Research Article Biophysical and RNA Interference ...

Hindawi Publishing CorporationJournal of ChemistryVolume 2013 Article ID 650610 11 pageshttpdxdoiorg1011552013650610

Research ArticleBiophysical and RNA Interference Inhibitory Propertiesof Oligonucleotides Carrying Tetrathiafulvalene Groupsat Terminal Positions

Soacutenia Peacuterez-Rentero1 Alvaro Somoza2 Santiago Grijalvo1 Jiliacute Janoušek3

Martin Bjlohradskyacute3 Irena G Staraacute3 Ivo Staryacute3 and Ramon Eritja1

1 Institute for Advanced Chemistry of Catalonia (IQAC) CSIC CIBER-BBN Networking Centre on BioengineeringBiomaterials and Nanomedicine Jordi Girona 18-26 08034 Barcelona Spain

2 IMDEA-Nanociencia 28049 Madrid Spain3 Institute of Organic Chemistry and Biochemistry vvi Academy of Sciences of the Czech Republic Flemingovo Namesti 216610 Prague 6 Czech Republic

Correspondence should be addressed to Ramon Eritja recgmacidcsices

Received 22 April 2013 Accepted 5 June 2013

Academic Editor Rachid Benhida

Copyright copy 2013 Sonia Perez-Rentero et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Oligonucleotide conjugates carrying a single functionalized tetrathiafulvalene (TTF) unit linked through a threoninol molecule tothe 31015840 or 51015840 endswere synthesized together with their complementary oligonucleotides carrying a TTF pyrene or pentafluorophenylgroup TTF-oligonucleotide conjugates formed duplexes with higher thermal stability than the corresponding unmodifiedoligonucleotides and pyrene- and pentafluorophenyl-modified oligonucleotides TTF-modified oligonucleotides are able to bind tocitrate-stabilized gold nanoparticles (AuNPs) and produce stable gold AuNPs functionalized with oligonucleotides Finally TTF-oligoribonucleotides have been synthesized to produce siRNA duplexes carrying TTF units The presence of the TTF molecule iscompatible with the RNA interference mechanism for gene inhibition

1 Introduction

There is a current interest in the preparation of oligonu-cleotide conjugates with functional 120587-electron systems sincetheir 120587-120587 interaction may provide additional binding energywithout perturbing the specific recognition with the comple-mentary sequences Several authors have described aromaticsystems that linked to the 31015840 end and 51015840 end of oligonu-cleotides inducing the stabilization of DNA duplexes by 120587-120587stacking interactions [1ndash6]

Recently we have reported that the introduction oftetrathiafulvalene (TTF) derivatives at the terminal positionof oligonucleotides induces a high stabilization of the DNAduplexes Moreover depending on the location of the TTFmolecules a strong aggregation was observed at high saltconcentrations yielding defined spherical nanostructures ofaround 190ndash210 nm of size [7] TTF molecules are strongelectron-donating molecules which have been used for the

construction of organic conducting materials [8] or as build-ing blocks in supramolecular chemistry [9]

In this study we aim to compare the duplex stabiliza-tion properties of TTF units paired with other donor oracceptor aromatic molecules To this end we synthesizedseveral oligonucleotides carrying a TTF unit functionalizedwith three aliphatic S-butyl groups (Scheme 1) at either 31015840or 51015840 ends of the oligonucleotide Their complementaryoligonucleotide sequences carrying TTF pyrene or pentaflu-orophenyl groups were used to form DNA duplexes with thedonoracceptor hydrophobic 120587-electron systems moleculesat the same or opposite termini of the duplex The relativethermal stability of the duplexes was analyzed demonstratinga distinct behavior of the TTF oligonucleotide conjugates thatis in agreement with the supramolecular properties of TTF

Moreover there is an interest in the preparation of stablegold nanoparticles (AuNPs) functionalized with oligonu-cleotides DNA oligomers carrying thiol and dithio groups

2 Journal of Chemistry

HOHO

NH2

O O

O

O

HN

HN HN

DMT

OO

HN

O

O

O

O

O

O

DMTDMT

OHOH OH

a b

c de

TTF1 (a)

TTF2 (b)

PFP (c)

PYR (d)S

S

S

S

S

S

S

S

S

S

BuS

BuS

SBu

SBu

SBu

SBu

FF F

FF

NCP N

HCPG

Ra

N

Randashd Randashd

Randashd

(4a)

(1andash1d)

(3andash3d)

(2andash2d)

Scheme 1 Preparation of the solids supports and phosphoramidite 4a needed for the assembly of oligonucleotides (a) R-COOH EDCI1-hydroxybenzotriazole and DIEA (b) dimethoxytrityl chloride DMAP (c) succinic anhydride DMAP (d) hemisuccinate NH

2-controlled

pore glass (CPG) TBTU triethylamine (e) O-2-cyanoethyl-NN-diisopropyl chlorophosphoramidite

are frequently used to functionalize citrate-stabilized goldnanoparticles [10ndash12] The presence of eight thioether func-tions in TTF units prompted us to investigate the use of TTF-oligonucleotides for the functionalization of gold nanopar-ticles As expected TTF-modified oligonucleotides are ableto bind to citrate stabilized gold nanoparticles (AuNPs) pro-ducing stable AuNPs functionalized with oligonucleotidesFinally TTF-oligoribonucleotides have been synthesized toproduce siRNA duplexes carrying TTF units Chemicalmodification of the 31015840 ends of siRNA is an active area ofresearch aimed at increasing the stability of modified siRNA[13 14] aswell as stabilizing the interaction of siRNAswith thePAZ domain of RISC [15] In this work we demonstrate thatthe presence of the TTFmolecule is compatible with the RNA

interference mechanism for gene inhibition especially if theTTF unit is located at the 31015840 ends of the passenger strand

2 Materials and Methods

21 General Information All the standard phosphoramiditesand reagents forDNAsynthesis were purchased fromAppliedBiosystems and from Link Technologies L-threoninol andother chemicals were purchased from Sigma-Aldrich andFluka TTF1-threoninol and pyrenyl-threoninol derivativeswere prepared as described [7 15] [41015840551015840-Tris(butylsulf-anyl)-221015840-bi-13-dithiol-4-yl]sulfanyl acetic acid was pre-pared as described previously [16] Dry solvents were pur-chased from Sigma-Aldrich and Fluka and used as received

Journal of Chemistry 3

NMR spectra were recorded at 25∘C on Varian Gemini300MHz Varian Mercury 400 or Varian Inova 500MHzspectrometers using deuterated solvents (Unitat de RMNServeis cientificotecnics de Barcelona) Tetramethylsilane(TMS) was used as an internal reference (0 ppm) for 1Hspectra recorded in CDCl

3and the residual signal of the

solvent (7716 ppm) for 13C-spectra For CD2Cl2(538 ppm)

and CD3OD (490 ppm) the residual signal of the solvent

was used as a reference The external reference for 19F-spectra was CCl

3F (0 ppm) Chemical shifts are reported in

part per million (ppm) in the 120575 scale coupling constantsin Hz and multiplicity as follows s (singlet) d (doublet) t(triplet) q (quadruplet) m (multiplet) and br (broad signal)Matrix-assisted laser desorption ionization time-of-flight(MALDI-TOF) mass spectra were recorded on a Voyager-DE-RP spectrometer (Applied Biosystems) in negative modeby using 246-trihydroxyacetophenone (THAP)ammoniumcitrate (CA) (1 1) as matrix and additive resp) Electrosprayionization mass spectra (ESI-MS) were recorded on a Micro-mass ZQ instrumentwith single quadrupole detector coupledto an HPLC and high-resolution (HR) ESI-MS on an Agilent1100 LCMS-TOF instrument (Servei drsquoEspectrometrıa deMasses Universitat de Barcelona)

22 3-[41015840551015840-Tris(butylsulfanyl)-221015840-bi-13-dithiol-4-yl]sulf-anylpropionic Acid (2-Cyanoethyl)sulfanyl TTF derivative[17] (12 g 22mmol) was dissolved in anhydrous DMF(50mL) and degassed A solution of CsOHsdotH

2O (043 g

258mmol and 117 equiv) in anhydrous MeOH (5mL) wasadded After stirring the reaction mixture at room tempera-ture for 30min a suspension of 3-bromopropionic acid (05 g33mmol and 15 equiv) and freshly annealed K

2CO3(18 g

13mmol and 59 equiv) in anhydrous DMF (60mL) wasaddedThe reactionmixture was stirred at room temperaturefor 2 h A solution of 1M HCl (20mL) was added and themixture was extracted with EtOAc (3 times 100mL)The organicphase was washed with water (3 times 100mL) dried over anhy-drous MgSO

4 and concentrated to dryness under reduced

pressure The crude residue was purified by chromatographyon silica gel (chloroform-methanol 100 0 to 90 10) Thepure compound was crystallized from dichloromethane andheptane providing red crystals (102 g 81) Melting point70-71∘C IR (CHCl

3) ] = 3512w 3091 w 2961 s 2932 s 2875 s

2864m 2739w 2656w 2581 w 1746w 1713 s 1583mbr1485w 1465m 1458m 1433m 1419m 1381m 1289m 1197w1100w 916w 887m cmminus1 UV-Vis (CHCl

3)120582max (log 120576) = 264

(418) 334 nm (414) 1H NMR 120575H (500MHz CD2Cl2) 092

(t 119869 = 74Hz 9H) 144 (h 119869 = 74Hz 6H) 161 (m 6H)273 (t 119869 = 68Hz 2H) 283 (t 119869 = 74 6H) and 305 (t119869 = 68Hz 2H) 13C NMR 120575C (126MHz CD

2Cl2) 1391

2219 3129 3237 3463 3654 12569 (=C-SCH2) 12844

(=C-SBu) 17500 (COOH) and (=CS2not detected) ESI

MS 571 ([M]minus) 499 ([M-(C3H5O2)]minus) HR ESI MS calcd for

C21H31O2S85710095 found 5710093

23 [41015840551015840-Tris(butylsulfanyl)-221015840-bi-13-dithiol-4-yl]sulfa-nyl-N-[(2R3R)-13-dihydroxybutan-2-yl]propanamide (1b)A solution of [41015840551015840-tris (butylsulfanyl)-221015840-bi-13-dith-

iol-4-yl]sulfanylpropanoic acid (061mmol) L-threoninol(061mmol) 1-ethyl-3-(3-dimethylaminopropyl)carbodiim-ide (EDCI) (091mmol) hydroxybenzotriazole (HOBt)(091mmol) and diisopropylethylamine (091mmol) inanhydrous DMF (10mL) was prepared under argonAfter stirring the reaction mixture at room temperatureovernight the mixture was concentrated to dryness underreduced pressure The residue was dissolved in toluene andconcentrated to dryness under reduced pressure (3x) Theresidue was dissolved in dichloromethane (100mL) andthe organic phase was washed with 10 aqueous NaHCO

3

(2times50mL) and saturated aqueous NaCl (50mL)The organicphase was dried and concentrated to dryness under reducedpressure The resulting TTF2-threoninol derivative was usedin the next step without further purification The desiredcompound was obtained as orange oil (338mg 83) TLC(ethyl acetate) 119877

119891= 022 1H NMR 120575H (400MHz CDCl

3)

638 (d 119869 = 68Hz 1H andNH) 420 (qd 119869 = 64 and 16Hz1H) 388minus383 (m 3H) 313 (t 119869 = 68Hz 2H) 287minus281(m 6H) 260 (td 119869 = 68Hz and 24Hz 2H) 166minus159(m 6H) 149minus140 (m 6H) 123 (d 119869 = 64Hz 3H) and093 (t 119869 = 76Hz 9H) 13C NMR 120575C (100MHz CDCl

3)

1715 (CO) 1307 (C) 1281 (C) 1279 (C) 1255 (C) 1114 (C)1094 (C) 692 (CH) 653 (CH

2) 550 (CH) 369 (CH

2) 363

(CH2) 362 (CH

2) 323 (CH

2) 320 (CH

2) 219 (CH

2) 218

(CH2) 208 (CH

3) and 138 (CH

3) HRMS (ESI+) mz calc

for C25H41NO3S8([M+H]+) 6600930 found 6600925

24 N-[(2R3R)-13-Dihydroxybutan-2-yl]-pentafluoroben-zamide (1c) To a solution of pentafluorobenzoic acid(11mmol) in DMF (2mL) at room temperature N-hydrox-ybenzotriazole (102mmol) and diisopropylcarbodiimide(102mmol) were added After stirring the mixture for 5minL-threoninol (093mmol) was added The mixture wasstirred at room temperature for 24 h and then quenchedwith methanol The solvent was evaporated under vacuumand the residue was purified by flash chromatography(cyclohexane-CH

2Cl2-MeOH 6 3 05) The desired com-

pound was isolated as a white solid (184mg 66) 1HNMR 120575H (400MHz CD

3OD) 410 (dq 119869 = 64 and 27Hz

1H) 403 (dt 119869 = 63 and 27Hz 1H) 370 (system ABX119869119860119861= 109Hz 119869

119860119883= 65Hz and 119869

119861119883= 62HZ 2H) 330

(m 1H) and 122 (d 119869 = 64Hz 3H) 13C NMR 120575119862(100Mz

CDCl3) 1581 (C) 1269 (C) 1267 (C) 1166 (C) 1114 (C)

691 (CH) 649 (CH2) 549 (CH) and 204 (CH

3) 19F NMR

120575F (376MHz CD3OD) and minus14402 (d 119869 = 192Hz 2F)

minus1563 (t 119869 = 199Hz 1F) minus1646 (m 2F) HRMS (ESI+)mz calc for C

11H11NO3F5([M+H]+) 3000653 found

3000660

25 N-[(2R3R)-1-(Bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxybutan-2-yl]-[41015840551015840-tris(butylsulfanyl)-221015840-bi-13-dithol-4-yl]sulfanylpropanamide (2b) The compound 1b(045mmol) was dried by evaporation of anhydrous acetoni-trile (ACN) under reduced pressure The product was dis-solved in anhydrous pyridine (5mL) and reacted with441015840-dimethoxytriphenylmethyl chloride (068mmol) diiso-pro-pyl ethylamine (102mmol) and 4-dimethylaminopyri-dine (006mmol DMAP) After stirring for 6 h at room


temperature 441015840-dimethoxyltritylchloride (068mmol)and DMAP (006mmol) dissolved in dry pyridine (5mL)were added and the mixture was stirred at room temperatureovernight The reaction was quenched with methanol(05mL) and the solvents were removed under reducedpressure The residue was dissolved in toluene (3 times 10mL)and evaporated to dryness Then the resulting material wasdissolved in dichloromethane (100mL) and the organicphase was washed with 5 aqueous NaHCO

3(50mL) and

with saturated aqueous NaCl (50mL) The solvent wasevaporated and the residue was purified by chromatographyon neutral aluminium oxide The product was eluted witha slow gradient of ethyl acetate from 50 to 100 in hexaneand then from 0 to 10 of methanol in ethyl acetate Thepure compound was obtained as an orange solid (226mg55) TLC (aluminium oxide) (ethyl acetate-hexane 2 1)119877119891= 054 1H NMR 120575H (400MHz CDCl

3) 740minus682 (m

13H) 620 (d 119869 = 88Hz 1H NH) 409 (qd 119869 = 64 and20Hz 1H) 397ndash392 (m 1H) 379 (s 6H) 342 (dd 119869 = 96and 44Hz 1H) 330 (dd 119869 = 96 and 34Hz 1H) 310 (t119869 = 68Hz 2H) 283minus278 (m 6H) 256 (t 119869 = 68Hz 2H)165ndash156 (m 6H) 147minus138 (m 6H) 115 (d 119869 = 64Hz3H) 094minus088 (m 9H) 13C NMR 120575C (100MHz CDCl

3)

1709 (CO) 1588 (C) 1445 (C) 1357 (C) 1355 (C) 1307(C) 1302 (CH) 1301 (CH) 1283 (CH) 1282 (CH) 1281(C) 1280 (C) 1273 (CH) 1256 (C) 1135 (CH) 1134 (CH)871 (C) 867 (CH) 653 (CH

2) 555 (CH

3) 538 (CH) 538

(CH) 370 (CH2) 363 (CH

2) 362 (CH

2) 322 (CH

2) 320

(CH2) 319 (CH

2) 219 (CH

2) 218 (CH

2) 202 (CH

3) 138

(CH3) HRMS (ESI+)mz calc for C

46H60NO5S8([M+H]+)

9622237 found 9622065

26 N-[(2R3R)-1-(Bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxybutan-2-yl]-pentafluorobenzamide (2c) To a solu-tion ofN-[(2R3R)-13-dihydroxybutan-2-yl]-pentafluoroben-zamide (053mmol) in pyridine (26mL) at 0∘C diisopropyl-ethylamine (079mmol) 441015840-dimethoxytritylchloride (079mmol) and 4-dimethylaminopyridine (01mmol) wereadded After 15min the mixture was allowed to reach roomtemperature then it was stirred for 24 h and finally thereaction was quenched with methanol The solvent wasevaporated under vacuum and the residue was purified byflash chromatography (cyclohexane-EtOAc 3 1) The desiredcompound was isolated as a yellowish solid (87mg 27)1H NMR 120575H (400Mz CDCl

3) 739 (d 119869 = 72Hz 1H)

729 (d 119869 = 89Hz 4H) 731ndash727 (m 2H) 724ndash718 (m1H) 683 (d 119869 = 89Hz 4H) 669 (d 119869 = 86Hz 1H) 413(d 119869 = 54Hz 1H) 415ndash410 (m 1H) 379 (s 6H) 349(system ABX 119869

119860119861= 98Hz 119869

119860119883= 38Hz and 119869

119861119883= 33Hz

2H) and 120 (d 119869 = 64Hz 3H) 13C NMR 120575C (100MzCDCl

3) 1587 (C) 1576 (C) 1441 (C) 1353 (C) 1350 (C)

1299 (CH) 1298 (CH) 1291 (C) 1281 (CH) 1277 (CH)1271 (CH) 1159 (C) 1147 (C) 1133 (CH) 1131 (CH) 870(C) 685 (CH

2) 654 (CH) 552 (CH

3) 541 (CH) 198

(CH3) 19F NMR 120575F (376MHz CDCl

3) minus1407 (m 2F)

minus1512 (t 119869 = 207Hz 1F) and minus1603 (m 2F) HRMS (ESI+)mz calc for C

32H28NO5F5Na ([M + Na]+) 6241779 found

6241784

27 Functionalization of CPG Solid Supports (3b-3c) TheDMT derivatives (2b-2c) were reacted with succinic anhy-dride and then incorporated on a long-chain alkylamine-controlled pore glass support (LCAA-CPG) as follows TheDMT derivative (009mmol) was dried by evaporation withanhydrous ACN under reduced pressure The residue wasdissolved in anhydrous pyridine (5mL) under argon Suc-cinic anhydride (022mmol) and DMAP (005mmol) weredissolved in 1mL of pyridine and added to the solutionAfter 4 h of stirring at room temperature succinic anhydride(022mmol) and DMAP (005mmol) were dissolved in 1mLof pyridine and added to the solution The reaction mixturewas allowed to react overnight The solvent was removedunder reduced pressure and the residue was dissolved intoluene (3 times 10mL) and evaporated to dryness The resultingmaterial was dissolved in dichloromethane (20mL) Theorganic phase was washed twice with 10 NaHCO

3(15mL)

and brine (15mL) dried over MgSO4 and evaporated to

dryness The resulting monosuccinate derivative was used inthe next step without further purification

Amino-LCAA-CPG (CPG New Jersey 73120583mol aminog300mg) was placed into a polypropylene syringe fitted witha polypropylene disc and washed sequentially with DMFmethanol THF DCM and ACN (2 times 5mL)Then a solutionof the appropriate homosuccinate (44 120583mol) and triethy-lamine (175120583mol) in anhydrous ACN (05mL) was preparedThe solution was added to the support and then a solu-tion of 2-(1H-benzotriazole-1-yl)-1133-tetramethyluroniumtetrafluoroborate (TBTU) (175 120583mol) in anhydrous ACN(300 120583L) was added The mixture was left to react for 1 h Thesupport was washed with DMF methanol DCM and ACN(2times5mL)The degree of functionalizationwas determined byanalysis of theDMT released upon the treatment of an aliquotof the support with a 3 solution of trichloroacetic acid inDCM The coupling procedure was repeated once more ifneeded and the functionality of the resin was determined byDMTquantification (119891 = 32 120583molg for 3b and 50 120583molg for3c DMT cation 120582max 498 nm 120576 = 71700Mminus1 cmminus1) Finallythe solid support was treated with the mixture of Ac

2ODMF

(1 1 500120583L) to cap free amino groups

28 Oligonucleotide Synthesis (DNA) The unmodified olig-onucleotide sequences 1 (51015840TAG AGG CTC CAT TGC31015840)2 (51015840GCA ATG GAG CCT CTA31015840) and 8 (51015840CGC GAATTC GCG31015840) were synthesized on a DNARNA synthe-sizer (Applied Biosystems 3400) using 200 nmol scale LV200polystyrene supports and commercially available chemicalsThe benzoyl (Bz) group was used for the protection of theamino group of C and A and the isobutyryl (iBu) group forthe protection of G The coupling yields were gt97 Thelast DMT group was removed at the end of the synthesisEach solid support was treated with aqueous concentratedammonia at 55∘C for 12 h to cleave the products from thesupports and remove the benzoyl and isobutyryl groupsThe mixtures were filtered and ammonia solutions wereevaporated to dryness Unmodified oligonucleotides weredesalted with Sephadex G-25 (NAP-10 column) and analyzedby HPLC Column XBridge OST C

18(46 times 50mm 25 120583m)


Solvent A 5 ACN in 100mM triethylammonium acetate(TEAA) (pH = 7) and solvent B 70 ACN in 100mMTEAA (pH = 7) Flow rate 1mLmin Conditions 10minof linear gradient from 0 to 30 B at room temperature foroligonucleotides 1 and 2 and at 60∘C for oligonucleotide 8The resulting oligomers were analyzed by mass spectrometry(MALDI-TOF) and UVVis spectroscopy and used withoutfurther purification

Oligonucleotides 3 and 4 (51015840TAGAGGCTCCATTGC31015840-TTF1 and 51015840GCAATGGAGCCTCTA31015840-TTF1 resp) con-taining the TTF1modification at the 31015840 ends were synthesizedon a 05 120583mol scale using the solid support functionalizedwith the TTF1 derivative and the oligonucleotide sequence5 (TTF1-51015840GCAATGGAGCCTCTA31015840) containing the modi-fication at the 51015840 ends was synthesized on a 200 nmol scaleemploying LV200 polystyrene supports using the TTF-1-phosphoramidite as described [7]

Oligonucleotides 6 and 7 (51015840TAGAGGCTCCATTGCX31015840)and 9 and 10 (51015840CGCGAATTCGCGX31015840) X being the pyrenyl(PYR) and the 23456-pentafluorophenyl (PFP) respec-tively were synthesized on a 1120583mol scale using the corre-sponding solid support functionalized with the appropriatethreoninol derivative prepared as described [15] The syn-theses were carried out as described above removing thelast DMT group at the end of the synthesis In this caseonly a 02120583mol of each solid support was treated withaqueous concentrated ammonia at 55∘C for 12 hThe resultingoligonucleotides were desalted with Sephadex G-25 (NAP-10 column) and purified by HPLC Column XBridge OSTC18

semipreparative column (10 times 50mm 25120583m) Flowrate 3mLmin Conditions 10min of linear gradient from0 to 30 of B at room temperature for oligonucleotides 6and 7 and at 60∘C for oligonucleotides 9 and 10 The purefractions were combined and evaporated to dryness Thepurified oligonucleotides were analyzed by HPLC ColumnXBridge OSTC

18(46times50mm 25 120583m) Flow rate 1mLmin

Conditions 10min of linear gradient from 0 to 30 of B atroom temperature for oligonucleotides 6 and 7 and at 60∘Cfor oligonucleotides 9 and 10 The resulting oligomers wereanalyzed by mass spectrometry (MALDI-TOF) and UVVisspectroscopy as well The yields obtained ranged from 25 to41

29 Oligoribonucleotide (RNA) Synthesis Theunmodified 21-nucleotide RNA sequences 11 (51015840aucugaagaaggagaaaaaTT31015840)12 (51015840uuuuucuccuucuucagauTT31015840) 13 (51015840gcuacagagaaaucu-cgauTT31015840) and 14 (51015840aucgagauuucucuguagcTT31015840) were syn-thesized on a DNARNA synthesizer (Applied Biosystems3400) using 200 nmol scale LV200 polystyrene supports21015840-TBDMS phosphoramidites thymidine phosphoramiditesand commercially available chemicals Guanosine was pro-tected with the dimethylaminomethylidene group cytidinewas protected with the acetyl group and adenosine withthe benzoyl group The last DMT group was left at theend of the synthesis Each solid support was treated witha mixture of aqueous concentrated ammonia and ethanol(3 1) at 55∘C for 1 h to cleave the products from the supportsand remove the base protecting groups The mixtures were

filtered and the solutions were evaporated to dryness Thena mixture of N-methylpyrrolidone (NMP 461 120583L) triethy-lamine (TEA 232 120583L) and triethylamine trihydrofluoride(TEAsdot3HF 307 120583L) was prepared and each dried samplewas treated with 250120583L of the mixture for 2 h 30min at65∘C Once the samples reached room temperature 175mLof a Tris Buffer solution (Glen-Pak RNA Quenching Buffer)was added and the oligonucleotides were purified with car-tridges (Glen-Pak RNA cartridge purification) following theprocedure described by the manufacturer The deprotectedoligonucleotides were desalted with Sephadex G-25 (NAP-10 column) and analyzed by HPLC Column XBridge OSTC18(46 times 50mm 25 120583m) Flow rate 1mLmin Conditions

10min of linear gradient from 0 to 30 of B The resultingoligomers were analyzed by mass spectrometry (MALDI-TOF) and UVVis spectroscopy (data not shown) and usedwithout further purification The yields obtained after car-tridge purification ranged from 29 to 40

Oligonucleotide sequences 15 (51015840aucugaagaaggagaaaa-aTT31015840-TTF2) and 16 (51015840uuuuucuccuucuucagauTT31015840-TTF2)(passenger and guide resp) containing the TTF2 modifi-cation at the 31015840 ends (3b) were synthesized on a 05 120583molscale using the solid support functionalized with the TTF2derivative The synthesis was carried out as described aboveremoving the last DMT groupThe cleavage and deprotectionsteps were performed as described above using 025 120583mol ofeach solid support The products were purified by HPLC onan XBridge OST C

18semipreparative column (10 times 50mm

25 120583m) Flow rate 3mLmin Conditions 3min of lineargradient from 0 to 9 B then 1min of linear gradient from9 to 60 B then 16min of linear gradient from 60 to 75 BThe purified oligomers were analyzed by mass spectrometry(MALDI-TOF) UVVis and HPLC Column XBridge OSTC18

(46 times 50mm 25 120583m) using the conditions describedabove except the flow rate that was 1mLmin The yieldsobtained after HPLC purification ranged from 5 to 8

210 Melting Experiments Performed with Nonself-Comple-mentary Oligonucleotides The melting experiments wereperformed in duplicate at 33120583M of duplex concentrationThe solutions were prepared either in 50mM or in 1M NaCland 10mM sodium phosphate buffer (pH = 7) The sampleswere heated at 90∘C for 5min allowed to cool slowly to roomtemperature to induce annealing and then kept overnight in arefrigerator at 4∘C Melting curves were recorded by heatingthe samples with a temperature controller from 15 to 80∘C orfrom 15 to 95∘C at a constant rate of 1∘Cmin by measuringthe absorbance at 260 nm When the temperature was below25∘C argon was flushed to prevent water condensation onthe cuvettesThe absorption spectra andmelting experiments(absorbance versus temperature) were recorded in 1 cm path-length cells The 119879

119898values were calculated with the Meltwin

32 software

211 Melting Experiments on Self-Complementary Oligonu-cleotides Themelting experiments were performed in dupli-cate at 66120583M oligonucleotide concentration as describedabove Melting curves were recorded by heating the samples


with a temperature controller from 15 to 95∘C at a constantrate of 1∘Cmin by monitoring the absorbance at 260 nm

212 Preparation of Oligonucleotide Gold Nanoparticle Con-jugates Citrate stabilized gold nanoparticles (97 nm) werepurchased from BBI Life Sciences and used as received Toprepare the conjugates 5 nmol of tetrathiafulvalene modifiedoligonucleotide (3 or 4) dissolved in 1mL of water wasadded to 1mL of the gold nanoparticle solution (94 nM)Themixture was shaken for 16ndash20 h prior to salt stabilizationThen the solution was brought to a final concentrationof 10mM sodium phosphate (pH = 72) The mixture wasthen allowed to equilibrate for 30min before bringing theconcentration to 015M NaCl over a 7 h 30min period in astepwise manner (005M NaCl increments each 2 h 30min)The solutions were sonicated for 20 s before each additionto keep the particles dispersed during the salting procedureThe salting process was followed by incubation overnight atroom temperature Finally to remove all unbound oligonu-cleotides the solution was centrifuged at 13200 rpm for30min The supernatant was removed and the reddish solidat the bottom of the centrifuge tube was dispersed in 1mL of a015MNaCl 10mMsodiumphosphate buffer (pH= 72)Thisprocedure was repeated three timesThe resulting conjugateswere resuspended in 1mL of a 015M NaCl 10mM sodiumphosphate (pH = 72) and 01 NaN

3solution Then each

solutionwas analyzed byUV-visible absorption spectroscopy(3-AuNP 743 nM and 4-AuNp 775 nM) The extinctioncoefficient used was the same as that used for unmodifiednanoparticles (120576

520= 76 times 10

7Mminus1 cmminus1 data providedby the manufacturer) Each solution was then stored in thefridge (4∘C) prior to use

213 Melting Experiments with Oligonucleotide-Gold Nano-particle Conjugates Melting experiments were performed induplicate at 37 nMof each oligonucleotide gold-nanoparticleconjugate by mixing 498 120583L of 3-AuNp 477 120583L of 4-AuNpand 25 120583L of the 015M NaCl and 10mM sodium phosphatebuffer The samples were heated at 90∘C for 5min allowedto cool slowly to room temperature to induce annealing andthen kept in a refrigerator at 4∘C for 48 h The solutionswere sonicated for 20 s before the melting experiment tokeep the particles dispersed Melting curves were recordedby heating the samples with a temperature controller from 15to 95∘C at a constant rate of 1∘Cmin and when monitoringthe absorbance at 523 nm During the experiment when thetemperature was below 25∘C argon was flushed to preventwater condensation on the cuvettes 119879

119898values were deter-

mined from the maxima or the minima of the first derivativeof the curves calculated with the Origin 80 software

214 Cell Culture HeLa cells were grown at 37∘C in Dul-beccorsquos modified Eaglersquos medium (DMEM GIBCO) supple-mented with 10 fetal bovine serum (FBS) in a humidifiedatmosphere consisting of 5CO

2and 95 air Cells were reg-

ularly passaged tomaintain exponential growth A day beforetransfection at 50ndash80 confluency cells were trypsinizedand diluted with fresh medium (sim1 times 105 cellsmL) and

transferred to 24-well plates (1mLplate) Four hours beforetransfection the medium was removed and 500120583L of freshmediumwas added Two luciferase plasmids firefly luciferase(pGL3) and Renilla luciferase (pRL-TK) from Promega wereused as control and reporter respectively For annealingof siRNAs 02 120583M of each strand was incubated using a100mM potassium acetate 30mM HEPES-KOH and 2mMmagnesium acetate buffer (pH = 74) The samples wereheated at 90∘C for 1min allowed to cool slowly to roomtemperature to induce annealing and then kept at 37∘C for1 hour Cotransfection of plasmids and siRNAs was carriedoutwith Lipofectamine 2000 (Life Technologies) as describedby the manufacturer for adherent cell lines Then 100120583Lcontaining the plasmids (10120583g pGL3 01 120583g pRL-TK) and thecorresponding siRNAs (18 nM) formulated into liposomeswere applied per well The final concentration of siRNAsper well was 03 nM The cells were harvested 20 hours aftertransfection and lysed using passive lysis buffer (100120583L perwell) according to the instructions of the Dual-LuciferaseReporter Assay System (Promega)The luciferase activities ofthe samples were measured using a MicroLuma Plus LB 96VLuminometer (Berthold Technologies) with a delay time of2 s and an integrate time of 10 s The following volumes wereused 20120583L of sample and 30 120583L of each reagent (LuciferaseAssay Reagent II and Stop and Glo Reagent) Ratios of targetto control luciferase were normalized to a buffer controlThe inhibitory effects generated by siRNAs duplexes wereexpressed as normalized ratios between the activities of thereporter Renilla reniformis (RL) and the control PhotinuspyralisGL3 (pGL3) luciferaseThe plotted data were averagedfrom three independent experiments plusmn sd

3 Results and Discussion

31 Synthesis of End-Capped Oligonucleotides L-threoninolwas selected as a linker to incorporate TTF pyrene orpentafluorophenyl moieties into DNA as illustrated inScheme 1 Threoninol has been used as a linker to intro-duce TTF pyrene or pentafluorophenyl groups at the ter-minal positions essentially as described [7 15 18] TwoTTF molecules were used with a methylene of differ-ence (Scheme 1) The synthesis of the threoninol deriva-tive of the [41015840551015840-tris (butylsulfanyl)-221015840-bis-13-dithiol-4-yl]sulfanylacetic acid (TTF1 [16]) was described previ-ously [7] Using a similar protocol the threoninol deriva-tive of the [41015840551015840-tris(butylsulfanyl)-221015840-bis-13-dithiol-4-yl]sulfanylpropionic acid (TTF2) was prepared in goodyields The threoninol derivative of the pentafluorophenyl(PFP) was prepared following the protocol described previ-ously for the threoninol derivative of pyrene (PYR) [15]

Oligonucleotide (DNA and RNA) sequences 1ndash16 shownin Table 1 were prepared using a DNARNA synthesizerFor the synthesis of TTF-containing RNA molecules 15 and16 the TTF2 derivative was selected As these oligonu-cleotides will be used in RNA interference experimentsthe extra methylene of the propionic acid derivative wasjudged to be potentially better in order to separate the TTFmolecule from the RNA sequence as this may influence inthe interaction with RISC [15] Using standard protocols


Table 1 Sequences of DNA and RNA oligonucleotides

No Sequence (51015840-31015840)(a)

1 TAG AGG CTC CAT TGC2 GCA ATG GAG CCT CTA3 TAG AGG CTC CAT TGC-TTF14 GCA ATG GAG CCT CTA-TTF15 TTF1-GCA ATG GAG CCT CTA6 TAG AGG CTC CAT TGC-PYR7 TAG AGG CTC CAT TGC-PFP8 CGC GAA TTC GCG9 CGC GAA TTC GCG-PYR10 CGC GAA TTC GCG-PFP11 auc uga aga agg aga aaa aTT12 uuu uuc ucc uuc uuc aga uTT13 gcu aca gag aaa ucu cga uTT14 auc gag auu ucu cug uag cTT15 auc uga aga agg aga aaa aTT-TTF216 uuu uuc ucc uuc uuc aga uTT-TTF2(a)TTF tetrathiafulvalene PYR pyrene PFP 23456-pentafluorophenylBase notation DNA = A C and G and T RNA = a g c and u

the desired conjugates were obtained in good yields TheHPLC profiles and mass spectrometry data are shown asSupporting Information

4 Melting Analysis

In a previouswork we described the effect of the TTF1moietylinked to the 51015840 or 31015840 ends of oligonucleotides on the stabilityof the correspondingDNAduplexes [7] Duplexes containinga TTF1 unit in each strand lead to either aggregation orstrong duplex stabilization depending on the relative positionof the TTF1 units and the concentration of salts Here weaimed to study the effect of the presence of the TTF1 uniton the melting temperatures of duplexes carrying a 120587-donorgroup such as pyrenyl or a 120587-acceptor group like 23456-pentafluorobenzyl Results are summarized in Table 2 The119879119898of the unmodified duplex (1+ 2) and duplexes containing

a single TTF1 unit or two TTF1 units were included in theTable for comparison purposes The melting experimentsperformed at 1MNaCl are shown in Figure 1 Interestingly noaggregationwas observed under any conditions except for theduplex 3+4 carrying TTF units in opposite ends as describedin reference [7] Duplexes 6 + 2 and 7 + 2 carrying a singlepyrenyl or 23456-pentafluorobenzyl group showed a clearincrease in stability as compared with 1 + 2 (Δ119879

119898ranging

between 28 and 42) Such a change is higher than thatobserved at the TTF1 duplexes 1 + 4 and 3 + 2 (Δ119879

119898ranging

between 08 and 28 Table 2)Therefore the stabilization wasslightly higher in the case of the pyrene-modified duplex6 + 2 probably owing to an end-capping mechanism [1ndash6]Duplexes 6+4 and 7+4 comprising both theTTF1 unit and thepyrenyl or 23456-pentafluorobenzyl group at the oppositeends maintained the degree of stabilization (Δ119879

119898ranging

between 49 and 54) which was found at the duplexes 6 + 2and 7 + 2 containing the aromatic moieties alone Whenthe TTF1 unit and the pyrenyl or 23456-pentafluorobenzyl

groups were placed at the same end of the duplexes 6 + 5 and7 + 5 a similar stability was observed (Δ119879

119898ranging between

28 and 49) than in the previous case of 6 + 2 and 7 + 2(Δ119879119898ranging between 28 and 42) In the same conditions

the duplex having two TTF1 units at the same end (3 + 4) hadthe highest increase on melting temperature (Δ119879

119898109 and

188) This indicates the strong interaction between two TTFmolecules

In addition we studied the effect on the thermal sta-bility of the duplexes carrying two pyrenyl groups or two23456-pentafluorobenzyl groups placed at opposite endsof a duplex To this end we synthesized the unmodifiedself-complementary oligonucleotides 8 and the modifiedself-complementary oligonucleotides 9 (a pyrenyl group atthe 31015840 ends) and 10 (a 23456-pentafluorobenzyl group atthe 31015840 ends) Results are summarized in Table 2 At lowsalt concentration (50mM NaCl) a broad transition wasobserved that can be due to the presence of two tran-sitions the duplex to hairpin transition followed by thedenaturation of the hairpin as described in a similar system[19] (Figure S5 C see Supplementary Material availableonline at httpdxdoiorg1011552013650160) At high saltconcentration (1M NaCl) a clear transition was observedPreviously if TTF1 was present at the end of this sequencethe formation of spherical aggregates was observed [7] Butnow with PYR and PFP groups we did not observe theformation of any aggregate So the formation of aggregates isa particular property of oligonucleotides bearing TTF units

When two PYR groups or two PFP groups were placed atopposite ends of the duplexes 9+9 and 10+10 we observed aclear increase in stability as compared with the nonmodifiedduplex (Δ119879

119898= 51 for the duplex with two pyrenyl units

and 35∘C for the duplex containing two PFP units) Againwe observed a higher stabilization in the case of the pyrene-modified duplex 9+ 9 probably owing to the aforementionedend-capping mechanism [1ndash6]

41 Gold Nanoparticle Functionalization with TTF1 Oligonu-cleotides In addition we studied the functionalization ofgold nanoparticles with oligonucleotides containing theTTF1 modification As oligonucleotides carrying thiol anddithio groups are used to functionalize citrate-stabilized goldnanoparticles [10ndash12] we consider that the presence of severalthioether functions in TTF will be also useful for anchoringTTF-oligonucleotides onto the surface of gold nanoparticlesFor this purpose oligonucleotides 3 and its complementary4 carrying a TTF1 modification at the 31015840 ends were usedImmobilization of the modified oligonucleotides on 10 nmcitrate-stabilized gold nanoparticles was carried out usingpreviously described protocols that consists in the addition ofthe TTF-oligonucleotides to the AuNp suspension followedby slow increase of the salt concentration to reach 015MNaCl [11] The resulting material is then centrifuged and theexcess of TTF-oligonucleotide is washed out The resultingfunctionalized gold nanoparticles were characterized by UV-vis spectroscopy (Figure 2(a)) After modification only asmall shift in the surface plasmon band was observed (from120582max 519 to 522 for 2-AuNp and from 120582max 519 to 523 for3-AuNp) The effect of the TTF1-modified oligonucleotides


Table 2 Melting temperatures of duplexes carrying pyrenyl (PYR) 23456-pentafluorophenyl (PFP) or tetrathiafulvalene (TTF1) units

Oligonucleotides 50mM119879119898(∘C) (Δ119879

119898)(b) 1M

119879119898(∘C) (Δ119879

119898)(b)

NaCl(a) NaCl(a)

1 + 2 Duplex 558 (0) Duplex 674 (0)

1 + 4 TTF1 Duplex 586 (28) Duplex 695 (21)

3 + 2TTF1

Duplex 572 (14) Duplex 682 (08)

3 + 4TTF1

TTF1 Duplex 589 (31) Aggregate mdash

3 + 5TTF1TTF1 Duplex 667 (109) Duplex 862 (188)

6 + 2 PYR Duplex 600 (42) Duplex 715 (41)

7 + 2PFP

Duplex 583 (30) Duplex 702 (28)

6 + 4PYRTTF1 Duplex 609 (51) Duplex 728 (54)

7 + 4PFP

TTF1 Duplex 607 (49) Duplex 726 (52)


7 + 5PFPTTF1 Duplex 586 (28) Duplex 710 (36)

8 Duplex Broad Duplex 627 (0)

9PYR

PYR Duplex Broad Duplex 678 (51)

10PFP

PFP Duplex Broad Duplex 662 (35)(a)The shape of the UV spectra ldquoDuplexrdquo indicates that a standard UV-vis spectrum of a duplex was observed and ldquoAggregaterdquo indicates that a broad UV-visspectrum with a large scattering was measured (b)Δ119879

119898= 119879119898of a modified duplex minus 119879

119898of an unmodified duplex under otherwise the same conditions

The arrows indicate the oligonucleotide orientation 51015840rarr 31015840 Broad indicates a broad transition (supporting information Figure S5)

on the stability of the conjugates was investigated followingprocedures described in the literature [20] Each conju-gate was treated with a DTT solution (10mM) at 40∘Cand left to aggregate Stability was evaluated by measuringthe increment of absorbance at 675 nm that resulted fromgold nanoparticle aggregation The half-time 119905

12(the time

required to reach half the value for complete aggregation)was less than 1min (data not shown) similar to othernanoparticles functionalized with disulfide bonds [20]

Moreover we performed an hybridization assay with goldnanoparticles functionalized with complementary oligonu-cleotides Assuming that the degree of gold nanoparticlefunctionalization was similar in each case we prepared a37 nM solution of each conjugate The solution was incu-bated at 4∘C for 48 h The UV-vis spectrum was mea-sured after annealing at 15∘C A plasmon band shift from522523 to 525 nm was observed indicating that there isplasmonic interaction among the assembled AuNPs in theannealed solution Then a melting experiment at 523 nmwas performed (Figure 2(b)) We observed a decrease in theabsorbance with increasing temperature We estimated themelting temperature as the midpoint of the curve (sim61∘C)TheUV-vis spectrumwasmeasured after themelting analysisat 85∘C (Figure 2(a)) Comparing with the UV-vis spectrumrecorded before annealing we observed a small decrease inthe absorbance and a shift of the plasmon band from 525 to523 nm This behaviour is different from gold nanoparticles

functionalized with thiol oligonucleotides that have largerchanges in the visible spectra after hybridization [10ndash12]but it is in agreement with the small changes in the spectraobservedwith gold-nanoparticles organized byDNAorigami[21] For these reasons we believe that hybridization of goldnanoparticles with TTF-oligonucleotides induces a smallnumber of contacts between AuNPs and for this reason thechanges in the plasmon band are small In any case thestability of the DNA-TTF-functionalized gold nanoparticlesis high as we do not observe changes in the aggregation stateof the nanoparticles over the time

42 Inhibition of the Luciferase Expression by siRNAsModifiedwith TTF2 Units The presence of aromatic groups at the 31015840ends of siRNAs has been previously investigated to stabilizethe interactions between the 31015840 ends of siRNA and thehydrophobic pocket of the PAZ domain of RISC [15] Inaddition aromatic groups at the 31015840end of siRNA efficientlyprotect siRNA from degradation of exonucleases present inthe serum [15] For these reasons we wanted to study theeffect of the TTF modification at the 31015840end of the siRNA Weselected the TTF2 modification at the 31015840 ends of the duplexsiRNAs and we analyzed the inhibition of the luciferase genein HeLa cell line As described before the extra methyleneof the propionic acid derivative was judged to be potentiallybetter to separate the TTF molecule from the RNA sequenceas this may influence in the interaction with RISC [15]


100

095

090

085

080

0 20 40 60 80 100

TTF1X

1 versus 23 versus 4


Abs

(260

nm) n

orm

aliz

ed

T (∘C)

(X = TTF1 PYR or PFP)

(a)

100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(b)

100

085

080

090

095

075

0 20 40 60 80 100

XX

(X = PYR or PFP)

8910

Abs

(260

nm) n

orm

aliz

ed

T (∘C)

(c)

Figure 1 Melting curves of unmodified 15mer duplex 1 + 2 and duplexes carrying aromatic groups (a) at opposite sites and (b) at the sameduplex site (c) Melting curves of unmodified self-complementary duplex 8 and duplexes carrying two pyrene (9) or two pentafluorophenyl(10) units at opposite sites Buffer 1M NaCl 10mM sodium phosphate buffer pH = 7

Theduplexes used in this studywereDUP1 (12+15 passengerstrand modified with TTF2) DUP2 (11 + 16 guide strandmodified with TTF2) and DUP 3 (15 + 16 both strandsmodified with TTF2) The siRNAs sequences were designedto inhibit Renilla reniformis luciferase In addition controlexperiments were performed with the unmodified duplex(WT 11 + 12) and with a scrambled siRNA (SCR 14 + 15)The Renilla luciferase inhibitory activity of modified siRNAswas evaluated using the dual-luciferase assay system afterincubation with the corresponding siRNAs for 20 hours at03 nM siRNAconcentrationThe inhibitory effects generatedby siRNAs duplexes were expressed as normalized ratios

between the activities of the reporter Renilla reniformis (RL)and the control Photinus pyralis GL3 (pGL3) luciferase

Figure 3 shows the amount of Renilla luciferase producedafter 20 hrs of transfection with 03 nM unmodified siRNA(WT) or 03 nM of the same siRNA duplex carrying TTF2 atthe 31015840 ends of passenger (DUP1) or guide strand (DUP2) ormodified in both strands (DUP3) A scrambled siRNAduplexcontrol sequence (Scr) was used as negative control siRNAmodified with TTF2 at the 31015840 ends of the passenger strandproduced an inhibition of the production ofRenilla luciferaseof 60 compared to the negative controls that was less effi-cient than unmodified siRNA (75 inhibition) By contrast


400 500 600 700 800

00

05

Abs

120582 (nm)

Gold colloid3-AuNp4-AuNp

After annealing at 15∘CAfter annealing at 85∘C

(a)

10 20 30 40 50 60 70 80 90 100041

042

043

044

045

046

047

048

At 523 nm

Abs

(523

nm)

T (∘C)

(b)

Figure 2 (a) UV-vis absorbance spectra of gold colloid (black) 3-AuNp 4-AuNp conjugates (red and blue resp) and 3-AuNp+4-AuNp afterannealing measured at 15∘C (green line) and at 85∘C (pink line) (b) Melting curve of 3-AuNp+4-AuNp measured at 523 nm

Bu Scr WT DUP1 DUP2 DUP300

02

04

06

08

10

12

siRNA

Nor

mal

ized

Rr-

lucPp

-luc

Figure 3 RNA interference activity of TTF2-modified siRNAsDUP 1 (siRNA carrying a TTF2 unit at the 31015840-end of passengerstrand) DUP2 (siRNA carrying a TTF2 unit at the 31015840-end of theguide strand) and DUP3 (siRNA carrying a TTF2 unit at both guideand passenger strands) WT (unmodified siRNA wild type) Scr(scrambled siRNA) Bu buffer alone Renilla reniformis luciferaseactivity (Rr-luc) (target) versus Photinus pyralis pGL3 luciferaseactivity (Pp-luc) (control) after 20 hrs incubation of siRNA (03 nM)designed against Renilla gene in transiently transfected Hela cellsRatios of target to control were normalized to a buffer control (Bu)

siRNAs containing TTF2 at the 31015840 ends of the guide strand orin both 31015840 ends of guide and passenger strands were clearlyless efficient (52ndash57 inhibition) This result is in agreementwith previous observations on other aromatic derivatives atthe 31015840 ends of the guide strand in the inhibition of a luciferasegene [15] In general it can be concluded that the introductionof TTF at the 31015840 ends can be tolerated by the RNAimachinery

only if the passenger strand of an RNA duplex is function-alized Although the results were not very promising thehydrophobicity of the TTFderivatives can provide some extraadvantage in the preparation of lipid formulations as it hasbeen described by cholesterol-modified conjugates Furtherwork in this direction is needed to preclude the use of theTTF-modified siRNA in RNA interference

5 Conclusions

In summary we described the effect on the hybridizationproperties of 51015840- or 31015840-end-modified oligonucleotides withseveral planar aromatic compounds such as tetrathiaful-valene (TTF) pyrene (PYR) and pentafluorophenyl (PFP)groups All these compounds induce the stabilization of DNAduplexes by end-capping mechanism as the PYR group themost stabilizing group followed by the PFP and being theTTF group the less stabilizing in duplexes carrying a singlemodification In contrast when two TTF groups are located atthe same terminus themodified duplex is highly stabilized asa result of a strong 120587-120587 stacking interaction between the twoTTF units This interaction is less stable with the PYRTTFpair and even less with the PFPTTF pair When two TTFgroups are located at opposite ends aggregate formationoccurs indicating interstrand 120587-120587 stacking interactionsThisbehaviour is only observed in duplexes carrying two TTFunits All these results show the strong 120587-120587 stacking interac-tions between TTF molecules in water which are visualizedby DNA duplex thermal denaturation experiments

In addition TTF-modified oligonucleotides are shown tobe able to interact with gold nanoparticles producing stableoligonucleotide-TTF-gold nanoparticle conjugates Finallythe synthesis and gene inhibitory properties of TTF-modifiedsiRNAs are described for the first time showing that the


modification of the 31015840 ends of the passenger strand generatesthe most active siRNAs derivatives

Acknowledgments

The authors thank Dr Anna Avino for providing the non-modified oligonucleotides 11ndash14This researchwas supportedby the European Commission (Grants FP7-FUNMOL 213382and NMP4-LA-2011-262943 MULTIFUN) by the SpanishMinistry of Education (grant CTQ2010-20541 SAF2010-15440) the Generalitat de Catalunya (2009SGR208) theCzech Science Foundation (P207102214) the Ministryof Education Youth and Sports of the Czech Republic(7E09054) and the Institute of Organic Chemistry andBiochemistry AS CR (RVO 61388963) The authors declareno conflict of interests

References

[1] KMGuckian B A Schweitzer R X-F Ren et al ldquoExperimen-tal measurement of aromatic stacking affinities in the context ofduplexDNArdquo Journal of the American Chemical Society vol 118no 34 pp 8182ndash8183 1996

[2] S Narayanan J Gall and C Richert ldquoClamping down on weakterminal base pairs oligonucleotides with molecular caps asfidelity-enhancing elements at the 51015840- and 31015840-terminal residuesrdquoNucleic Acids Research vol 32 no 9 pp 2901ndash2911 2004

[3] Z Dogan R Paulini J A R Stutz S Narayanan andC Richert ldquo51015840-Tethered stilbene derivatives as fidelity- andaffinity-enhancing molulators of DNA duplex stabilityrdquo Journalof the AmericanChemical Society vol 126 no 15 pp 4762ndash47632004

[4] S Egetenmeyer and C Richert ldquoA 51015840-cap for DNA probesbinding RNA target strandsrdquo Chemistry vol 17 no 42 pp11813ndash11827 2011

[5] A Zahn and C J Leumann ldquoRecognition properties of donor-and acceptor-modified biphenyl-DNArdquo Chemistry vol 14 no4 pp 1087ndash1094 2008

[6] A Avino S Mazzini R Ferreira and R Eritja ldquoSynthesisand structural properties of oligonucleotides covalently linkedto acridine and quindoline derivatives through a threoninollinkerrdquo Bioorganic and Medicinal Chemistry vol 18 no 21 pp7348ndash7356 2010

[7] S P Perez-Rentero I Gallego A Somoza et al ldquoInterstrandinteractions on DNA duplexes modified by TTF units at the 31015840or 51015840-endsrdquo RSC Advances vol 2 no 10 pp 4069ndash4071 2012

[8] J Yamada and T Sugimoto TTF Chemistry Fundamentals andApplications of Tetrathiafulvalene Springer Berlin Germany2004

[9] J L Segura and N Martın ldquoNew concepts in tetrathiafulvalenechemistryrdquo Angewandte Chemie no 8 pp 1372ndash1409 2001

[10] S J Hurst A K R Lytton-Jean and C A Mirkin ldquoMaximizingDNA loading on a range of gold nanoparticle sizesrdquo AnalyticalChemistry vol 78 no 24 pp 8313ndash8318 2006

[11] Z Li R Jin C A Mirkin and R L Letsinger ldquoMultiple thiol-anchor capped DNA-gold nanoparticle conjugatesrdquo NucleicAcids Research vol 30 no 7 pp 1558ndash1562 2002

[12] L M Demers C A Mirkin R C Mucic et al ldquoA fluorescence-based method for determining the surface coverage andhybridization efficiency of thiol-capped oligonucleotides bound

to gold thin films and nanoparticlesrdquo Analytical Chemistry vol72 no 22 pp 5535ndash5541 2000

[13] J K Watts G F Deleavey and M J Damha ldquoChemicallymodified siRNA tools and applicationsrdquoDrug Discovery Todayvol 13 no 19-20 pp 842ndash855 2008

[14] Y-L Chiu andTMRana ldquosiRNA function inRNAi a chemicalmodification analysisrdquo RNA vol 9 no 9 pp 1034ndash1048 2003

[15] A SomozaM Terrazas and R Eritja ldquoModified siRNAs for thestudy of the PAZ domainrdquo Chemical Communications vol 46no 24 pp 4270ndash4272 2010

[16] B-T ZhaoM-J Blesa F Le Derf et al ldquoCarboxylic acid deriva-tives of tetrathiafulvalene key intermediates for the synthesisof redox-active calixarene-based anion receptorsrdquo Tetrahedronvol 63 no 44 pp 10768ndash10777 2007

[17] H Asanuma K Shirasuka T Takarada H Kashida and MKomiyama ldquoDNA-dye conjugates for controllable Hlowast aggrega-tionrdquo Journal of the American Chemical Society vol 125 no 8pp 2217ndash2223 2003

[18] J Rybacek M Rybackova M Hoslashj et al ldquoTetrathiafulvalene-functionalized triptycenes synthetic protocols and elucidationof intramolecular Coulomb repulsions in the oxidized speciesrdquoTetrahedron vol 63 no 36 pp 8840ndash8854 2007

[19] A Avino S Perez-Rentero A V Garibotti M A Siddiqui V EMarquez and R Eritja ldquoSynthesis and hybridization propertiesofmodified oligodeoxynucleotides carrying non-natural basesrdquoChemistry and Biodiversity vol 6 no 2 pp 117ndash126 2009

[20] S P Perez-Rentero S Grijalvo R Ferreira and R EritjaldquoSynthesis of oligonucleotides carrying thiol groups using asimple reagent derived from threoninolrdquo Molecules vol 17 no9 pp 10026ndash10045 2012

[21] B Ding Z Deng H Yan S Cabrini R N Zuckermannand J Bokor ldquoGold nanoparticle self-similar chain structureorganized by DNA origamirdquo Journal of the American ChemicalSociety vol 132 no 10 pp 3248ndash3249 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


HOHO

NH2

O O

O

O

HN

HN HN

DMT

OO

HN

O

O

O

O

O

O

DMTDMT

OHOH OH

a b

c de

TTF1 (a)

TTF2 (b)

PFP (c)

PYR (d)S

S

S

S

S

S

S

S

S

S

BuS

BuS

SBu

SBu

SBu

SBu

FF F

FF

NCP N

HCPG

Ra

N

Randashd Randashd

Randashd

(4a)

(1andash1d)

(3andash3d)

(2andash2d)

Scheme 1 Preparation of the solids supports and phosphoramidite 4a needed for the assembly of oligonucleotides (a) R-COOH EDCI1-hydroxybenzotriazole and DIEA (b) dimethoxytrityl chloride DMAP (c) succinic anhydride DMAP (d) hemisuccinate NH

2-controlled

pore glass (CPG) TBTU triethylamine (e) O-2-cyanoethyl-NN-diisopropyl chlorophosphoramidite

are frequently used to functionalize citrate-stabilized goldnanoparticles [10ndash12] The presence of eight thioether func-tions in TTF units prompted us to investigate the use of TTF-oligonucleotides for the functionalization of gold nanopar-ticles As expected TTF-modified oligonucleotides are ableto bind to citrate stabilized gold nanoparticles (AuNPs) pro-ducing stable AuNPs functionalized with oligonucleotidesFinally TTF-oligoribonucleotides have been synthesized toproduce siRNA duplexes carrying TTF units Chemicalmodification of the 31015840 ends of siRNA is an active area ofresearch aimed at increasing the stability of modified siRNA[13 14] aswell as stabilizing the interaction of siRNAswith thePAZ domain of RISC [15] In this work we demonstrate thatthe presence of the TTFmolecule is compatible with the RNA

interference mechanism for gene inhibition especially if theTTF unit is located at the 31015840 ends of the passenger strand

2 Materials and Methods

21 General Information All the standard phosphoramiditesand reagents forDNAsynthesis were purchased fromAppliedBiosystems and from Link Technologies L-threoninol andother chemicals were purchased from Sigma-Aldrich andFluka TTF1-threoninol and pyrenyl-threoninol derivativeswere prepared as described [7 15] [41015840551015840-Tris(butylsulf-anyl)-221015840-bi-13-dithiol-4-yl]sulfanyl acetic acid was pre-pared as described previously [16] Dry solvents were pur-chased from Sigma-Aldrich and Fluka and used as received










2O (043 g


2CO3(18 g




3) ] = 3512w 3091 w 2961 s 2932 s 2875 s


3)120582max (log 120576) = 264

(418) 334 nm (414) 1H NMR 120575H (500MHz CD2Cl2) 092


2Cl2) 1391

2219 3129 3237 3463 3654 12569 (=C-SCH2) 12844



C21H31O2S85710095 found 5710093



3


119891= 022 1H NMR 120575H (400MHz CDCl

3)


3)

1715 (CO) 1307 (C) 1281 (C) 1279 (C) 1255 (C) 1114 (C)1094 (C) 692 (CH) 653 (CH

2) 550 (CH) 369 (CH

2) 363

(CH2) 362 (CH

2) 323 (CH

2) 320 (CH

2) 219 (CH

2) 218

(CH2) 208 (CH

3) and 138 (CH






3OD) 410 (dq 119869 = 64 and 27Hz


119860119883= 65Hz and 119869

119861119883= 62HZ 2H) 330


CDCl3) 1581 (C) 1269 (C) 1267 (C) 1166 (C) 1114 (C)

691 (CH) 649 (CH2) 549 (CH) and 204 (CH

3) 19F NMR



11H11NO3F5([M+H]+) 3000653 found

3000660




3(50mL) and


3) 740minus682 (m


3)


2) 555 (CH

3) 538 (CH) 538

(CH) 370 (CH2) 363 (CH

2) 362 (CH

2) 322 (CH

2) 320

(CH2) 319 (CH

2) 219 (CH

2) 218 (CH

2) 202 (CH

3) 138


46H60NO5S8([M+H]+)

9622237 found 9622065


3) 739 (d 119869 = 72Hz 1H)


119860119861= 98Hz 119869

119860119883= 38Hz and 119869

119861119883= 33Hz


3) 1587 (C) 1576 (C) 1441 (C) 1353 (C) 1350 (C)


2) 654 (CH) 552 (CH

3) 541 (CH) 198


3) minus1407 (m 2F)


32H28NO5F5Na ([M + Na]+) 6241779 found

6241784


3(15mL)




2ODMF



18(46 times 50mm 25 120583m)

















32 software





3solution Then each


520= 76 times 10














No Sequence (51015840-31015840)(a)



4 Melting Analysis



119898ranging


119898ranging


119898ranging






119898109 and










119898)(b) 1M

119879119898(∘C) (Δ119879

119898)(b)

NaCl(a) NaCl(a)

1 + 2 Duplex 558 (0) Duplex 674 (0)


3 + 2TTF1

Duplex 572 (14) Duplex 682 (08)

3 + 4TTF1




7 + 2PFP

Duplex 583 (30) Duplex 702 (28)


7 + 4PFP





9PYR


10PFP






12(the time






100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(a)

100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(b)

100

085

080

090

095

075

0 20 40 60 80 100

XX

(X = PYR or PFP)

8910

Abs

(260

nm) n

orm

aliz

ed

T (∘C)

(c)






400 500 600 700 800

00

05

Abs

120582 (nm)



(a)

10 20 30 40 50 60 70 80 90 100041

042

043

044

045

046

047

048

At 523 nm

Abs

(523

nm)

T (∘C)

(b)



02

04

06

08

10

12

siRNA

Nor

mal

ized

Rr-

lucPp

-luc




5 Conclusions





Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










2O (043 g


2CO3(18 g




3) ] = 3512w 3091 w 2961 s 2932 s 2875 s


3)120582max (log 120576) = 264

(418) 334 nm (414) 1H NMR 120575H (500MHz CD2Cl2) 092


2Cl2) 1391

2219 3129 3237 3463 3654 12569 (=C-SCH2) 12844



C21H31O2S85710095 found 5710093



3


119891= 022 1H NMR 120575H (400MHz CDCl

3)


3)

1715 (CO) 1307 (C) 1281 (C) 1279 (C) 1255 (C) 1114 (C)1094 (C) 692 (CH) 653 (CH

2) 550 (CH) 369 (CH

2) 363

(CH2) 362 (CH

2) 323 (CH

2) 320 (CH

2) 219 (CH

2) 218

(CH2) 208 (CH

3) and 138 (CH






3OD) 410 (dq 119869 = 64 and 27Hz


119860119883= 65Hz and 119869

119861119883= 62HZ 2H) 330


CDCl3) 1581 (C) 1269 (C) 1267 (C) 1166 (C) 1114 (C)

691 (CH) 649 (CH2) 549 (CH) and 204 (CH

3) 19F NMR



11H11NO3F5([M+H]+) 3000653 found

3000660




3(50mL) and


3) 740minus682 (m


3)


2) 555 (CH

3) 538 (CH) 538

(CH) 370 (CH2) 363 (CH

2) 362 (CH

2) 322 (CH

2) 320

(CH2) 319 (CH

2) 219 (CH

2) 218 (CH

2) 202 (CH

3) 138


46H60NO5S8([M+H]+)

9622237 found 9622065


3) 739 (d 119869 = 72Hz 1H)


119860119861= 98Hz 119869

119860119883= 38Hz and 119869

119861119883= 33Hz


3) 1587 (C) 1576 (C) 1441 (C) 1353 (C) 1350 (C)


2) 654 (CH) 552 (CH

3) 541 (CH) 198


3) minus1407 (m 2F)


32H28NO5F5Na ([M + Na]+) 6241779 found

6241784


3(15mL)




2ODMF



18(46 times 50mm 25 120583m)

















32 software





3solution Then each


520= 76 times 10














No Sequence (51015840-31015840)(a)



4 Melting Analysis



119898ranging


119898ranging


119898ranging






119898109 and










119898)(b) 1M

119879119898(∘C) (Δ119879

119898)(b)

NaCl(a) NaCl(a)

1 + 2 Duplex 558 (0) Duplex 674 (0)


3 + 2TTF1

Duplex 572 (14) Duplex 682 (08)

3 + 4TTF1




7 + 2PFP

Duplex 583 (30) Duplex 702 (28)


7 + 4PFP





9PYR


10PFP






12(the time






100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(a)

100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(b)

100

085

080

090

095

075

0 20 40 60 80 100

XX

(X = PYR or PFP)

8910

Abs

(260

nm) n

orm

aliz

ed

T (∘C)

(c)






400 500 600 700 800

00

05

Abs

120582 (nm)



(a)

10 20 30 40 50 60 70 80 90 100041

042

043

044

045

046

047

048

At 523 nm

Abs

(523

nm)

T (∘C)

(b)



02

04

06

08

10

12

siRNA

Nor

mal

ized

Rr-

lucPp

-luc




5 Conclusions





Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



3(50mL) and


3) 740minus682 (m


3)


2) 555 (CH

3) 538 (CH) 538

(CH) 370 (CH2) 363 (CH

2) 362 (CH

2) 322 (CH

2) 320

(CH2) 319 (CH

2) 219 (CH

2) 218 (CH

2) 202 (CH

3) 138


46H60NO5S8([M+H]+)

9622237 found 9622065


3) 739 (d 119869 = 72Hz 1H)


119860119861= 98Hz 119869

119860119883= 38Hz and 119869

119861119883= 33Hz


3) 1587 (C) 1576 (C) 1441 (C) 1353 (C) 1350 (C)


2) 654 (CH) 552 (CH

3) 541 (CH) 198


3) minus1407 (m 2F)


32H28NO5F5Na ([M + Na]+) 6241779 found

6241784


3(15mL)




2ODMF



18(46 times 50mm 25 120583m)

















32 software





3solution Then each


520= 76 times 10














No Sequence (51015840-31015840)(a)



4 Melting Analysis



119898ranging


119898ranging


119898ranging






119898109 and










119898)(b) 1M

119879119898(∘C) (Δ119879

119898)(b)

NaCl(a) NaCl(a)

1 + 2 Duplex 558 (0) Duplex 674 (0)


3 + 2TTF1

Duplex 572 (14) Duplex 682 (08)

3 + 4TTF1




7 + 2PFP

Duplex 583 (30) Duplex 702 (28)


7 + 4PFP





9PYR


10PFP






12(the time






100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(a)

100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(b)

100

085

080

090

095

075

0 20 40 60 80 100

XX

(X = PYR or PFP)

8910

Abs

(260

nm) n

orm

aliz

ed

T (∘C)

(c)






400 500 600 700 800

00

05

Abs

120582 (nm)



(a)

10 20 30 40 50 60 70 80 90 100041

042

043

044

045

046

047

048

At 523 nm

Abs

(523

nm)

T (∘C)

(b)



02

04

06

08

10

12

siRNA

Nor

mal

ized

Rr-

lucPp

-luc




5 Conclusions





Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

















32 software





3solution Then each


520= 76 times 10














No Sequence (51015840-31015840)(a)



4 Melting Analysis



119898ranging


119898ranging


119898ranging






119898109 and










119898)(b) 1M

119879119898(∘C) (Δ119879

119898)(b)

NaCl(a) NaCl(a)

1 + 2 Duplex 558 (0) Duplex 674 (0)


3 + 2TTF1

Duplex 572 (14) Duplex 682 (08)

3 + 4TTF1




7 + 2PFP

Duplex 583 (30) Duplex 702 (28)


7 + 4PFP





9PYR


10PFP






12(the time






100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(a)

100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(b)

100

085

080

090

095

075

0 20 40 60 80 100

XX

(X = PYR or PFP)

8910

Abs

(260

nm) n

orm

aliz

ed

T (∘C)

(c)






400 500 600 700 800

00

05

Abs

120582 (nm)



(a)

10 20 30 40 50 60 70 80 90 100041

042

043

044

045

046

047

048

At 523 nm

Abs

(523

nm)

T (∘C)

(b)



02

04

06

08

10

12

siRNA

Nor

mal

ized

Rr-

lucPp

-luc




5 Conclusions





Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




3solution Then each


520= 76 times 10














No Sequence (51015840-31015840)(a)



4 Melting Analysis



119898ranging


119898ranging


119898ranging






119898109 and










119898)(b) 1M

119879119898(∘C) (Δ119879

119898)(b)

NaCl(a) NaCl(a)

1 + 2 Duplex 558 (0) Duplex 674 (0)


3 + 2TTF1

Duplex 572 (14) Duplex 682 (08)

3 + 4TTF1




7 + 2PFP

Duplex 583 (30) Duplex 702 (28)


7 + 4PFP





9PYR


10PFP






12(the time






100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(a)

100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(b)

100

085

080

090

095

075

0 20 40 60 80 100

XX

(X = PYR or PFP)

8910

Abs

(260

nm) n

orm

aliz

ed

T (∘C)

(c)






400 500 600 700 800

00

05

Abs

120582 (nm)



(a)

10 20 30 40 50 60 70 80 90 100041

042

043

044

045

046

047

048

At 523 nm

Abs

(523

nm)

T (∘C)

(b)



02

04

06

08

10

12

siRNA

Nor

mal

ized

Rr-

lucPp

-luc




5 Conclusions





Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



No Sequence (51015840-31015840)(a)



4 Melting Analysis



119898ranging


119898ranging


119898ranging






119898109 and










119898)(b) 1M

119879119898(∘C) (Δ119879

119898)(b)

NaCl(a) NaCl(a)

1 + 2 Duplex 558 (0) Duplex 674 (0)


3 + 2TTF1

Duplex 572 (14) Duplex 682 (08)

3 + 4TTF1




7 + 2PFP

Duplex 583 (30) Duplex 702 (28)


7 + 4PFP





9PYR


10PFP






12(the time






100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(a)

100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(b)

100

085

080

090

095

075

0 20 40 60 80 100

XX

(X = PYR or PFP)

8910

Abs

(260

nm) n

orm

aliz

ed

T (∘C)

(c)






400 500 600 700 800

00

05

Abs

120582 (nm)



(a)

10 20 30 40 50 60 70 80 90 100041

042

043

044

045

046

047

048

At 523 nm

Abs

(523

nm)

T (∘C)

(b)



02

04

06

08

10

12

siRNA

Nor

mal

ized

Rr-

lucPp

-luc




5 Conclusions





Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




119898)(b) 1M

119879119898(∘C) (Δ119879

119898)(b)

NaCl(a) NaCl(a)

1 + 2 Duplex 558 (0) Duplex 674 (0)


3 + 2TTF1

Duplex 572 (14) Duplex 682 (08)

3 + 4TTF1




7 + 2PFP

Duplex 583 (30) Duplex 702 (28)


7 + 4PFP





9PYR


10PFP






12(the time






100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(a)

100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(b)

100

085

080

090

095

075

0 20 40 60 80 100

XX

(X = PYR or PFP)

8910

Abs

(260

nm) n

orm

aliz

ed

T (∘C)

(c)






400 500 600 700 800

00

05

Abs

120582 (nm)



(a)

10 20 30 40 50 60 70 80 90 100041

042

043

044

045

046

047

048

At 523 nm

Abs

(523

nm)

T (∘C)

(b)



02

04

06

08

10

12

siRNA

Nor

mal

ized

Rr-

lucPp

-luc




5 Conclusions





Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(a)

100

095

090

085

080

0 20 40 60 80 100

TTF1X



Abs

(260

nm) n

orm

aliz

ed

T (∘C)


(b)

100

085

080

090

095

075

0 20 40 60 80 100

XX

(X = PYR or PFP)

8910

Abs

(260

nm) n

orm

aliz

ed

T (∘C)

(c)






400 500 600 700 800

00

05

Abs

120582 (nm)



(a)

10 20 30 40 50 60 70 80 90 100041

042

043

044

045

046

047

048

At 523 nm

Abs

(523

nm)

T (∘C)

(b)



02

04

06

08

10

12

siRNA

Nor

mal

ized

Rr-

lucPp

-luc




5 Conclusions





Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


400 500 600 700 800

00

05

Abs

120582 (nm)



(a)

10 20 30 40 50 60 70 80 90 100041

042

043

044

045

046

047

048

At 523 nm

Abs

(523

nm)

T (∘C)

(b)



02

04

06

08

10

12

siRNA

Nor

mal

ized

Rr-

lucPp

-luc




5 Conclusions





Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Research Article Biophysical and RNA Interference ...

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