G-Quadruplex-Forming Oligonucleotide Conjugated to MagneticNanoparticles: Synthesis, Characterization, and Enzymatic StabilityAssaysDomenica Musumeci,*,†,‡ Giorgia Oliviero,† Giovanni N. Roviello,‡ Enrico M. Bucci,‡
and Gennaro Piccialli†
†Dipartimento di Chimica delle Sostanze Naturali, Universita di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy‡Istituto di Biostrutture e Bioimmagini − CNR, via Mezzocannone 16, I-80134 Napoli, Italy
*S Supporting Information
ABSTRACT: In the present work, we report the conjugation of super-paramagnetic nanoparticles to a fluorescently labeled oligodeoxyribonucleo-tide (ODN) able to fold into stable unimolecular guanine quadruple helixunder proper ion conditions by means of its thrombin-binding aptamer (TBA)sequence. The novel modified ODN, which contained a fluorescent dUPy unitat 3′-end and a 12-amino-dodecyl spacer (C12−NH2) at 5′ terminus, wascharacterized by ESI-MS and optical spectroscopy (UV, CD, fluorescence),and analyzed by RP-HPLC chromatography and electrophoresis. From CDand fluorescence experiments, we verified that dUPy and C12−NH2incorporation does not interfere with the conformational stability of the G-quadruplex. Subsequently, the conjugation of the pyrene-labeled ODN withthe magnetite particles was performed, and the ODN-conjugated nano-particles were studied through optical spectroscopy (UV, CD, fluorescence)and by enzymatic and chemical assays. We found that the nanoparticles enhanced the stability of the TBA ODN to enzymaticdegradation. Finally, we evaluated the amount of the TBA-conjugated nanoparticles immobilized on a magnetic separator in viewof the potential use of the nanosystem for the magnetic capture of thrombin from complex mixtures.
■ INTRODUCTIONDue to their promising properties, nanoscaled iron oxideparticles covalently linked to drugs, proteins, enzymes,antibodies, or nucleic acids have been widely investigated fortheir great potential in biomedical applications1−4 such asmagnetic bioseparation,5,6 drug delivery,7−10 MRI,11−14 mag-netofection7,15−21 and cancer treatment by hyperthermia.22−25
Generally, most of these applications require that thesenanoparticles have high magnetization values and sizes smallerthan 100 nm, with an overall narrow particle size distributionsuitable to ensuring uniform physical and chemical properties.In this regard, iron oxide nanoparticles with magnetic corediameter <100 nm exhibit superparamagnetic phenomena, witheach particle acting as a single magnetic domain and notretaining any residual magnetism upon removal of the magneticfield.26 In addition, biomedical applications require a suitablecoating of the magnetic particles, which has to bebiocompatible, providing colloidal stability and allowing thenanoparticle transport and interaction with biological tis-sues.1,2,4,26 Polysaccharides such as dextran have been usedfor this purpose due to their biocompatibility, low toxicity, andversatility for nanoparticle suspension in cell culture media.Dextran enhances blood circulation time and stabilizes thecolloidal solution. Homogeneous suspensions of magneticnanoparticles with proper surface coating into a suitable
solvent are called ferrofluids. This type of suspension caninteract with an external magnetic field and be positioned to aspecific area. For example, magnetic nanoparticles conjugatedto anti-inflammatory drugs could be delivered to the exact areaof inflammation reducing drug dosages and potential sideeffects to healthy tissues, and increasing the rapidity of action.Particles ranging from about 10 to 100 nm are optimal forintravenous injection and are characterized by prolonged bloodcirculation times. The particles in this size range are smallenough to evade reticuloendothelial system (RES) of the bodyas well as penetrate the very small capillaries within the bodytissues and, therefore, may offer the most effective distributionin certain tissues.In the present work, we report the conjugation of 50 nm
superparamagnetic nanoparticles to a fluorescently labeled G-rich oligodeoxyribonucleotide (ODN), able to fold intoquadruple helix under proper ion conditions. The selectedparticles have a single-domain magnetite core (Fe3O4) of 20nm diameter, thus possessing a superparamagnetic magnet-ization, and a carboxymethyl dextran-biocompatible coating of15 nm thickness. A pyrene fluorescent group was attached to
Received: June 12, 2011Revised: December 6, 2011Published: January 12, 2012
the 3′-end of the ODN in order to follow the ODN fate whenanchored to the nanoparticles.27,28 As ODN, we selected thethrombin-binding aptamer (TBA) sequence, which exists asunstructured in buffers like TRIS, but folds into a stableunimolecular guanine quadruple helix in the presence of ionssuch as K+ and of the thrombin protein, recognized withnanomolar affinity.29−33 Thus, this is clearly the reason for awide number of scientific studies on the TBA sequence34−42 inview of its possible use as a highly potent inhibitor of thrombinclotting activity43−46 or as a probe for detection of K+
concentrations in living organisms.47,48 However, to the bestof our knowledge, this is the first report in the literature on theconjugation of this particular sequence to magnetite nano-particles. The potential in biomedical applications of thisconjugate, as magnetic biosensor or therapeutic agent, can beunderstood considering the combination of the attractiveproperties of both the TBA sequence and the magneticnanoparticles. Indeed, since each nanoparticle carries more thanone ODN, this system could be very useful to obtain high localconcentrations of both the aptamer and the magneticnanoparticles, especially when high doses of the therapeuticare needed to accumulate at the target site.The novel TBA−nanoparticle conjugate was studied through
optical spectroscopy (UV, CD, fluorescence), and its resistanceto enzymatic degradation was evaluated in 76% murine plasma.Furthermore, we evaluated the amount of the TBA-
conjugated nanoparticles immobilized on a steel matrix-filledcolumn placed in a strong permanent magnet estimating in thisway the potential capture capacity of thrombin by thisnanosystem, in view of its use for the magnetic capture ofthrombin from complex mixtures.
■ EXPERIMENTAL SECTION
Materials, Apparatus, and General Methods. CPGUniversal Support 500 (49 μmol/g loading), 5′-dimethoxytrityl-N-acyl-2′-deoxyribonucleoside, 3′-[(2-cyanoethyl)-(N,N-diiso-propyl)]-phosphoramidites (dN-CE Phosphoramidites), 5′-dimethoxytrityl-5-(pyren-1-yl-ethynyl)-2′-deoxyUridine, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Pyrene-dU-CE Phosphoramidite), 12-(4-monomethoxytritylamino)-dodecyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphorami-dite (5′-Amino-Modifier C12), and standard DNA-synthesisreagents (Amidite diluent, Activator solution, Cap A, Cap B,Acetonitrile wash B, Oxidizer) were purchased from Primm(Milan, Italy). Acetonitrile external wash (wash A) was fromBiosolve (<10 ppm water). The ferrofluid fluidMAG-CMX waspurchased from Chemicell GmbH (Berlin, Germany) andconsisted of an aqueous dispersion (25 mg/mL, 1.3 × 1013
particles per mg) of 50-nm-diameter magnetite nanoparticles(80%, w/w; single-domain 20 nm diameter magnetite core; 40kDa dextran coating of 15 nm thickness) with a COOH surface.All other reagents and solvents were from Sigma-Aldrich (EDC,NHS, MES-buffer, CH3CN for HPLC, Acrylamide/BisAcrylamide 19:1 - 40%, TRIS, Boric acid, EDTA, TEMED,APS, Glycerol, Bromophenol blue-Xylenecyanol solid mixture[gel tracking dyes], KCl, MgCl2, CaCl2, Urea, Formamide,Ethidium bromide). DNase I from bovine pancreas (1 mg/mL)was kindly provided by Dr. Sonia Di Gaetano, IBB-CNR,Napoli (Italy).DNA solid phase synthesis was performed on an ABI
Expedite 8909 oligosynthesizer. HPLC chromatographicanalyses and purifications were performed using a Jasco PU-
2089 Plus gradient pump, equipped with the Jasco UV-2075Plus UV/vis detector.Desalting of purified oligomers was performed on NAP-25
columns (GE Healthcare). Samples were lyophilized in a Alpha1−4 LD Freeze-Dryer (Martin Christ) for 16 h.UV measurements were obtained on a JASCO V-550 UV/vis
spectrophotometer equipped with a Peltier block by using 1 cmquartz cells of both 0.5 and 3 mL internal volume (Hellma).Oligomer quantification was achieved measuring the
absorbance (λ = 260 nm) at 80 °C, using the molar extinctioncoefficients calculated for the unstacked oligonucleotides. Themolar extinction coefficients used for the calculations were asfollows: A, 15.06; T, 8.56; G, 12.18; C, 7.10; U, 9.66 m−1
M−1.49 Annealing of all the duplexes was performed bydissolving equimolar amounts of the two complementarystrands in Milli-Q water, heating the solution at 85 °C (5 min)and then allowing to slowly cool to room temperature.LC/ESI-MS analyses were performed on a MSQ mass
spectrometer (ThermoElectron, Milan, Italy) equipped with anESI source operating at 3 kV needle voltage (T = 320 °C), andwith a complete Surveyor HPLC system, comprising a MSpump, an autosampler, and a PDA detector. Elution wasperformed on a Phenomenex Jupiter C18 300 Å (5 μm, 4.6 ×150 mm) column, monitoring at 260 nm, by building up agradient starting with H2O (0.03% TFA) and applyingincreasing amounts of CH3CN (0.03% TFA), with a flowrate of 0.8 mL/min.All CD spectra were collected on a Jasco J715 spectropo-
larimeter equipped with a Peltier element for temperaturecontrol, using a Teflon stoppered 1 cm quartz cuvette of 3 mLinternal volume (Hellma), and a tandem cell (2 × 4.375 mm,Suprasil quartz, Hellma). CD parameters were the following:spectral window 200−320 nm, data pitch 1 nm, bandwidth 2nm, response 4 s, scanning speed 50 nm/min, 3 accumulations.The CD binding studies in the tandem cell were obtained byrecording the “sum” spectrum (buffered solution of 1 or 2 inone chamber and buffered solution of 3 in the other) and the“complex” spectrum (after mixing of the two solutions) at 15°C.UV melting curves were recorded by following the
absorbance changes at a determined wavelength as thetemperature was increased (melting parameters: heating rate0.5 °C/min, monitoring wavelength 29550 or 260 nm,bandwidth 2 nm, response 16 s). Tm values were calculatedas the maxima of the plots of the first derivative of theAbsorbance versus temperature (error values, ±0.5 °C).Fluorescence spectra were obtained on a Varian Cary Eclipse
fluorimeter (Varian, Oxford, UK). We registered emissionfluorescence spectra exciting at 335 nm (unless otherwisespecified) and maintaining the excitation and emission slits at 5nm.Electrophoretic analysis was performed on a Scie-Plas mini-
gel unit (Standard TV100, twin-plate 10 × 10 cm2) equippedwith a APELEX PS 503/500 V Power Supply (Eppendorf srl,Italy). Gels were exposed to UV light (254 nm) after ethidiumbromide staining and photographed by digital camera (NikonCoolpix S5).
ODN Synthesis, Purification, and Characterization.The ODN sequences were the following: NH2−C12-G G T T G G T G T G G T T G G - d U P y − O H ( 1 ) ,GGTTGGTGTGGTTGG (2), and CCAACCACACCAACC(3). DNA solid phase synthesis was performed on a CPGUniversal Support (35 mg, 1.4 μmol) using 1 μmol scale
standard protocol, with the DMT-OFF option for oligomers 2and 3, and the DMT-ON one for the pyrene-labeled oligomer1. Coupling efficiency during the automated synthesis wasestimated spectrophotometrically by the DMT cation releasedduring the detritylation steps. Double coupling and 6 mincoupling time were employed for the Pyrene-dU-CE and 5′-amino-modifier C12 phosphoramidites.The oligos were deprotected and cleaved from the solid
support with 1.5 mL 30% ammonium hydroxide: for 16 h at 55°C for oligomer 2 and 3, and for 1 h at room temperaturefollowed by 17 h at 40 °C for oligomer 1. After evaporation ofthe solvent, the resulting crude mixtures of 1−3 werechromatographed on C18 analytical column (Thermo Hyper-sil-100, 4.6 × 250 mm, 5 μm) using a linear gradient of CH3CNin 0.1 M TEAB solution (flow rate 1 mL min−1). UV detectionwas achieved at 260 nm. 1: 8% (2 min) to 40% CH3CN in 0.1M TEAB over 30 min, tR = 16.3 min (23.2% CH3CN). 2: 8% to20% CH3CN in 0.1 M TEAB over 25 min, tR = 13.4 min(14.4% CH3CN). 3: 8% (2 min) to 28% CH3CN in 0.1 MTEAB over 26 min, tR = 13.1 min (16.5% CH3CN).Purified oligonucleotides 1−3 were lyophilized, and then
ODN 1 was treated with 2 mL acetic acid/water (80:20) atroom temperature for 2 h in order to remove the MMT group,followed by evaporation of the solvents under vacuum.Subsequently, all the oligomers were dissolved in 2.5 mLH2O and desalted using prepacked NAP-25 columns. Desaltedoligos were lyophilized, dissolved in a known amount of Milli-Qwater and quantified by UV measurements. The epsilon valuesused for the quantification of the oligos are as follows: 1(G9T6U) ε260 = 170.64 m−1 M−1; 2 (G9T6) ε260 = 160.98 m−1
M−1; 3 (C9A6) ε260 = 154.26 m−1 M−1. UV quantification of theoligos provided the following values: 1 = 252 nmol (18%yield); 2 = 448 nmol (32% yield); 3 = 490 nmol (35% yield).For LC/ESI-MS characterization, the ODN 1−3 stock
solutions (200 μM) were diluted to 20 μM in an aqueoussolution of 1% CH3COOH, and 50 μL aliquots of each samplewere injected into the LC/MS system by the autosampler andacquired in positive ion mode. ESI-MS data were the following:1 = m/z 1374.3 (found), 1376.9 (expected for [M+4H]4+),1100.6 (found), 1101.7 (expected for [M+5H]5+); 2 = m/z1577.0 (found), 1576.4 (expected for [M+3H]3+), 1181.1(found), 1182.5 (expected for [M+4H]4+), 944.9 (found),946.2 (expected for [M+5H]5+); 3 = m/z 1105.2 (found),1106.0 (expected for [M+4H]4+), 882.9 (found), 885.0(expected for [M+5H]5+).Conjugation to Nanoparticles. An aqueous suspension of
fluidMAG-CMX (40 mg in 1.6 mL) was dialyzed two times(for 15 and 7 h) against MES buffer (0.5 M, pH = 6.3) in a 5mL Float-A-Lyzer G2 column (Spectrumlab.com) with amolecular weight cutoff of 100 kDa. Subsequently, a 0.52 Msolution (0.4 mL) of EDC (40 mg) and a 100 μM solution (1mL) of ODN 1, both in 0.5 M MES buffer, were added to thenanoparticle suspension, and the reaction mixture was gentlystirred at room temperature. After 15 h the crude was dialyzedagainst water to remove residual EDC and ODN molecules,and then against 20 mM TRIS HCl (pH 7.4) to cap theeventual unreacted carboxylate groups on the nanoparticles bythe aminomethane derivative.Enzymatic Assay with DNase. To 500 μL of the
conjugated fluidMAG suspension (8 mg/mL) in 20 mMTRIS HCl (pH 7.4), 50 μL of 200 mM TRIS and 10 μL bovinepancreatic DNase I (1 mg/mL) were added, and the mixturewas allowed to gently stir at room temperature. After 1 h, 28 μL
MgCl2 (600 mM) and 14.7 μL CaCl2 (200 mM) were added,obtaining a final volume of 602.7 μL and the following finalconcentrations: 6.6 mg/mL nanoparticles, 33 mM TRIS,0.0166 mg/mL DNase, 28 mM MgCl2, 4.9 mM CaCl2. After20 h, the reaction mixture was dialyzed against water 3 times.The dialyzed fractions were concentrated, lyophilized, dissolvedin 1 mL Milli-Q water, and loaded on a equilibrated Sep-packC18 column (Millipore). Elution: 2.6 mL water (fraction 1), 3mL 4:6 H2O/CH3CN (fraction 2), 3.4 mL CH3CN (fraction3). Emission fluorescence spectra were performed on the 3eluted fractions (λex = 340 nm; excitation slits = 5 nm, PNTDetector Voltage = 800 V): only fraction 2 showedfluorescence signal. The same experiment was conducted onthe unconjugated fluidMAG suspension.
Acidic Treatment of the Nanoconjugate. In order tofurther confirm the presence of the fluorescent ODN on thenanoparticle surface, we treated 500 μL of the conjugatedfluidMAG suspension (8 mg/mL) with 0.4 M HCl (500 μL) at65 °C for 15 h. The reaction crude was divided in two aliquots.One aliquot was dialyzed against water, and the dialyzedfractions were concentrated under vacuum and analyzed viafluorescence spectroscopy. On the other aliquot, DCM/waterextraction of the reaction mixture was performed: the aqueousand organic phases were dried, dissolved in a small volume(450 μL) of water and CH3CN, respectively, and analyzed byUV. The chemical assay was also performed on the sameamount of nanoparticles from the fluidMAG stock solutionwhich was subsequently analyzed as described before.
Enzymatic Stability Assays. 60 μL oligonucleotidesequence 1 (30.6 μM) in H2O was added to a plastic vialcontaining 190 μL 100% murine plasma, and the mixture wasincubated at 37 °C (final conc.: 7.2 μM ODN, 76% plasma).13.5 μL samples, withdrawn from the reaction mixture atdifferent times (0, 10, 20, 30, 40, 60, 120, 180, 240, 300, 360min), were added to 10 μL electrophoretic loading buffer (TBE1×, 50% glycerol, 40% formamide, 0.2% gel tracking dyes) ineppendorf tubes, and the mixtures were kept for 3 min at 90 °Cand frozen until further analysis. Samples (10 μL), preheated at90 °C for 1 min, were loaded on a 15% denaturingpolyacrylamide gel containing 7 M urea, and the gel was runon the electrophoretic system for 1 h at 95 V in TBE 1×/7 Murea running buffer.600 μL of the conjugated nanoparticles (8 mg/mL) in 20
mM TRIS HCl, pH 7.4, were added to 1.9 mL 100% murineplasma in a plastic falcon tube, and the mixture was incubatedat 37 °C (final conc.: 1.9 mg/mL nanoparticles, 76% plasma).After gentle stirring of the falcon for 20 s, 150 μL samples werewithdrawn from the degradation mixture at various times (0,20, 40 min; 1, 2, 3, 4, 5, 6, 7, 8, 22, 24, 26 h). Each sample wasdiluted to 1 mL and dialyzed against water. The dialyzedfractions were concentrated to 450 μL and analyzed byfluorescence spectroscopy.
Magnetic Separation of the TBAPy-Conjugated Nano-particles. The TBA-nanoparticle conjugate solution (11.7 μL,8.00 mg/mL) was diluted 85.5-fold to 1 mL (0.0936 mg/mLiron content) and loaded on a spherical steel matrix column(LD-MACS, Miltenyi Biotec), pre-equilibrated with 3 × 2 mLH2O, placed in the MidiMACS Magnetic Separator (MiltenyiBiotec). Elution by gravity on the magnetic separator: 5 × 2 mLH2O, fractions 1−5 (2 mL each). Elution out of the magneticseparator by using the plunger: 3 × 2 mL H2O, fractions 6−8(2 mL each). Fractions 1−3 (6 mL in total) and fractions 6−7
(4 mL in total) were separately unified and analyzed by UVspectroscopy.
■ RESULTS AND DISCUSSION
Design, Synthesis, and Characterization of the NH2−C12-TBA-dU
Py Oligomer and Description of SomeMagnetic Nanoparticle Properties. We designed andsynthesized a fluorescently labeled ODN conjugated tomagnetic particles. The magnetic nanoparticles selected by usconsisted of a 80% (w/w) single-domain magnetite core 20 nmin diameter with a 40 KDa dextran coating of 15 nm thicknessbearing methylene carboxylic groups. In particular, theferrofluid fluidMAG-CMX used by us consisted of an aqueousdispersion of magnetic iron oxide nanoparticles (25 mg/mL)with hydrodynamic diameter of 50 nm and, thus, withsuperparamagnetic properties. Approximately, there were 1.3× 1013 particles per mg of the solid component.The ODN sequence selected by us was GG TT GG TGT
GG TT GG, which is the thrombin-binding aptamer (TBA)sequence, and the fluorescent group chosen was the pyrene.Initially, UV spectra of the ferrofluid at various dilutions,
starting from 100 to 3200-fold, was performed covering awavelength range from 220 to 700 nm (SupportingInformation, Figure S1).From these experiments we concluded that to observe an
absorbance below 1 (or between 1 and 2), in the 220−400 nmrange, where the ODN and pyrene group contribute, thefluidMAG dispersion should be diluted 1600-fold (magentaline, Figure S1) (or 800-fold, red line) corresponding to a0.0156 mg/mL (or 0.0312 mg/mL) iron concentration. Atthese dilutions, considering the binding capacity of the particleswith the ODN-pyrene molecules, there would be an ODN-Pyconcentration of about 4 nM (or 8 nM). Thus, considering themolar absorption coefficients (ε), the high UV absorption ofiron over the whole range of visible light greatly predominatesover the ODN-pyrene UV contribution which would probablynot be appreciable by the UV spectrophotometer.
In this respect, we intended to monitor the ODN anchoredto the nanoparticles through the pyrene fluorescence. Thepyrene group not only provides fluorescence spectra whichresult very sensitively to its concentration, and thus to theODN concentration, but is also a very sensitive fluorescentprobe for conformational changes of the oligomer to which isattached.27,28 Iron oxides do not exhibit any fluorescence signal,as we verified by performing several fluorimetry experiments.We registered emission fluorescence spectra of fluidMAG from400 to 600 nm at various dilutions (800, 1600, 3200, 6400-fold)exciting at different wavelengths (340, 350, 360, 370, 380 nm),taking into consideration the fluorescence properties of thepyrene group: no fluorescence was detected.Furthermore, iron oxides are not expected to interfere with
the pyrene fluorescence. Indeed, in a recent work on thesynthesis of magnetite nanoparticles with polymeric shellincluding pyrene groups, it was demonstrated that fluorescentspectra of pyrene-polymeric shell particles in the presence orabsence of the magnetic core were almost identical.51
Hence, we proceeded with the synthesis of the pyrene-labeled ODN, constituted by a 5-(pyren-1-yl-ethynyl)-2′-deoxyUridine (dUPy) at the 3′ end, the TBA sequence (GGTT GG TGT GG TT GG) and a 12-amino-dodecyl spacer (5′-Amino-Modifier C12) at the 5′ terminus, useful for theconjugation to the nanoparticles. The modified ODN wassynthesized on solid phase at 1 μmol scale by phosphoramiditechemistry using an automatic synthesizer. After cleavage/deprotection, the oligo was purified by reverse phase HPLC ona C18 analytical column (Supporting Information, Figure S2),and the MMT group was removed by mild acidic treatment ofthe purified ODN.The amino-free ODN (NH2-C12-TBA-dU
Py, 1) was desaltedby gel filtration, and characterized by ESI-MS and opticalspectroscopy (UV, CD, fluorescence), and analyzed by RP-HPLC chromatography and electrophoresis. UV spectrum,shown in Figure S3 (Supporting Information), revealed thecharacteristic absorptions of both the TBA sequence, with thedouble-hump band between 230 and 300 nm, and the pyrene
Figure 1. Overlapped CD spectra of (a) 2 μM 5′OHTBA, in 20 mM TRIS (black line) and in 20 mM TRIS/150 mM KCl (blue line); (b) 2 μM NH2-C12-TBA-dU
Py, in 20 mM TRIS (red line) and in 20 mM TRIS/150 mM KCl (green line); (c) 5′OHTBA (black) and NH2-C12-TBA-dUPy (red), 2
μM in 20 mM TRIS; (d) 5′OHTBA (blue) and NH2-C12-TBA-dUPy (green), 2 μM in 20 mM TRIS/150 mM KCl.
group, with the characteristic UV contribution in the range320−450 nm (inset of Figure S3). Absorbance measurementsat 260 nm, considering the molar extinction coefficient of theODN afforded a 18% yield of the purified oligomer.Electrophoretic mobility of the new NH2-C12-TBA-dU
Py
molecule was evaluated on a denaturing polyacrylamide gel(15%) and compared with that of the 5′OHTBA, synthesized ascontrol: as evidenced by the band position in Figure S4(Supporting Information), in these conditions, the two ODNshave almost the same mobility, differing only for the color ofthe bands revealed by UV light (254 nm) after ethidiumbromide staining.Circular dichroism properties of the ODN containing the
dUPy and C12−NH2 modifications were investigated andcompared with that of the natural 5′OHTBA (2, Figure 1a,b).We also detected and compared the conformational changefrom unstructured to G-quadruplex of both ODNs after addingKCl (Figure 1c,d). From these CD data we verified that theincorporation of dUPy and C12−NH2 does not interfere withthe formation of the G-quadruplex.Then, we studied the conformational change of NH2-C12-
TBA-dUPy from unstructured to G-quadruplex also byfluorescence. Hyperchromic and bathochromic shifts wereobserved, as reported in Figure 2. The random coil structure
showed an emission band at λmax = 430 nm (black line), whichshifted to 475 nm on transition to the G-quadruplex structureafter adding KCl (red line).Finally, we verified the binding of the modified TBA ODN 1
to its complementary sequence (CCAACCACACCAACC, 3)and compared the stability of the resulting duplex with that ofthe nonmodified one. CD experiments performed in a tandemcell evidenced the formation of a complex between 1 and 3(Figure 3a) which is certainly a duplex structure, alsoconsidering the similarity of the mix CD spectrum of the 1−3 complex with that of the duplex formed between the naturalTBA 2 and the complementary strand 3 (Figure 3b,c). The 1−3 duplex is formed also by mixing preformed TBA quadruplex 1(in 150 mM KCl) with complementary strand 3 (Figure 3d), inanalogy to the behavior of the natural TBA 2 (data notshown).52
Melting temperatures (Tm) of the modified quadruplex 1 (in150 mM KCl) and the 1/3 duplex, obtained by UV
measurements (Tm = 52.5 and 57.4, respectively), were almostidentical to the natural counterparts, quadruplex 2 and duplex2/3 (Tm = 47.3 and 54.8, respectively, Figure 4), denoting thatthe two modifications (dUpy and C12-linker) do not interferewith the stability of the TBA-forming structures, but ratherstabilize the resulting structures, probably thanks to the dUpy-duplex or dUpy-quadruplex interaction (stacking effect).53
Conjugation of the NH2-C12-TBA-dUPy Oligomer with
the Magnetic Nanoparticles. After the chemical−physicalcharacterization of the NH2-C12-TBA-dU
Py ODN, we per-formed its conjugation with the magnetic nanoparticles. Thecovalent coupling between the ODN amino groups and theCOOH on the nanoparticles was achieved by carbodiimideactivation method using EDC (25 °C, 15 h). Unbound ODNand EDC derivatives were removed from the conjugatesuspension by a series of dialyses against water. By selectingthe proper membrane pore cutoff of the dialysis column (100KDa), unconjugated ODN, EDC excess, and EDC derivativespassed outside the membrane, whereas iron nanoparticlesremained inside. Dialyzed fractions were checked by UVmeasurements until no further UV signal was detected on theconcentrated fraction.UV spectrum of the conjugation mixture, in comparison with
the starting fluidMAG, is shown in Figure 5. As expected, nosubstantial difference in the spectra between conjugated andunconjugated nanoparticles was revealed.Subsequently, both the FluidMAG stock solution and the
ODN-conjugated nanoparticles were subjected to thermalanalysis, following the UV absorbance value at 260 and 650nm upon increasing the temperature from 15 to 70 °C. In theoverall temperature range, no change in the A260 and A650 wasdetected, and the UV spectra of both the samples, recordedafter each thermal experiment, were superimposable on thatobtained before the analysis, at the same temperature (15 °C).Thus, these results suggest that both the FluidMAG stocksolution and the ODN-conjugated nanoparticles are stable totemperature growth up to 70 °C, since the optical properties ofboth samples are not affected by changes in temperature.Furthermore, no CD signal of the ODN−nanoparticle
conjugate solution was detected (indeed spectra resultedperturbed by the high potential), even at various dilutions(40, 100, 400, 800-fold), and even by adding KCl (150 mM) ateach diluted sample in order to allow the formation of the G-quadruplex on the nanoparticle, which, possessing a higher CDsignal, would have been able to increase the CD sensitivity.Unexpectedly, also fluorescence spectra of the ODN−
nanoparticle conjugate did not reveal any signal. In particular,perturbed signals were detected even at different dilutions ofthe conjugated mixture from 100- to 1660-fold, and exciting atdifferent wavelengths (340, 350, 360, 370, 380 nm). Thus, ourdata apparently disagree with a previous report in which themagnetite core of nanoparticles with the pyrene-containingpolymeric coating did not perturb the pyrene fluorescence.51
This apparent discordance can be explained on the basis of thedifferent pyrene/magnetite ratio which was much higher in thecited work than in our case.Subsequently, we verified the presence of the fluorescent
ODN on the nanoparticle by treating a known amount of theconjugated fluidMAG with bovine pancreatic deoxyribonu-clease I (DNase I) in proper conditions. DNase I is a DNA-specific endonuclease able to hydrolyze the phosphodiesterlinkages of double-stranded or single-stranded DNA to amixture of oligo- and mononucleotides. Since the ODN was
Figure 2. Overlapped fluorescence spectra of 0.4 μM NH2-C12-TBA-dUPy in 20 mM TRIS (black line) and in 20 mM TRIS/150 mM KCl(red line).
linked to the nanoparticles through its 5′-end, whereas thepyrene fluorophore was positioned at the 3′-terminus, far fromthe steric hindrance of the nanoparticle, we expected to findODN fragments also containing the fluorophore after DNasedegradation. Actually, after dialysis of the DNase reactionmixture, we found a clear fluorescent signal in the concentrateddialyzed fractions (Figure 6), which was absent in the case ofthe dialyzed fraction from the fluidMAG stock solution treatedwith DNase in the same conditions used for the conjugatedfluidMAG. From this experiment we were also able todetermine the approximate number of ODN molecules per
particle, as described in the Supporting Information (FigureS5).In order to further confirm the presence of the fluorescent
ODN on the nanoparticle surface, we treated another knownamount of conjugated fluidMAG with 0.4 M HCl at 65 °C for15 h. In these conditions, we expected depurination andprobably loss of the pyrene group to occur. The reaction crudewas divided into two aliquots. One was subjected to dialysisagainst water: we found fluorescent signal in the concentrateddialyzed fractions (Figure 7a), which was absent in the case ofthe dialyzed fraction from the fluidMAG stock solution treated
Figure 3. (a) Overlapped sum and mix spectra of ODNs 1 and 3, 2 μM each strand, 20 mM TRIS, tandem cell. (b) Overlapped sum and mix spectraof ODNs 2 and 3, 2 μM each strand, 20 mM TRIS, tandem cell. (c) Comparison of the CD spectra of the 1−3 and 2−3 complexes. (d) Quadruplex-Duplex competition experiment in tandem cell: sum (magenta) and mix (green) spectra of TBA quadruplex 1 and cTBA 3, 2 μM each strand in 20mM TRIS/150 mM KCl.
Figure 4. Overlapped melting curves of (a) 2 μM NH2-C12-TBA-dUPy (1, straight line) and 2 μM 5′OHTBA (2, dashed line) in 20 mM TRIS and 150
mM KCl, at 295 nm, (b) 1 μM 1−3 duplex (straight line) and 1 μM 2−3 duplex (dashed line) in 20 mM TRIS and 150 mM KCl, at 260 nm.
Figure 5. UV spectra of the conjugated nanoparticle (red line) andstarting fluidMAG (black line) solutions, at 1:800 and 1:1600dilutions, respectively.
Figure 6. Fluorescence spectrum of the dialyzed fractions after DNasetreatment of the conjugated fluidMAG.
with HCl in the same conditions as the conjugated one. On theother aliquot DCM/water extraction of the reaction mixturewas performed: the aqueous and organic phases were dried,dissolved in a small volume of water and CH3CN, respectively,and analyzed by UV. The aqueous phase revealed the usualstrong iron absorption, while the organic one showed bands of
both nucleotidic and pyrene nature, shifted at higher wave-lengths in CH3CN and characterized by low intensity due tothe small amount of starting nanoparticle mixture (Figure 7b).No significant UV absorbance was detected in the organicphase relative to the HCl treatment of the fluidMAG stocksolution, performed as a control.The resistance to enzymatic degradation of the TBAPy-
conjugated nanoparticles was explored and compared with thatof the free TBAPy. We expected a protection of the ODN fromenzymatic degradation thanks to the presence of the nano-particles, in analogy to recent works.54,55 Thus, unconjugatedTBA ODN 1 (7.2 μM) was incubated, as a control, in 76%murine plasma at 37 °C, and its degradation was monitored byelectrophoretic analysis of several aliquots, withdrawn from thereaction mixture at different times (0, 10, 20, 30, 40, 60, 120,180, 240, 300, 360 min; Figure 8a). In these conditions theODN exhibited an half-life of about 30 min. The sameexperiment was carried out by incubating the conjugatednanoparticles (1.9 mg/mL) in 76% murine plasma. The analysisof the samples, withdrawn from the degradation mixture of theconjugate at various times (0, 20, 40 min; 1, 2, 3, 4, 5, 6, 7, 8,22, 24, 26 h) was performed by dialyzing the samples againstwater and recording the fluorescence spectra of the dialyzedand concentrated fractions. Fluorescence instead of electro-phoresis analysis was performed on the nanoconjugatedegradation mixture in order to increase the sensitivity of themethod: indeed the dialysis, carried out on every samplewithdrawn with the aim of separate the iron components fromthe oligonucleotide samples, required the analysis of largevolumes. No degradation products was detected within 5 h.The first fluorescence signal was revealed for the samplecollected at 6 h (gray line), while the last one was for the 22 hsample (red line, Figure 8b).
Magnetic Separation of the TBAPy-Conjugated Nano-particles. In view of the potential use of the TBA-conjugatednanoparticles for the capture of thrombin from a complexmixture we characterized with respect to our conjugatesolution, the LD-MACS column (Miltenyi Biotec), associatedwith the MidiMACS Separator and generally used for themagnetic cell separation. When a MACS column, composed ofa spherical steel matrix, is placed in a MACS Separator,consisting of a strong permanent magnet, a high gradientmagnetic field is induced within the column, and a certainamount of magnetically labeled cells can be efficiently retained.
Figure 7. (a) Fluorescence spectrum of the dialyzed fraction after HCltreatment of the conjugated fluidMAG; (b) UV spectra of the DCMand water extracts after HCl treatment of the conjugated fluidMAG.The inset shows enlargements of the 320−480 nm region of thespectrum.
Figure 8. (a) Gel electrophoresis image of TBAPy degradation in plasma; (b) fluorescence spectra of the dialyzed fractions derived from thedegradation of the TBAPy-conjugated nanoparticles in plasma withdrawn at 6 (gray), 7 (blue), 8 (black), and 22 h (red).
The TBA−nanoparticle conjugate solution was diluted 85.5-fold (0.0936 mg/mL iron content) to 1 mL and UV spectrumwas recorded (black line, Figure 9). This sample was then
loaded on the LD-MACS column, placed in the MagneticSeparator, and 5 × 2 mL H2O washings were applied. Somebrown solution was collected in the fractions 1−3 (2 mL each),while fractions 4 and 5 contained clear water solutions. Then,the column was removed from the magnetic field and theretained nanoparticles were flushed out with 3 × 2 mL in threeseparated fractions (6−8) by using the plunger. The first twofractions (6 and 7) contained magnetite nanoparticles, whilethe third one was a clear solution. Subsequently, fractions 1−3(unretained nanoparticles, 6 mL in total) and fractions 6−7(retained nanoparticles, 4 mL in total) were separately unified,and UV spectra were recorded. In Figure 9, we report the UVspectra of the retained (blue line) and unretained (red line)nanoparticles, normalized with respect to the dilution of theloaded sample.From the UV analysis of the spectra reported in Figure 9, it
was deduced that (1) the amount of the magnetite nano-particles loaded in the MACS column was 0.0936 mg,corresponding to 1.22 × 1012 particles, (2) the amount of theunretained particles was 0.0514 mg (6.69 × 1011 particles), (3)the amount of the retained particles was 0.0421 mg (5.47 ×1011 particles). Considering that there are approximately 10TBA oligomers per particle (Supporting Information, FigureS5), the number of TBA molecules retained on the column wasabout 5.5 × 1012 (∼10 pmol). Thus, the magnetic separation ofour TBA−nanoparticle conjugate, immobilized on the LD-MACS column, could permit at maximum the capture, from acomplex mixture, of about 10 pmol thrombin each time. Thecaptured protein could then be easily released from the TBAimmobilized on the column, for example, by adding the TBAcomplementary sequence (CC AA CC ACA CC AA CC), asreported in a recent literature report.56
■ CONCLUSIONS
In this paper we reported the conjugation of superparamagneticnanoparticles to an oligodeoxyribonucleotide (ODN), able tofold into stable unimolecular guanine quadruple helix underproper ion conditions thanks to its nucleobase sequence (TBAsequence). The high potential in biomedical applications of thisconjugate, as magnetic nanosensor or therapeutic agent, can be
understood considering the combination of the attractiveproperties of both the TBA sequence and the magneticnanoparticles. A pyrene fluorescent group, by means of a dUPy
unit, was attached to the ODN 3′-end before the conjugation tonanoparticles, in order to follow the ODN fate when anchoredto the nanoparticle, whereas a 12-amino-dodecyl spacer (5′-Amino-Modifier C12) was incorporated at the 5′-end, in orderto allow the coupling of the amino group on the ODN with theCOOH groups on the nanoparticles. The novel pyrene-labeledODN was characterized by ESI-MS and optical spectroscopy(UV, CD, fluorescence), and analyzed by RP-HPLCchromatography and electrophoresis. Then, we performed theconjugation of the pyrene-labeled ODN with the magnetitenanoparticles, optimizing the workup protocol, which tookadvantage of repeated dialyses against water. Subsequently, westudied the ODN-conjugated magnetic fluid through opticalspectroscopy (UV, CD, fluorescence). None of thesetechniques, directly performed on the conjugate, revealed thepresence of the ODN. As expected, the UV and CD opticalproperties of the ODN were obscured by the high magnetiteabsorption over the whole range of visible light. Unexpectedly,the fluorescence measurements on the ODN−nanoparticleconjugate also did not reveal any signal, in contrast with aprevious report in which the magnetite core of nanoparticleswith a pyrene-contained polymeric coating did not perturb thepyrene fluorescence. This discordance could be explained byconsidering the different pyrene/magnetite ratio, which wasvery high in the cited work, whereas it was approximately 10 inour case. However, we demonstrated indirectly the presence ofthe fluorescent ODN on the nanoparticles by enzymatic(DNase) and chemical (HCl) assays.Furthermore, we investigated the enzymatic degradation
stability of the ODN−nanoparticle conjugate in 76% murineplasma. No degradation products were detected within 5 h,while unconjugated TBA were completely digested after 3 h(half-life of about 30 min). We found that TBA oligomers werestill present on the nanoparticles until 8 h, and no moredegradation products were detected after 22 h. Thus, as weexpected, the nanoparticles enhanced the stability of ODN toenzymatic degradation.Finally, in view of the potential use of the ODN-conjugated
nanoparticles for the capture of a target protein from a complexmixture, we evaluated the amount of TBA-magnetite nano-particles retained on a Magnetic Separator based on a steelmatrix-filled column placed in a strong permanent magnet.From UV analysis, we were able to estimate the maximumcapture capacity of our system relative to its target, in this casethe thrombin protein.
■ ASSOCIATED CONTENT
*S Supporting InformationUV spectra of the magnetic nanoparticle solution. Syntheticconditions and purification of the modified ODN 1. UVspectrum of the pyrene-labeled ODN 1. Electrophoreticanalysis of natural and modified TBA ODN. Determinationof the nanoparticle loading by fluorescence measurements. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.
Figure 9. Overlapped UV spectra of the conjugate TBA-magneticnanoparticles solution. Black line: 0.0936 mg/mL nanoparticle beforeloading on LD-MACS column; red line: not-retained nanoparticles(spectrum normalized with respect to the dilution of the loadedsample); blue line: retained nanoparticles (spectrum normalized withrespect to the dilution of the loaded sample).
We acknowledge the University of Naples “Federico II” for thefinancial support under the PRIN 2007 Project. We are alsograteful to Giuseppe Perretta for his invaluable technicalassistance.
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