Proc. Natl. Acad. Sci. USAVol. 95, pp. 11047–11052, September 1998Chemistry, Biochemistry

Synthesis, biophysical properties, and nuclease resistanceproperties of mixed backbone oligodeoxynucleotidescontaining cationic internucleoside guanidinium linkages:Deoxynucleic guanidineyDNA chimeras

(antisenseyhybrid duplexycationic oligonucleotideyDNA cappingyexonuclease)

DINESH A. BARAWKAR AND THOMAS C. BRUICE*Department of Chemistry, University of California, Santa Barbara, CA 93106

Contributed by Thomas C. Bruice, July 27, 1998

ABSTRACT The synthesis of mixed backbone oligode-oxynucleotides (18-mers) consisting of positively charged gua-nidinium linkages along with negatively charged phosphodi-ester linkages is carried out. The use of a base labile-protecting group for guanidinium linkage offers a syntheticstrategy similar to standard oligonucleotide synthesis. Thenuclease resistance of the oligodeoxyribonucleotides cappedwith guanidinium linkages at 5* and 3* ends are reported. Thehybridization properties and sequence specificity of binding ofthese deoxynucleic guanidineyDNA chimeras with comple-mentary DNA or RNA are described.

The use of antisense oligodeoxyribonucleotides (ODNs) toregulate gene products requires the development of modifiedODNs possessing the properties of enhanced cellular uptake,nuclease resistance, and sequence specific hybridization tocomplementary RNAs. Numerous DNA structural analogueswith modified heterocycle, sugar, and phosphodiester back-bone moieties have been synthesized (1–3). Substantialprogress has been made toward successful backbone modifi-cations by using phosphorus and non-phosphorus groups (4,5). A number of modifications or replacements of phosphodi-ester linkages such as 29-f luoro-N-39-P59-phosphoramidates(6), 39-thioformacetals (7, 8), 29-O-Me methylene(methyl-imino) (9), 29-O-Me amide (10), 29-O-methylribonucleosidemethylphosphonate (11), and peptide nucleic acid (PNA) (12)have been shown to complement with DNA and RNA withsimilar or higher stability while maintaining the sequencespecificity. Except for the 29-f luoro-N39-P59-phosphorami-dates, these analogues are neutral and thus eliminate theelectrostatic repulsion of negative charges present in naturalDNA and RNA. An alternative approach, involves replace-ment of anionic phosphodiester groups by cationic linkages(13–17) or the use of oligonucleotides conjugated with posi-tively charged groups to provide zwitterionic DNA analogues(18–20). These ODNs show increased binding with comple-mentary DNA or RNA. Conceptually, replacement of anionicphosphodiester linkage by neutral or positively charged link-ages can modulate the net charge of antisense ODN complexand thereby may enhance its antisense properties (4).

Our ongoing research in this area is focused on the devel-opment of deoxynucleic guanidine (DNG) in which the neg-atively charged OOO(PO2

2)OOO backbone of DNA is re-placed by positively charged, achiralONHOC(ANH2

1)ONHO linkage (15–17) to provide very stable complexes (21, 22).As DNG is positively charged, it binds effectively to target

DNA or RNA because the repulsive electrostatic effects indouble-stranded DNA (dsDNA) would be replaced by attrac-tive electrostatic interactions in DNG:DNA or DNG:RNAduplexes. On the other hand, if electrostatic binding betweenpolycationic and polyanionic structures becomes more signif-icant than the specific interactions between heterocyclic bases,then binding becomes nonspecific and independent of com-plementary base pairing. To overcome this possible limitation,we propose to synthesize mixed backbone ODNs, to reduce thenet positive charge of poly(DNG). For this purpose, we havesynthesized fully protected guanidinium-linked dinucleosideincorporable into oligonucleotide (23). This allows us to insertpositive point charges into otherwise negatively charged ODNsproviding a net reduced charge to DNGyDNA chimeras (Fig.1) and rendering increased binding ability.

In this paper we report solid phase synthesis of chimericguanidiniumyphosphodiester (DNGyDNA) oligonucleotides.The thermal stability of duplexes and sequence-specific bind-ing of these DNGyDNA chimeras to their complementaryDNA or RNA strands are reported. The resistance of synthe-sized DNGyDNA chimeras to nucleolytic hydrolysis by exo-nuclease I has been studied. Importantly, DNA capped withguanidinium linkages proves to be stable to exonucleases.

MATERIALS AND METHODS

General. 1H, 13C, and 31P NMR spectra were recorded at400, 50, and 161.9 MHz, respectively; chemical shifts arereported in d (ppm) relative to CHCl3 (7.24 ppm) andDMSO-d6 (2.49 ppm), and for 31P NMR are given from H3PO4as an internal standard. IR spectra were measured by usingneat samples over NaCl plate for liquids and using the KBrtechnique for solids. Mass spectra were obtained by usingeither fast atom bombardment (FAB) or electrospray ioniza-tion (ESI) conditions. TLC was carried out on aluminium-backed silica gel 60 (F254) 0.25-mm plates (Merck). Columnchromatography was performed by using silica gel 230–400mesh from ICN.

Acetylisothiocyanate (Compound 3). Potassium thiocyanatewas finely powdered and dried in an oven at 100°C for 24 hr.To a suspension of dried potassium thiocyanate (15.71 g, 161.7mmol) in anhydrous benzene (15 ml), acetyl chloride (10 ml,140.6 mmol) was added. The suspension was refluxed for 5 hrat 90°C and was allowed to cool to room temperature (RT).Reaction was monitored by gas chromatography, whichshowed the disappearance of starting material. The superna-tant was decanted and then purified by vacuum distillation.The first fraction boiling at 60°C was discarded. The second

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Abbreviations: ODNs, oligodeoxyribonucleotides; DNG, de-oxynucleic guanidine; Rt, retention time; MMTr, monomethoxytrityl.*To whom reprint requests should be addressed. e-mail: tcbruice@

bioorganic.ucsb.edu.

11047

fraction boiling at 90°C contained the pure product. Yield was5.8 g (40.7%). The IR spectrum had peaks at 1965, 1740, 1230,and 1145 cm21. 1H NMR (CDCl3) 2.37, S, CH3. This singlet inisothiocyanate moved upfield compared with CH3COCl (2.66p). 13C NMR (CDCl3) 165.3, CO; 146.9, NCS, and 27.7, CH3.HRCI. myz 101.12 calculated for C3H3NOS (M 1 H)1

102.1361.N-Acetyl-N*-(3*-deoxythymidine-3*-yl)-5*-O-(4-methoxy-

phenyl)-diphenyl methyl thiourea (Compound 4). The solutionof 59-O-MMTr-39-amino-39-deoxythymidine 2 (1.02 g, 2 mmol)in anhydrous dicholoromethane (15 ml) was cooled in ice bath.To this solution, acetylisothiocyanate 3 (0.185 ml, 2.2 mmol)was added slowly. The reaction mixture was allowed to attainRT and stirred for 3 hr. Reaction was monitored by TLC,evaporated to dryness, and then purified by column chroma-tography by using CH2Cl2:MeOH solvent system. Yield was0.760 g (62%). TLC (CH2Cl2:MeOH, 95:5) Rf 5 0.35; IR (KBr)spectrum had peaks at 3330, 3200, 2106, 1678, 1473, 1277, and1085 cm21. 1H NMR (400 MHz DMSO-d6): d (ppm) 1.5 (s, 3H,CH3 Thiourea), 2.0 (s, 3H, CH3 Thym), 2.25–2.35 (m, 2H, 29and 299 H2), 3.2–3.4 (m, 2H, 59 and 599 H2), 3.8 (s, 3H,OOCH3MMTr), 4.1 (m, 1H, 49H), 5.1 (m, 1H, 39H), 6.2 (t, 1H, 19H),6.9 (d, 2H, MMTr), 7.2–7.4 (m, 12H, MMTr), 7.6 (s, 1H, 6-H),10.8 (d, 1H, exch NH Thiourea), 11.28 (s, 1H, exch NHThiourea), and 11.34 (s, 1H, exch NH Thym). (FAB) myz614.2119, calculated for C33H34N4O6S (M 1 H)1 615.2277.

N*-Acetyl-N-(3*-deoxythymidine-3*-yl)-N**-(5*-deoxythymi-dine-5*-yl)guanidine (Compound 7). To solution of 4 (0.400 g,0.65 mmol) and 59-amino-59-deoxythymidine (6) (0.156 g, 0.65mmol) in anhydrous dimethylformamide (DMF; 5 ml) wasadded anhydrous triethylamine (0.22 ml, 1.62 mmol) andHgCl2 (0.211 gm, 0.78 mmol). Reaction mixture was allowed tostir overnight, when black precipitate (ppt) of Hg2S separates

out. Reaction was monitored by TLC and filtered over celite,and filtrate was evaporated to dryness under reduced pressureresulting in a oily residue. This oil was then purified by silicagel column chromatography by using CH2Cl2:MeOH solventsystem. Pure compound elutes at 10% MeOH. Yield was 0.365g (68%). TLC (CH2Cl2:MeOH, 9:1) Rf 5 0.4; 1H NMR (400MHz DMSO-d6): d (ppm) 1.4 (s, 3H, CH3 Guan), 1.7 (s, 3H,CH3 Thym), 1.8 (s, 3H, CH3 Thym), 2–2.4 (m, 4H, 2 3 29-H2),3.2–3.4 (m, 4H, 2 3 59H2), 3.75 (s, 3H,OOOCH3 MMTr), 3.8(m, 1H, 49H), 3.95 (m, 1H, 49H), 4.15 (m, 1H, 39H), 4.8 (m, 1H,39H), 5.4 (m, 1H, exch, 39-OH), 6.2 (t, 1H, 19H), 6.4(t, 1H,19H), 6.9 (d, 2H, MMTr), 7.2–7.5 (m, 12H, MMTr), 7.6 (s, 1H,6-H), 10.3 (s, 1H, exch, NH Thym), 11.4 (2s, 2H, NH Guan).High-resolution mass spectrometry (FAB) myz 822.3384, cal-culated for C43H48N7O10 (M 1 H)1 822.3479.

Preparation of Amidite Building Block (Compound 1). To asolution of 7 (0.6 g, 0.73 mmol) in anhydrous dichloromethane(10 ml), diisopropylethylamine (0.5 ml, 2.92 mmol) and [Chlo-ro(diisopropylamino)-b-cyanoethoxyphosphine] was added.Reaction mixture was kept stirring at RT for 3 hr. Reaction wasmonitored by TLC and evaporated to dryness under vacuum.This amidite was coevaporated several times over anhydrousacetonitrile and was used without further purification. 31PNMR (DMSO-d6): d (ppm), 147.7 and 148.5. (FAB) myz1022.10, calculated for C52H64N9O11P (M 1 H)1 1021.4462.

Oligonucleotide Synthesis and Purification. RNA oli-gomers were obtained from the Integrated DNA Technologies(Coralville, IA). All of the oligodeoxynucleotides were syn-thesized on 1.3 mmol scale on a Pharmacia GA Plus DNAsynthesizer by using CPG support and base protected 59-O-(4,49-dimethoxytrityl)deoxyribonucleoside-3[-O-(diisopro-pylamino)-b-cyanoethylphosphoramidite] monomers andphosphoramidite dimer 1. Standard synthesis cycle with anextended coupling time (15 min) was used during coupling ofmodified phosphoramidite dimer 1; coupling efficiency of.95% was observed for this step. The final trityl was kept onfor purification purpose, and oligonucleotides were depro-tected with NH4OH at 60°C. Oligonucleotides containingguanidinium linkages were subjected to longer NH4OH treat-ment (48 hr, 60°C) so that guanidinium deprotection is com-plete. All oligonucleotides were purified by RP-HPLC onpreparative Alltech C-8 RP column. Solvent system used wassolvent A: 0.1 M triethylammoniumacetate, pH 7.0 and solventB: CH3CN. The gradient used was 0–2 min, 15% B; 2–12 min,25% B, and 12–45 min, 25% B at a flow rate of 4 mlymin. TheHPLC-purified oligonucleotides were then detritylated byusing 1 ml of 80% acetic acid (1 hr, RT), evaporated to drynessunder reduced pressure, redissolved in double distilled water,and further purified by size exclusion chromatography.

Thermal Denaturation Studies. The concentrations of nu-cleotide solutions were determined by using the extinctioncoefficients (per mol of nucleotide) calculated according tonearest neighbor approximation for the DNA. (24). «260 M21

cm21; 9–12, 40880; 13, 50750; 15, 50751, and 16, 50750. Allexperiments were conducted in 10 mM Na2HPO4 buffer pH7.1, and ionic strength was adjusted with NaCl (0, 10, and 100mM). The final concentration of oligonucleotide strands was 3mM. The solutions were heated to 95°C for 5 min and allowedto cool to RT slowly before being stored at 4°C overnight.Spectrophotometric measurements were performed at 260 nmon a Cary 1E UVyvis spectrophotometer equipped withtemperature-programming and a thermal-melting softwarepackage, using 1-cm path length quartz cuvettes at a heatingrate of 0.5°Cymin over the range of 10–80°C. Dry nitrogen gaswas flushed in the spectrophotometer chamber to preventmoisture condensation at temperatures below 15°C. Meltingtemperatures were taken as the temperature of half dissocia-

FIG. 1. Structure of internucleoside phosphodiester (DNA) andguanidium linkage (DNGyDNA). p indicates guanidinium linkage inDNGyDNA chimeras 9–11.

11048 Chemistry, Biochemistry: Barawkaw and Bruice Proc. Natl. Acad. Sci. USA 95 (1998)

tion and were obtained from first derivative plots. The Tmvalues are accurate to 6 0.5°C over reported values.

Stoichiometry of Binding. The stoichiometry of binding wasdetermined by the method of continuous variation (25). So-lutions containing the different molar ratios of DNGyDNA 10and complementary DNA 13 were heated to 90°C and allowedto cool slowly to 15°C. The total concentration of duplex wasalways 3 mm. The pH was maintained at 7.1 with 10 mMNa2HPO4 buffer while the ionic strength was held constant at10 mM with NaCl. The A at 260 nm of each solution wasmeasured by Cary 1E UVyvis spectrophotometer.

Base Composition Analysis. The base composition analysisof modified oligonucleotides were confirmed by enzymatichydrolysis (26). Oligonucleotides 9–12 (0.2 A254 unit) weredissolved in 20 mM TriszHCl (200 ml, pH 8.9) and treated withsnake venom phosphodiesterase (0.02 unit) and alkaline phos-phatase (0.8 unit) at 37°C for 12 hr. This hydrolyzate (30 ml)was analyzed on analytical C-8 RP-HPLC column and elutedby using 0.5%ymin gradient of CH3CN in 0.1 M TEAA, pH 7.0for 50 min at a flow rate of 1 mlymin. The retention times for(1) standard nucleosides were dC, 4.59 min; T, 7.12 min; dG,7.77 min; dA, 11.47 min, and for dinucleoside containingguanidinium linkage 8, 14.27 min (2) for enzymatic hydroly-zate of 9 were dC, 4.58 min; T, 7.13 min; dG, 7.77 min; dA,11.69 min, and TgT 8, 14.13 min. The corresponding peak areaswere used to calculate the base composition of oligonucleo-tides.

Exonuclease I Digestion Experiment. Oligonucleotides 9–12(0.2 OD) with phosphodiester or guanidinium linkages weretreated with exonuclease I (500 units) in 10 mM TriszHCl, pH9.0 (0.2 ml) containing 50 mM KCl. Reaction mixtures wereanalyzed on C-8 RP-HPLC column by using 0.5%ymin gradi-ent of CH3CN in 0.1 M tetraethylammonium acetate, pH 7.0,for 50 min at a flow rate of 1 mlymin. Time points were 0, 1,2, 6, and 12 hr for DNGyDNA oligonucleotides (9-11) and only0 and 1 hr for phosphodiester oligomer 12 because this wascompletely digested by that time.

RESULTS AND DISCUSSION

Synthesis. For incorporation of guanidinium internucleo-side linkages into ODN, we synthesized the phosphoramidite1 (Scheme 1). The guanidinium linkage of 1 remained pro-tected during ODN synthesis and can be deprotected at the endof the synthesis to give a positively charged guanidiniumlinkage. There are reports of the synthesis of N-substitutedguanidinium internucleoside linkages that are neutral andcannot be deblocked to give charged guanidinium (27, 28). Thesynthesis of 1 involves coupling of the 59-amino group of59-amino-59-deoxythymidine 6 with in situ-generated carbodi-imide 5, obtained from reaction of the acetyl-protected thio-urea 4 with mercury (II) in the presence of Triethylamine (29).The acetyl protected thiourea 4 was synthesized by using59-O-monomethoxytrityl-39-amino-39-deoxythymidine 2 andacetylisothiocyanate 3, in dichloromethane. The electron with-drawing nature of acetyl group on thiourea of 4 activates thecarbodiimide intermediate 5, facilitating the attack by the59-amine, and then this acetyl group acts as a protecting groupon the resulting guanidinium linkage of dinucleoside 7.

The acetyl protection of internucleoside guanidinium re-mains stable to conditions required for DNA solid-phasesynthesis, and the acetyl is removed during usual final depro-tection conditions of DNA synthesis. To confirm deprotectionof guanidinium linkage, 7 was treated with 35% ammoniumhydroxide at 55°C for 45 hr followed by detritylation to providethe deprotected guanidinium dinucleoside 8, in quantitativeyield (as determined by HPLC and characterized by FAB MS[(M 1 H)1 5 508]). The phosphotylation of 7, using [chloro-(diisopropylamino)-b-cyanoethoxyphosphine] provided the fi-nal phosphoramidite 1, which exhibited the characteristic 31P

NMR signals at 147.7 and 148.5 ppm. Thus, the ability toprotect the guanidinium linkage with an acetyl function andremove this function at completion of synthesis of DNGyDNAchimera makes it suitable for standard automated solid phasesynthesis.

The guanidinium linkage was incorporated into ODNs 9–11(Fig. 1) by using Pharmacia GA Plus DNA synthesizer. The‘‘standard synthesis cycle’’ with an extended time (15 min) wasused during the coupling of phosphoramidite 1. After com-pletion of the synthesis, ODNs 9–11 were purified by RP-HPLC and then detritylated and again purified by size exclu-sion. Electrospray mass spectroscopic analysis for ODN 59-T*TAGGGT*TA-39 (C92H113N39O46P6, 2686.9) indicated theexpected masses for the doubly charged C92H113N39O46P6 (M1 2H)21: 1343.4 and triply charged C92H113N39O46P6 (M 13H)31: 895.4 species confirming the presence of modifiedmoiety into purified DNGyDNA chimera.

To further insure that the guanidinium-linked nucleobaseshave survived the synthetic chemistry of solid phase synthesisby phosphoramidite approach, enzymatic hydrolysis of 9–11were carried out by using snake venom phosphodiesterasefollowed by alkaline phosphatase (26). The enzyme digest wasanalyzed by RP-HPLC, which indicated the presence ofdinucleoside 8, with guanidinium internucleoside linkage. Thecorresponding peak areas showed the correct base composi-tion of ODNs 9–11.

Duplex Formation by DNGyDNA Chimeras. The DNGyDNA chimeric ODNs 9–11 are 18-mers (Fig. 1) with eitherthree (at 59, 39, and center), two (at 59 and 39) or one (at center)guanidinium linkages. The duplexes were formed betweenDNGyDNA chimeric ODNs 9–11 with complementary DNA13 or RNA 15. The 18-mer ODNs containing all four nucleo-bases are non self-complementary and form only antiparallelduplexes. Before performing thermal denaturation experi-ments the stoichiometry of binding of DNGyDNA chimericODN and DNA was determined by the method of continuousvariation (25) to generate mixing curves of the absorbance vs.mol fraction of 10 and 13 (Fig. 2). Increasing mol fraction of13 to the 10 (in 10 mM Na2HPO4, pH 7.1 at 15°C) lowered theUV A at 260 nm an inflection point at 0.5 mol fractionindicated the formation of expected 1:1 stoichiometry forDNGyDNA:DNA duplex (10:13).

Tm values for unmodified DNA:DNA (12:13) andDNA:RNA (12:15) duplexes were determined. These Tmvalues serve for comparative purposes with the Tm values ofDNGyDNA chimeras (Table 1). As expected, the Tm wasobserved to increase with increase in salt concentration withDNA:DNA and DNA:RNA. The duplexes of DNGyDNAchimera with complementary DNA or RNA showed increasein A at 260 nm upon increasing temperature and showed thecharacteristic sigmoidal melting pattern (Fig. 3). Incorporationof three or one guanidinium linkages (ODN 9 and 11) has noeffect on hybridization properties with complementary DNA13, when there is no salt (cf. Exps. 3 and 5 with 1). As saltconcentration was increased (0–100 mM NaCl) stability ofduplexes 9:13 and 11:13 was found to decrease when comparedwith DNA:DNA (cf. Exps. 3 and 5 with 1). The duplex ofDNGyDNA chimeras 9 and 11 with complementary RNA 15showed destabilization at all salt concentrations (cf. Exps. 6and 8 with 2). When there are two guanidinium linkages, as inthe duplex of ODN 10 with complementary DNA 13, and inabsence of salt, there is 2°C and 2.8°C increase in Tm comparedwith DNA:DNA (12:13) and DNA:RNA (12:15) hybrid, re-spectively (cf. Exp. 4 with 1 and 2). In absence of salt, theduplex of DNGyDNA chimera 10 with complementary RNA15 exhibits similar stability as does the DNA:RNA duplex(compare Exp. 7 with 2). With increasing salt concentrationthe duplexes of 10 with complementary DNA as well as RNAexhibit a decrease in Tm (cf. Exp. 4 with 1 and Exp. 7 with 2).

Chemistry, Biochemistry: Barawkaw and Bruice Proc. Natl. Acad. Sci. USA 95 (1998) 11049

Our thermal denaturation results show that incorporation ofguanidinium internucleoside linkages into ODNs, either sep-arated by a few nucleotide units (as in 9) or at the centerposition (11), in absence of salt, has no effect on duplex

stability. On the other hand, incorporating guanidinium link-ages at 59 and 39 ends (10) shows stabilization of duplex withcomplementary DNA as well as RNA (with no salt). Thisshows the importance of the position of guanidinium linkages

11050 Chemistry, Biochemistry: Barawkaw and Bruice Proc. Natl. Acad. Sci. USA 95 (1998)

in ODN. As expected, increasing salt concentration (0–100mM NaCl) destabilizes duplexes consisting of DNGyDNAchimeras 9–11, with complementary DNA as well as RNA (cf.Exps. 3–5 with 1 and Exps. 6–8 with 2). This salt effect isopposite to that seen with DNA:DNA (12:13) and DNA:RNA(12:15) duplexes, where increasing salt concentration providesstability by masking the electrostatic opposition of negativecharges. Decreasing salt concentration allows the positivelycharged guanidinium of DNGyDNA chimera to become inti-mately salt paired with negatively charged phosphodiester ofcomplementary DNA or RNA. Thus, duplexes of DNGyDNAwith DNA and RNA are stabilized by lowering of the ionicstrength.

Sequence Specificity. To study the sequence specificity ofbinding of DNGyDNA chimera with complementary DNA orRNA, DNGyDNA chimeric ODN was allowed to form duplexwith complementary DNA 14 or RNA 16 containing one basemismatch (TyU instead of dAyA) at the center and then thestability of the duplex was monitored by thermal denaturation.The duplexes 12:14 (DNA:DNA) and 12:16 (DNA:RNA)exhibit (Table 1) 9.1°C and 4.2°C decrease in Tm, respectively,in comparison to fully complementary 12:13 and 12:15 du-plexes (cf. Exp. 9 with 1 and 10 with 2). Approximately thesame mismatch discrimination was observed for DNGyDNA:DNA hybrid (DTm 210 to 211°C, cf. Exps. 11–13 with3–5) whereas for DNGyDNA:RNA hybrid base mismatch,discrimination was almost double (DTm 29 to 211°C, cf. Exps.14–16 with 6–8). This clearly demonstrates that binding of

DNGyDNA chimera with complementary DNA and RNA issequence specific.

Stability of DNGyDNA Toward Exonuclease. ODNs cappedwith guanidinium internucleoside linkages are expected to beresistant to the cleavage by exonucleases. To investigate thisDNGyDNA, ODNs 9–11 were subjected to nucleolytic diges-tion by exonuclease I. The hydrolyzate was then analyzed byRP-HPLC. The control ODN 12 was found to be completelyhydrolyzed after 1 hr of incubation. The DNGyDNA chimera9 and 10 were found to be absolutely stable toward exonuclease1 digestion even after 12 hr of incubation. The DNGyDNAchimera 11, which contains only one guanidinium linkage atthe center of ODN, was found to be partially hydrolyzed after1 hr (at 0 hr ODN 11, on RP-HPLC gave Rt 32.8 min, and after1 hr Rt 29.7 min) and there was no further hydrolysis even after12 hr (Rt 29.7 min). This clearly shows that DNGyDNA ODNs9 and 10 having guanidinium linkages at 59 and 39 ends arecompletely stable to exonuclease 1. The partial hydrolysis ofODN 11, having guanidinium at the center indicates thatphosphodiester linkages around guanidinium are stable toexonuclease cleavage. Further investigations are in progress.

Conclusions. In this paper, we successfully have demon-strated the insertion of cationic internucleoside guanidiniumlinkage in place of negative phosphodiester linkages in DNA.For this purpose, we have used standard phosphoramiditechemistry and automated solid phase synthesis. The DNGyDNA chimera 10, with two terminal positive charges, showedenhanced binding to complementary DNA and RNA in ab-sence of salt. The binding of 10, with complementary DNA at10 mM NaCl (close to physiological conditions), was similar tounmodified DNA:DNA. The binding of DNGyDNA chimerasis highly sequence specific. The 59 and 39 capping of the DNAwith gaunidinium linkage provides protection to hydrolysis byexonuclease 1. The lower net negative charge of DNGyDNAchimera, arising from insertion of positive guanidinium link-ages, may assist the cellular uptake of these ODNs.

This work was initiated under support by the Office of NavalResearch (N00014-96-1-01232). We gratefully acknowledge continu-ing support by the National Institutes of Health (3 R37 DK09171–3451).

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Table 1. Duplex melting* temperatures (Tm°C)

Exp no. Duplex No NaCl 10 mM NaCl 100 mM NaCl

1 12:13 34.8 48.6 58.52 12:15 34.0 49.7 59.93 9:13 34.8 46.6 53.54 10:13 36.8 48.6 57.55 11:13 34.8 47.6 56.56 9:15 30.1 41.8 50.97 10:15 34.2 45.9 57.18 11:15 31.2 43.9 54.09 12:14 — 39.5 —

10 12:16 — 45.5 —11 9:14 — 35.5 —12 10:14 — 38.5 —13 11:14 — 37.6 —14 9:16 — 30.5 —15 10:16 — 35.5 —16 11:16 — 34.6 —

*Buffer used: 10 mM Na2HPO4, pH 7.1 with differant NaCl concen-tration (0–100 mM).

Chemistry, Biochemistry: Barawkaw and Bruice Proc. Natl. Acad. Sci. USA 95 (1998) 11051

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