. They weredetermined in multiple turnover reactions and represent a mean of three independent experiments.
9) and can induce bending or enhance curvature in DNA (10).
with self-complementary ends (11). These structural effects of Furthermore, Mg2⫹ and other divalent cations enhance end-to- cations may be even more profound in a single-stranded and end DNA interactions, particularly in the case of fragments flexible DNAzyme molecule.

Structural Transitions Induced in the DNAzyme by Mg2⫹ Mg2⫹-dependent cleavage has special relevance to biology in vivo (12). However, at the present time, it is hard to because it is compatible with intracellular conditions, raising explain their intracellular catalytic activity, keeping in mind the possibility that DNA enzymes might be made to operate the catalytic dependence upon high concentrations of Mg2⫹,which is unlikely to be achieved in cytoplasm. To address thequestion of their intracellular catalytic activity, we at-tempted to correlate changes in the catalytic activity andconfiguration of the DNAzyme induced by gradually boundMg2⫹. To characterize structural changes in the DNAzyme,we performed fluorescence resonance energy transfer (FRET)analysis, which allowed us to monitor general folding of themolecule based on the measurements of distances betweenfluorophores linked to 5⬘ and 3⬘ side bases, and surface plas-mon resonance analysis of the DNAzyme binding to its RNAsubstrate. Structural changes induced by cations inDNAzymes were also monitored by circular dichroism andfluorescence anisotropy analysis.
EXPERIMENTAL PROCEDURES Synthesis of DNAzyme to 3 mRNA—DNAzyme was chemically syn- thesized on a solid support using an ABI-394 DNA synthesizer, asdescribed previously (13). This particular DNA sequence (5⬘-GAGTCCCATAg g c t a g c t a c a a c g a AAGACTTGAG-3⬘) was used previously to analyze the enzymatic activity, specificity, exo-nuclease resistance, and ability to inhibit expression of ␤3 integrins inendothelial cells (2). For BIAcore experiments, the inactive DNAzyme, FIG. 2. Biological activity of Fluo-MeO-3DE-Rhod. A,
, with a single substitution (G6 3 A) in the reactive loop and DNAzyme activity of the fluorophore-labeled construct identical to that antisense oligodeoxynucleotide ␤3(1245–1265) containing both flank- used for the FRET analysis. The cleavage activity was examined after ing arms of the ␤3DE (5⬘-GAGTCCCATACAAGACTTGAG-3⬘) were incubation of Fluo-MeO-␤3DE-Rhod with a 20-fold excess of 5⬘-32P- synthesized. Two analogues of ␤3DE and ␤3(1245–1265) were labeled RNA substrate in the presence of 15 mM MgCl at 37 °C. A produced as well, which contained the modified oligonucleotides such sample was removed at different time points (lanes 1-5). B, the fluores- as phosphorothioates or 2⬘-O-methyl-substituted residues introduced cence image of endothelial cells treated with Fluo-MeO-␤3DE-Rhod.
at both 5⬘ and 3⬘ sides. Hence, S-␤3DE and S-␤3(1245–1265) have Endothelial cells, exposed to 0.5 ␮M of the fluorophore-labeled construct two phosphorothioate substitutions, whereas MeO-␤3DE for 24 h at 37 °C and processed as described under ‘‘Experimental MeO-␤3(1245–1265) contain two 2⬘-O-methyl-substituted residues at Procedures,'' were analyzed by confocal fluorescence microscopy. The both their 5⬘ and 3⬘ sides, respectively. An additional mutated punctate fluorescence distribution within the cytoplasm was detected DNAzyme (MeO-␤3DE ) with a downsized catalytic loop of a by monitoring both fluorescein and rhodamine attached to DNAzyme.
C, the reduced expression of ␤3 mRNA in HUVECs treated with MeO- TAg g c t a c a a c g a AAGACTTGAG-3⬘) was synthesized ␤3DE when compared with unchanged expression in untreated cells. ␤3 and used as a control. DNAzymes with the 11-mer catalytic loop were mRNA was evaluated by relative quantitative reverse transcriptase-PCR using glyceraldehyde-3-phosphate dehydrogenase mRNA as an described to be Ca2⫹-dependent deoxyribozymes and showed signifi- intrinsic control.
cantly reduced binding affinities and catalytic activities in the pres- FIG. 3. Fluorescence resonance energy transfer between fluorescein and rhodamine attached to MeO-3DE at 3and 5ends,
respectively. Fluo-MeO-␤3DE-Rhod (40 nM) had two peaks at 520 nm and 580 nm upon excitation at 494 nm. Inset, autoradiogram of a gel after
electrophoresis of the Fluo-MeO-␤3DE-Rhod digested with endonuclease from S. marcescens. Degradation of the fluorophore-labeled DNAzyme
resulted in a significant increase in fluorescence intensity at 520 nm and a disappearance of the 580 nm maximum.
Structural Transitions Induced in the DNAzyme by Mg2⫹ ence of Mg2⫹ (14). All deoxyoligonucleotides were purified by HPLC matically corrected for lamp intensity variations. Buffers were de- and ion exchange chromatography (to 98%), and their purity was gassed by bubbling nitrogen to prevent quenching of fluorescence by checked by PAGE under denaturing conditions.
dissolved oxygen. The fluorescence emission signals were stable to The doubly labeled ␤3 DNAzymes with the 15-mer and 11-mer cat- photobleaching under the experimental conditions of measurement.
alytic loops were synthesized by the solid-state phosphoramidite ap- The apparent interchromophore separation, R, the distance separat- proach on an ABI 392 synthesizer, starting from fluorescein CPG sup- ing the energy donor and acceptor was calculated by the Forster port (CPG, Inc.). The 2⬘-O-Me RNA cyanoethyl phosphoramidites (Glen equation: r R (1/E ⫺ 1)1/6, where E is the efficiency of energy Research) were used for introduction of 2⬘-O-Me nucleotide units flank- transfer from donor to acceptor. E ⫽ 1 ⫺ F ing the sequence from the 3⬘ and 5⬘ ends. The synthesis was terminated distance for 50% transfer efficiency E. F with the addition of rhodamine cyanoethyl phosphoramidite (CPG, cence intensities of the donor in the presence and absence of the Inc.). Oligonucleotides were cleaved from the solid support and depro- acceptor, respectively. F were measured at 520 nm, the tected by brief (4 h at 55 °C) treatment with 30% ammonia. Pure emission maximum for fluorescein.
product was isolated by preparative HPLC on a Hamilton PRP1 col- The fluorescence anisotropy of the fluorescein and rhodamine probes umn. The peak fractions were evaporated to dryness, redissolved in attached to the DNAzyme (Fluo-MeO-␤3DE water, and then ethanol-precipitated.
␤3DE -Rhod) was monitored in an LS-50 spectrofluorometer The synthetic, biotinylated (at the 3⬘ end) 21-mer RNA (biotin-CU- (PerkinElmer Life Sciences) equipped with an automatic anisotropy CAGGGUAUGUUCUGAACUC) used in BIAcore experiments was pur- measuring device. The anisotropy r is defined as chased from Bionovo (Poland). Its sequence corresponded to the 1245–1265 fragment of ␤3 mRNA. After synthesis, the product was purified r ⫽ (I G IVH) / (IVV 2 ⫻ G IVH) by HPLC and its purity was checked by PAGE.
where I is fluorescence intensity. The first and second indices refer to Preparation of Target RNA Substrates and Kinetic Analysis—Ali- the orientation of excitation and emission polarizers, respectively. G is quots of RNA substrates (20 ␮l, 5 ␮M) dissolved in a T4 polynucleotide the correction factor. The cell holder was thermostated at 21 °C.
kinase buffer were mixed with [␥-32P]ATP (2 ␮l, 20 ␮Ci) and T4 polynu- Analysis of Fluorescence Data—Efficiencies of energy transfer were cleotide kinase (3 units). Reaction was carried out for an h at 37 °C. All determined from enhancement acceptor fluorescence (17, 18). The reported kinetic values were determined in multiple turnover reactions.
emission at a given wavelength (v1) of a double-labeled sample ex- values were determined from the y intercept and slope, cited primarily at the donor wavelength (v⬘) contains emission from respectively, of the best-fit line to a Lineweaver-Burke plot of 1/V versus the donor, emission from directly excited acceptor, and emission from 1/[S]. Reactions (10 min at 37 °C; total volume ⫽ 20 ␮l) were carried out acceptor excited by energy transfer from the donor, i.e. in 50 mM Tris, pH 8.0, containing 15 mM MgCl , 0.01% SDS, DNAzyme (0.0125 ␮M) with the radiolabeled RNA substrate used in a wide range F(v1,v⬘) ⫽ [S]䡠⑀D(v⬘)䡠␾D(v1)䡠d⫹䡠{(1 ⫺ EFRET)䡠a⫹ ⫹ a⫺ of concentrations. The cleavage reaction was stopped by the addition of5 ␮l of 0.5 M EDTA, and the products were separated by electrophoresis ⫹ ⑀A(v⬘)䡠␾ A(v1)䡠a⫹ ⫹ ⑀D(v⬘)䡠␾A(v1)䡠EFRET d⫹ ⫹ a⫹} in 20% polyacrylamide gels under denaturing conditions. Amounts ofthe product were evaluated by use of a PhosphorImager (Amersham ⫽ FD(v1,v⬘) ⫹ FA(v1,v⬘) (Eq. 2) Cell Culture—Human umbilical vein endothelial cells (HUVEC) where [S] is the concentration of DNAzyme, d⫹ and a⫹ are the molar were isolated from freshly collected umbilical cords by collagenase fraction of DNAzyme molecules labeled with donor and acceptor respec- treatment (15, 16). Cells were grown in gelatin-coated 75-cm2 tissue tively, and a⫺ is the molar fraction of DNAzyme molecules unlabeled culture flasks and were maintained at confluence in RPMI 1640 with acceptor. Superscripts D and A refer to donor and acceptor, re- medium supplemented with streptomycin (100 ␮g/ml), penicillin (100 spectively. ⑀D(v⬘) and ⑀A(v⬘) are the molar absorption coefficients of units/ml), fungizone (2.5 mg/ml), heparin (90 ␮g/ml), donor and acceptor, respectively, and ␾ (v ) and ␾ (v ) are the fluores- cent quantum yields of donor and acceptor, respectively. Thus the M), sodium bicarbonate (2 mg/ml), 20% fetal bovine serum, and epidermal growth factor (40 ng/ml) at 37 °C in a humidified 5% spectrum contains the components due to donor emission [FD(v ,v⬘), i.e. atmosphere. Primary cultures were harvested at confluence the first term containing ␾ (v )] and those due to acceptor emission with trypsin/EDTA and transferred into gelatin-coated dishes. For [FA(v ,v⬘), i.e. the latter two terms containing ␾ (v )]. The first stage of the experiments, confluent cultures were used at the second the analysis involves subtraction of the spectrum of DNA labeled only with donor, leaving just the acceptor components, i.e. FA(v ,v⬘). The For microscopic examination, cells were plated at a density of 5 ⫻ 104 pure acceptor spectrum thus derived is normalized to one from the same cells/well on Thermanox cover-slips in 8-well tissue culture chamber sample excited at a wavelength (v⬙) at which only the acceptor is slides (NUNC) with detachable chambered upper structures. Before excited, with emission at v2. We then obtain the acceptor ratio performance of assays, the serum-containing medium was changed to a serum-free medium (Opti-MEM). The cultures were gently rinsed three 1,v⬘) / FA(v2,v⬙) times with the medium and preincubated with fluorophore-labeled FRET d⫹䡠[⑀D(v⬘) / ⑀A(v⬙) ⫹ ⑀A(v⬘) / ⑀A(v⬙)]}䡠[␾A(v1) / ␾A(v2)] M) for 6 h in the presence of Lipofectin (5 ␮g/ml).
After that time, the transfection mixture was replaced by normal se- is directly proportional to (ratio) and can be easily calculated rum-containing medium, and cells were grown for another 18 h. At- because ⑀D(v⬘)/⑀A(v⬙) and ⑀A(v⬘)/⑀A(v⬙) are measured from absorption tached, treated intact cells were maintained in a CO incubator at spectra, and ␾ (v )/␾ (v ) is unity when v ⫽ v .
37 °C. Two control assays were carried out using either untreated cells Analysis of Circular Dichroism—The circular dichroism spectra of or cells exposed to 0.25 ␮M fluorescein. After incubation, cells were MeO-␤3DE (1 ␮M), free or in the complex with the substrate (2 ␮M), was washed three times with phosphate-buffered saline, fixed with freshly measured in a solution of 10 mM Tris-HCl, pH 7.5, containing increasing prepared 3.5% paraformaldehyde for 15 min at room temperature, concentrations of MgCl at 21 °C. In control experiments, 0.1 washed three times with phosphate-buffered saline, mounted in 2.5% NaCl were used. Before measurement, the complex was allowed to form DABCO™ in glycerol, and processed for microscopy.
by heating the solution at 95 °C for 2.5 min followed by gradual cooling.
Fluorescence Spectroscopy—Fluorescence emission spectra were Measurements were made in a quartz cuvette (5-mm path length) with measured on an LS-50 spectrofluorometer (PerkinElmer Life Sciences), a CD spectrometer (model CD6, Jobin Yvon) from 200 to 320 nm in and spectra were corrected for lamp fluctuations and instrumental triplicate. The spectra were obtained by smoothing the averaged spec- variations. Polarization artifacts were avoided by setting excitation and tra with a calculator.
emission polarizers to magic angle conditions (54.74°). All of the fluo- Surface Plasmon Resonance—The kinetic parameters (association rescence measurements were performed at the temperature of 23 ⫾ and dissociation rate constants, k and k , respectively) and the af- 1 °C. Emission spectra, excitation spectra, and luminescence intensity finity constant (K ) between DNAzyme and the mRNA substrate were were recorded with 5-nm band passes for both the excitation and measured by surface plasmon resonance using a BIAcoreX (Amersham emission monochromators. A cut-off filter in the emission beam was Biosciences). Briefly, avidin was covalently attached to carboxymethyl used to eliminate second-order wavelength interference. The excita- dextran chips (CM5, BIAcore) previously activated with N-hydroxysuc- tion wavelengths used were 494 nm and 560 nm for fluorescein- and cinimide and N-ethyl-N⬘-dimethylaminopropyl carbodiimide, according rhodamine-conjugated constructs, respectively. Emission spectra to the manufacturer's instructions. Experiments were performed at were corrected for the blank contribution and for the instrument 37 °C using 50 mM Tris, pH 8.0, containing divalent cations at the response and normalized to the DNA concentration in a quartz cell indicated concentrations. 15 ␮l of 5⬘ end-biotinylated RNA at 100 nM in with a 1-cm path length. Excitation and emission spectra were auto- 50 mM Tris, pH 8.0, was injected at the flow rate of 5 ␮l/min and Structural Transitions Induced in the DNAzyme by Mg2⫹ consequently immobilized on the bound avidin to give a response of same as those recently reported for another 10 –23 DNAzyme ⬃500 resonance units, an arbitrary unit specific for the BIAcore instru- (21). Cellular transport of the Fluo-MeO-␤3DE ment. The levels of immobilized RNA were within the low levels that function of the external oligonucleotide concentration was non- have to be used to ensure that the observed binding rate will be limitedby the reaction kinetics rather than by the mass transport effects of the linear, being more efficient at concentrations below 2 ␮M. The injected DNAzyme (19). In typical experiments, DNAzyme flowed in punctate fluorescence distribution observed even after 24 h of two channels of the sensor; the first one contained the RNA substrate exposure to the DNAzyme seems to suggest that endosomal attached to avidin, and the second was without the RNA substrate. The vesicles are the primary targets of the probes under study (Fig.
latter was used to correct SPR traces and remove the background -Rhod could be detected intracellu- binding between DNAzyme and the immobilized avidin on the dextran.
larly, both when emission of either fluorescein or rhodamine The concentration of the injected DNAzyme was in the range 20 –200nM, and the flow rate was 5 ␮l/min. The amount of ligand bound to was measured and both fluorophores showed full colocaliza- immobilized RNA substrate was monitored by measuring the variation tion. Thus, the attached fluorophores did not influence the of the surface plasmon resonance angle as a function of time. Results enzymatic activity and biological properties of the DNAzyme, were expressed in resonance units. In preliminary experiments, the including the ability to interact with cellular components re- data obtained for at least three different concentrations of DNAzymes sponsible for its transport. Transfection of endothelial cells were fitted to several models; the best fits (␹2 ⫽ 1.4) were obtained byassuming a one-to-one interaction. Then, the association rate constant, with the DNAzyme (5 ␮M) efficiently reduced expression of ␤3 k , and dissociation rate constant, k , were determined separately integrin subunit measured at the level of ␤3 mRNA by reverse from individual association and dissociation phases, respectively. The transcriptase-PCR (Fig. 2C) and at the cellular surface by flow overall affinity constant, K , was derived from k /k . The sensor chip cytometry (not shown). Although MeO-␤3DE was regenerated with three 10-␮l pulses of 12.5% formamide.
same cellular distribution as MeO-␤3DE -Rhod, it did not show any biological activity detectable at the level of ␤3 mRNA or ␤3 expression at the cell surface. These data provide the Enzymatic Characteristics of the DNAzyme to 3 Integrin evidence that the 10 –23 DNAzyme has an intracellular cata- Subunit—The 10 –23 DNAzyme used in these studies was de- lytic or antisense activity, even at the much lower cation con- signed to cleave ␤3 integrin subunit mRNA, and preliminary centrations than those normally used in in vitro analysis.
characterization was done in our recent work (2). Standard conditions for kinetic measurements of the catalytic activity of Rhod were next used to measure energy transfer resulting from DNAzyme in vitro include 25 mM MgCl a dipolar coupling between the transition moments of the two conditions, the 10 –23 DNAzyme shows optimal enzymatic ac- fluorophores, fluorescein as the energy donor and rhodamine as tivity, whereas its mutant MeO-␤3DE with the shortened the energy acceptor. When the fluorescence spectrum of one catalytic loop is inactive (Fig. 1A). In this experiment, enzy- fluorophore (the donor) overlaps with the excitation spectrum matic reactions were performed in 50 mM Tris-HCl, pH 8.0, of another fluorophore (the acceptor), the excitation of the containing 0 –25 mM MgCl , under multiple turnover condi- donor induces fluorescence of the acceptor, although its own tions. The 32P-labeled mRNA substrate was mixed with the fluorescence decreases. The extent of FRET is extremely sen- DNAzyme ␤3DE in the molar ratio of 80:1 and incubated at sitive to the distance between the donor and the acceptor, being 37 °C; aliquots were withdrawn after 10 min. The cleavage inversely proportional to the sixth power of the distance. At- reaction was stopped by the addition of 5 ␮l of 0.5 M EDTA, and tachment of rhodamine to Fluo-MeO-␤3DE products were separated by electrophoresis in 20% polyacryl- decrease in the fluorescence emission at 520 nm characteristic amide gels under denaturing conditions. Amounts of the prod- for fluorescein, and the fluorescence spectrum of the resulting uct were evaluated by use of a PhosphorImager. Fig. 1B showsa composition of cleavage mixtures obtained after a 60-min -Rhod, had two peaks at 520 nm incubation of 32P-labeled mRNA substrates with 0.025 ␮ and 580 nm upon excitation at 494 nm (Fig. 3). The mutated ␤3DE added at a molar ratio ranging from 5:1 to 80:1. Each of ␤3DE, Fluo-MeO-␤3DE -Rhod, showed the same fluores- the DNAzymes, unmodified (␤3DE) and modified (MeO-␤3DE), cence properties. These spectra clearly indicate that consider- cleaved the substrate at the predicted site. Interestingly, able energy from the excited fluorescein was transferred to DNAzyme with the 2⬘-MeO residues showed a significantly rhodamine, providing the evidence that both fluorophores are higher enzymatic activity than ␤3DE, as evidenced by the in close proximity. Cleavage of the Fluo-MeO-␤3DE catalytic efficiency k of 3.62 ⫻ 106 and 1.09 ⫻ 106 ⫺1 with endonuclease from Serratia marcescens resulted in a sig- nificant increase of the fluorescence intensity at 520 nm, which Study of Ion-induced Folding of the DNAzyme by Fluores- approaches the level of fluorescein alone, indicating that both cence Resonance Energy Transfer—To follow the folding tran- fluorophores are separated to the distance enabling the energy sitions of the DNAzyme induced upon binding of Mg2⫹ ions, FRET was utilized. For this purpose, ␤3DE with the 15-mer Variation in End-to-end Distances during the Ion-induced and 11-mer catalytic loops was modified by attachment of the Folding of the DNAzyme—According to the model for the fold- donor and acceptor fluorophores, rhodamine and fluorescein, to ing of the DNAzyme, the length of oligodeoxynucleotide should the 5⬘ and 3⬘ ends of the flanking arms, respectively. Next, a shorten over the full range of Mg2⫹ concentration. Experimen- series of experiments were designed to analyze whether the tally, we find that E increases rapidly upon the addition of fluorophores attached to the terminal bases affect the ability of cations to Fluo-MeO-␤3DE -Rhod and reaches a plateau the oligodeoxynucleotide construct to function as an active value by 5 mM Mg2⫹. Assuming a Forster critical distance (R ) DNAzyme species. As seen in Fig. 2A, incubation of the Fluo- of 5.5 nm for donor-fluorescein (22), the distance between donor -Rhod with the 32P-labeled substrate in the pres- (fluorescein) and acceptor (rhodamine) in the absence of Mg2⫹ ence of 25 mM Mg2⫹ under multiple-turnover conditions at was calculated to be 7.82 ⫹ 0.39 nm and was not significantly 37 °C leads to cleavage at the correct site. When such a con- dependent upon the DNAzyme concentration. The distance struct was incubated with endothelial cells, it remained resist- between two fluorophores shortens over this range from 7.82 ant to intracellular nucleases and, even after 24 h, was located nm to 6.22 nm and further addition of Mg2⫹ ions does not exclusively within the cytoplasm, particularly in the perinu- essentially change it (Fig. 4A). At 25 mM Mg2⫹, the R value clear organelles. The cellular uptake, intracellular distribu- reaches 6.11 nm and is almost identical to that characteristic tion, and stability of the Fluo-MeO-␤3DE for the Fluo-MeO-␤3DE-Rhod in the complex with its mRNA

Structural Transitions Induced in the DNAzyme by Mg2⫹ FIG. 4. FRET analysis for the MeO-3DE as a function of Mg2concentration. A, calculated interfluorophore distances based on
measured efficiency of energy transfer presented as a function of MgCl concentration. The plot shows the variation in FRET efficiency as a function of Mg2⫹ concentration up to 25 mM when Fluo-MeO-␤3DE -Rhod (–) or Fluo-MeO-␤3DE -Rhod (f–f) were tested. In accordance with the model, the FRET efficiency is found to increase (indicating a reducing end-to-end distance) over this complete range. B, scheme with theexpected behavior of the free DNAzyme or complexed with its RNA substrate in the presence of Mg2⫹. The distances (R) were calculated based onFRET analysis.
substrate. Essentially the same changes in the interfluoro- bozyme by means of high affinity binding to sites (Fig. 4B).
phore distance were induced in the Fluo-MeO-␤3DE-Rhod To evaluate overall changes induced in the DNAzyme by upon binding of other divalent cations, such as Ca2⫹ and Mn2⫹ cations, the fluorescence anisotropy of both fluorophores in (Table I). Such folding of the DNAzyme does not result simply Fluo-MeO-␤3DE-Rhod was measured in the presence of in- from the neutralization of the polyanionic nature of the oligode- creasing concentrations of Mg2⫹, Ca2⫹, Li⫹, Na⫹, and K⫹. The oxynucleotide, because neither Na⫹ nor K⫹ added in place of fluorescence anisotropy r reflects the local and global motions Mg2⫹, even at 1 M, showed any effect. In the case of DNAzyme of the fluorophore and is close to zero for a freely rotating with the 11-mer loop, Fluo-MeO-␤3DE fluorophore. The theoretical upper limit of 0.4 corresponds to a matically increased upon the addition of 0.5 mM Mg2⫹, indicat- totally non-rotating fluorophore (23). The fluorescein- and rho- ing that these cations induce folding of the mutated deoxyri- damine-labeled DNAzyme has a flexible single-stranded mole-

Structural Transitions Induced in the DNAzyme by Mg2⫹ cule characterized by a low r value ⫽ 0.016 and 0.037, as or Ca2⫹ concentration reached 5 mM or 1 mM, respectively, measured for fluorescein and rhodamine, respectively (Fig. 5).
indicating the increased condensation state of the molecule.
In both cases, the fluorescence anisotropy doubles when Mg2⫹ However, there was no change in the fluorescence anisotropywhen monovalent cations were used even at much higher con- centrations (Fig. 5). These results are fully consistent with the The estimated interfluorophore distance based on proposed mechanism involving the divalent cation-induced FRET analysis of the Fluo-MeO-3 DE-Rhod folding of the DNAzyme and indicate that its molecule becomes The fluorophore-labeled MeO-␤3DE (40 nM) was incubated with dif- more compact upon Mg2⫹ binding.
ferent divalent cations used in the concentration range from 0 to 25 mM,and the interfluorophore distance R was evaluated based upon the Complex Formation between DNAzyme and its RNA Sub- Forster equation. Data represent a mean value of three separate deter- strate—The conformational changes of the DNAzyme induced by Mg2⫹ were next analyzed by CD spectroscopy (Fig. 6). CD Interfluorophore distance [nM] spectra of an ion-free DNAzyme/RNA complex were reported earlier (24). We have been interested in examining how far CDspectroscopy (being sensitive to the structure helicity and fold) could be applicable to probing binding of metal ions to single- and double-stranded species of interest. As the reference, CD spectra related to a regular complex formation (Fig. 6A), re- flecting an antisense mechanism, were inspected first. The CD FIG. 5. Effect of Mg2on the fluores-
cence anisotropy of Fluo-MeO-3DE-
MgCl (–), CaCl (E–E), KCl
(Œ–Œ), and NaCl (‚–‚) were added step-wise to 0.5 ␮M Fluo-MeO-␤3DE and incubated for 5 min before measuringthe fluorescence anisotropy. To monitorthe fluorescence anisotropy of fluorescein(A) or rhodamine (B), the samples wereexcited at 494 nm or 560 nm and fluores-cence emission was measured at 520 nmor 580 nm, respectively.

Structural Transitions Induced in the DNAzyme by Mg2⫹ FIG. 6. Effect of Mg2on CD spectra
of the free MeO-3DE and its com-
plex with the target RNA.
CD spectra
MeO-␤3(1245–1265)/RNA complex (A),the single stranded MeO-␤3DE (B), andthe MeO-␤3DE/RNA complex (C) weretaken in the presence of Mg2⫹ concentra-tions ranging from 0 to 25 mM. However,because there was no change when con-centrations higher than 5 mM Mg2⫹ wereused, those spectra were deleted for clar-ity of the plot. Spectra of the single-stranded antisense oligodeoxynucleotideMeO-␤3(1245–1265) and MeO-␤3DE areshown as a gray line in A and C, respec-tively. Spectra were taken at the follow-ing Mg2⫹ concentrations: 0 mM (—), 0.5mM (----), 1.0 mM (䡠 䡠 䡠 䡠), 2.0 mM (-䡠 -䡠 -䡠 -䡠),and 5.0 mM (-䡠 䡠 -䡠 䡠 -䡠).
spectrum of a non-enzymatic, single 21-mer DNA strand is strand of MeO-␤3DE, most probably close to that predicted by irregular and of low magnitude. Addition of a complementary the DNA-folding algorithm (26). An addition of Mg2⫹ at the RNA strand resulted in raising a regular positive Cotton effect initial concentration level (0.5 mM) led to a formation of a more at 269 nm, i.e. a region typical of the DNA/RNA hybrids (25).
regular and higher amplitude positive band at 276 nm. Further Influence of an increased Mg2⫹ concentration on a double hel- Mg2⫹ additions (up to 25 mM) had no practical effect on the ical structure is not strong but clearly visible, as indicated by DNAzyme strand spectra. The binding of the Mg2⫹-free MeO- the increase in amplitude of the Cotton effect. An initial addi- ␤3DE strand to the target RNA leads to the formation of a new tion of Mg2⫹ (0.5 mM) resulted in both a higher Cotton effect type of spectrum with a positive Cotton band at 270 nm of than that produced by 1 mM Mg2⫹ and the formation of two higher amplitude than that of the DNAzyme strand (Fig. 6C).
characteristic, discrete peaks (265 nm and 269 nm). At concen- Both the lack of the symmetry for this band and the appearance trations higher than 1 mM Mg2⫹, the height of these peaks is of the weaker negative effect at 244 nm and positive effect at reversed and kept practically unchanged, even at higher Mg2⫹ 223 nm are characteristic for the spectrum. Upon addition of an concentrations of up to 25 mM. The positive CD band of the initial amount of Mg2⫹ (0.5 mM), the amplitude of the positive Mg2⫹-free DNAzyme, although rather broad (276 –278 nm), is Cotton effect rises substantially, and the peak is shifted down much more regular (Fig. 6B) than that of the 21-mer oligode- to 268 nm. No further increase of the Cotton effect was ob- oxynucleotide. The spectrum also contains a negative effect at served above 1 mM Mg2⫹. The results presented above indicate 249 nm and a weak, positive effect at 221 nm. This result that, upon binding of Mg2⫹, the global geometry of the indicates that some secondary structure exists for the 35-mer DNAzyme adopts a compact structure projecting flanking arms Structural Transitions Induced in the DNAzyme by Mg2Kinetic parameters for binding of 3DE or antisense oligodeoxynucleotide 3(1245-1265) to immobilized RNA substrate and their dependence upon Mg2concentration The 3⬘ end biotinylated RNA substrate (GUUCCACUCGUUAUCUUC) was immobilized on a BIAcore™ CM5 sensor chip coated with avidin (⌬RU ⫽ 5000). The analyte was injected at a flow of 5 ␮l/min, and measurements were done at 37 °C using 50 mM Tris, pH 8.0, containing divalentcations at the indicated concentrations. MeO-␤3DE used in these experiments was inactive due to a single base substitution (G6 3 A). Theinteraction of the corresponding oligodeoxynucleotide ␤3(1245-1265) containing both flanking arms of the ␤3DE with the same RNA substrate wasanalyzed under the same conditions. This oligodeoxynucleotide was modified as in MeO-␤3DE. The k were determined from the association and dissociation phases, respectively, with four different concentrations of the DNAzyme and the oligodeoxynucleotide. Apparent KAcorresponds to k /k ratio. Data are shown as a mean value of four separate analyses.
kon (1/Ms) koff (1/s) (8.23 ⫾ 0.53) ⫻ 104 (1.98 ⫾ 0.94) ⫻ 10⫺3 (2.25 ⫾ 0.43) ⫻ 107 (8.47 ⫾ 0.77) ⫻ 104 (2.99 ⫾ 1.05) ⫻ 10⫺3 (3.16 ⫾ 1.23) ⫻ 107 (1.23 ⫾ 0.28) ⫻ 105 (2.84 ⫾ 1.53) ⫻ 10⫺3 (6.91 ⫾ 2.60) ⫻ 107 1.20 ⫾ 0.11) ⫻ 105 3.22 ⫾ 0.94) ⫻ 10⫺3 2.74 ⫾ 0.84) ⫻ 107 9.51 ⫾ 0.15) ⫻ 104 3.09 ⫾ 0.86) ⫻ 10⫺3 3.05 ⫾ 1.01) ⫻ 107 8.55 ⫾ 0.79) ⫻ 104 2.84 ⫾ 0.94) ⫻ 10⫺3 5.71 ⫾ 2.71) ⫻ 107 (3.44 ⫾ 1.04) ⫻ 104 (1.41 ⫾ 0.49) ⫻ 10⫺4 (1.64 ⫾ 0.29) ⫻ 108 (6.04 ⫾ 2.10) ⫻ 104 (3.76 ⫾ 0.53) ⫻ 10⫺4 (1.78 ⫾ 0.88) ⫻ 108 (8.49 ⫾ 2.15) ⫻ 104 (5.90 ⫾ 0.18) ⫻ 10⫺4 (1.46 ⫾ 0.40) ⫻ 108 5.22 ⫾ 1.46) ⫻ 104 8.40 ⫾ 1.88) ⫻ 10⫺4 1.19 ⫾ 0.84) ⫻ 108 7.84 ⫾ 1.48) ⫻ 104 6.11 ⫾ 1.02) ⫻ 10⫺4 1.37 ⫾ 0.52) ⫻ 108 9.41 ⫾ 2.94) ⫻ 104 7.21 ⫾ 1.50) ⫻ 10⫺4 1.48 ⫾ 0.83) ⫻ 108 at right position and angles to bind substrate mRNA.
the divalent cation dependence and kinetic parameters of RNA To further test this concept, the binding kinetics were di- cleavage. Exhaustive studies on chimeric DNAzymes and sub- rectly measured by surface plasmon resonance analysis in the strates composed of DNA and RNA showed that both types of presence of increasing concentrations of Mg2⫹. In these exper- enzymes have a very similar catalytic mechanism (5). The iments, biotinylated RNA substrate was attached to the avidin- reactions have an identical dependence on pH, both demon- coated sensor. To avoid cleavage of the RNA substrate, the strate an inverse correlation between the pK of metal hydrates inactive DNAzyme containing a single nucleotide substitution and activity and solvent isotope effects, and thio effects on the in the catalytic domain of ␤3DE was used. The antisense oli- reactions are identical (24). The crystal structure of several godeoxynucleotide ␤3(1245–1265) consisting of both flanking unmodified and modified hammerhead RNA in the absence of arms (and thus antisense to ␤3 integrin subunit mRNA) was divalent metal ions has been solved (28, 30 –32). Cation binding tested as a control. To clarify the effect of cations on association sites and the mechanism by which they control enzymatic and dissociation processes between the DNAzyme and its RNA activity have been elucidated (30, 33). Five Mg2⫹ sites are seen substrate, we determined the parameters of k , k , and K in in the crystal structure of the Mg2⫹-soaked freeze-trapped con- the binding reactions between RNA substrate and either formational intermediate of the hammerhead ribozyme, which DNAzyme or the antisense oligodeoxynucleotide (Table II). The could be divided into two groups based on their roles in cata- binding affinity of ␤3 DNAzyme to the RNA substrate was lytic activity. The first group consists of Mg2⫹ sites that upon dependent upon the concentration of Mg2⫹. The association binding of cations induce folding of the enzyme into its active constant determined in the presence of 15 mM Mg2⫹ was sig- conformation. The second group includes sites occupied by nificantly higher (p ⬍ 0.001) than that observed at 5 mM of Mg2⫹ bound directly to the optically active oxygen showing R Mg2⫹. However, it was still much lower than the K describing diastereomeric form (pro-R) at the cleavage site. The hammer- the interaction of the antisense oligodeoxynucleotide with the head ribozyme can cleave its own RNA, and this activity re- same RNA fragment. Interestingly, the binding affinity of the quires one or more catalytic divalent metal ions, one of which antisense oligodeoxynucleotide to the RNA substrate did not ionizes the 2⬘-hydroxyl at the cleavage site. The newly gener- depend upon cation concentration, and regardless of the Mg2⫹ ated nucleophile attacks the adjacent phosphate by an in-line presence, it was almost an order of magnitude higher than that mechanism. The same metal ion, or perhaps another, stabilizes of the DNAzyme.
the pentacoordinated phosphate transition state by binding directly to the pro-R phosphate oxygen. The reaction generates The intracellular ability of various 10 –23 DNAzymes to in- 5⬘-hydroxyl and 2⬘,3⬘-cyclic phosphate termini at the cleavage hibit expression of the targeted proteins was evidenced by several in vitro and in vivo studies (27), indicating their poten- The structural effect of Mg2⫹ is well established in the ham- tial advantages as biocatalysts in oligonucleotide therapy. De- merhead ribozyme (35). In the absence of divalent metal ions, spite tremendous therapeutic potential, the ability of the the hammerhead structure is extended, with a disordered core, DNAzyme to influence biological processes has not been deter- but upon addition of metal ions, folding occurs in two distinct mined at the molecular level. Because of its high flexibility, the steps. Both events are well described by two-state transitions three-dimensional structure of the DNAzyme molecule is not induced by the non-cooperative binding of Mg2⫹. One can as- yet known. Therefore, even a basic knowledge about the mech- sume that similarly to the hammerhead ribozyme, metal ions anism by which Mg2⫹ and other divalent cations regulate en- will induce the folding of the DNAzyme molecule into the zymatic activity of the 10 –23 DNAzyme is at present specula- geometry required to facilitate the pathway into the transition tive. The hypothetical mechanism for catalysis of RNA cleavage state and will also bind at a specific location(s), where they can by the DNAzyme is essentially based on assumptions that it participate directly in the chemistry of the cleavage reaction.
behaves similarly to the hammerhead ribozyme, which also is Data presented in this report show that binding of Mg2⫹ to active in the presence of various divalent metal cations (28).
the 10 –23 DNAzyme induces significant rearrangement of the Despite different compositions, the 10 –23 DNAzyme and the catalytic loop, which leads to optimal folding of the molecule.
hammerhead ribozyme show many common features, including This folding may occur in several distinct stages. The first Structural Transitions Induced in the DNAzyme by Mg2⫹ transition induced by 0.5 mM Mg2⫹ results in the formation of ribozyme (29, 30, 38). Interestingly, the binding affinity of the a compact structure of the DNAzyme. The DNAzyme in such a DNAzyme to RNA increased linearly in the presence of divalent state binds weakly to its RNA substrate and lacks catalytic cations when they were used in the range from 0 to 15 mM, but activity. In the next stage observed at concentrations up to 5 it was still much lower than that of the antisense oligode- mM Mg2⫹, the flanking arms are projected into the proper oxynucleotide consisting of the DNAzyme flanking arms. (v) position to bind the RNA substrate. Under such conditions, the Essentially the same effect on the folding, binding affinity to DNAzyme binds efficiently to the substrate and shows substan- RNA substrate and catalytic activity of the DNAzyme were tial catalytic activity. Further increase of Mg2⫹ leads to the found when other divalent cations such as Ca2⫹ and Mn2⫹ were final transition, involving formation of the completely orga- used in the same concentration range. Monovalent cations such nized catalytic domain of the DNAzyme. Such a mechanism is as Na⫹, K⫹, and Li⫹ added in place of Mg2⫹ even at much supported by the following observations: (i) E higher concentrations of up to 1 M did not show any effect.
-Rhod rapidly increases in the range from 0 to 5 Acknowledgments—We thank Dr. A. Okruszek for synthesis of oli- M cations and reaches a plateau value by 5 mM Mg2⫹, indi- cating at this concentration the shortest distance between the energy donor and acceptor. In the absence of Mg2⫹, theDNAzyme is inactive and its catalytic core is essentially un- folded. With the addition of 5 mM Mg2⫹, the orientation of the 1. Santoro, S. W., and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94,
catalytic loop changes, and the distance between 5⬘ and 3⬘ ends almost reaches the value characteristic for the DNAzyme in 2. Cieslak, M., Niewiarowska, J., Nawrot, M., Koziolkiewicz, M., Stec, W. J., and Cierniewski, C. S. (2002) J. Biol. Chem. 277, 6779 – 6787
complex with its RNA substrate. Under these conditions, the 3. Liu, C., Cheng, R., Sun, L. Q., and Tien, P. (2001) Biochem. Biophys. Res. DNAzyme shows substantial enzymatic activity. A significant Commun. 284, 1077–1082
4. Zhang, L., Gasper, W. J., Stass, S. A., Ioffe, O. B., Davis, M. A., and Mixson, increase in the catalytic activity of the DNAzyme is observed A. J. (2002) Cancer Res. 62, 5463–5469
when the Mg2⫹ concentration is increased from 5 to 15 mM, 5. He, Q. C., Zhou, J. M., Zhou, D. M., Nakamatsu, Y., Baba, T., and Taira, K.
suggesting that additional structural alterations within the (2002) Biomacromolecules 3, 69 – 83
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catalytic loop have occurred, even though there was no further 7. Chiu, T. K., and Dickerson, R. E. (2000) J. Mol. Biol. 25, 915–945
change in E . The hyperbolic concentration dependence of 8. Welche, J. B., Duckett, D. R., and Lilley, D. M. (1993) Nucleic Acids Res. 21,
the end-to-end distance with a midpoint of ⬃2 mM Mg2⫹, which 9. Soyfer, V. N., and Potaman, V. N. (1966) Triple-helical Nucleic Acids, p. 360, is significantly lower than that characteristic for the chemical Springer, New York cleavage step (Fig. 2), indicates that structural changes in- 10. Brukner, I., Susic, S., Dlakic, M., Savic, A., and Pongor, S. (1994) J. Mol. Biol. 11, 26 –32
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nm) and further structural stabilization upon binding to the 26. Zuker, M. (2003) Nucleic Acids Res. 31, 1–10
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29. Pley, H. W., Flaherty, K. M., and McKay, D. B. (1994) Nature, 372, 68 –74
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32. Ruffner, D. E., Stormo, G. D., and Uhlenbeck, O. C. (1990) Biochemistry 29,
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A Provider's Guide for the Care of Women with Physical Disabilities and Chronic Suzanne C. Smeltzer, RN, EdD, FAAN Professor & Director, Nursing Research Director, Health Promotion for Women with Disabilities Project Villanova University College of Nursing 800 Lancaster Avenue Villanova, PA 19085 Phone: 610-519-6828 Fax: 610-519-7650 Nancy C. Sharts-Hopko, RN, PhD, FAAN