Oligonucleotide duplexes containing 2′-amino-2′-deoxycytidines: thermal stability and chemical reactivity

20-24 Nucleic Acids Research, 1994, Vol. 22, No. 1 ©1994 Oxford University Press Oligonucleotide duplexes containing 2'-amino-2'-deoxycytidines: thermal stability and chemical reactivity Helle Aurup, Thomas Tuschl, Fritz Benseler, Janos Ludwig+ and Fritz Eckstein* Max-Planck-lnstitut fur Experimented Medizin, Hermann-Rein-Stra(3e 3, D-37075 Gottingen, Germany Received November 1, 1993; Revised and Accepted December 6, 1993 ABSTRACT INTRODUCTION The chemical modification of oligonucleotides has gained considerable importance. This is mainly due to their potential application as modulators of gene expression either as antisenseoligonucleotides, triple helices, RNA-decoys or ribozymes (for reviews see (1-6)) Modifications have been introduced at the bases, the phosphate and the sugar moieties of the oligomers, primarily to increase stability against degradation by nucleases but also in an effort to improve the thermal stability of the resultant duplexes, with either DNA or RNA. Although there is considerable information on the thermal stability of duplexes of oligonucleotides containing 2'-O-alkyl or 2'-fluorine substitutions (7-11) there is very little information on 2'-amino derivatives. We wish to report here on the thermal stability of duplexes consisting of oligodeoxy- or oligoribonucleotides containing 2'-amino-2'-deoxycytidines in one strand and natural 2'-deoxy- or ribonucleotides in the other strand. The chemical reactivity of the 2'-amino group for the attachment of dyes is also explored. MATERIALS AND METHODS I3 C-NMR spectra were recorded at 90.55 MHz on a Bruker WH360 spectrometer with d(4)-3-(trimethylsilyl)propionic acid sodium salt as the internal standard, 5 1.7 ppm. 31P spectra were recorded on the same instrument at 145.79 MHz with 85% aq. H3PO4 as external standard. For the pKa determination by 13C- NMR spectroscopy 50 mM solutions of the dinucleotide in 750 mM of either phosphate, citrate or glycine buffer adjusted to the pH given in fig. 2 were measured. Preparation of 2'-amino-2'-deoxyuridyl(3'—5')thymidyl phosphate N2'HTifluoroacetyl-2'-amino-2'-deoxyuridine (12) was converted to the 5'<)-(4,4'-dmiethoxytrityl)-N2'-trifluoroacetyl-2'-amino2'-deoxyuridine-3'-0-(/3-cyanoethyl N,N-diisopropylphosphoramidite) by standard methods (13). This compound (210.5 mg, 0.25 mmol) together with 3'-O-acetylthymidine (85.3 mg, 0.3 mmol) was dissolved in a solution of tetrazole in CH3CN (0.5 M, 5 ml). After 60 min an iodine solution (6 ml, 0.05 M in THF/pyridine/H2O 7:2:1, v/v) was added dropwise to the reaction mixture until the solution remained coloured. The reaction mixture was then stirred for 20 min and the excess iodine destroyed by addition of 5% aqueous NaHSO3 Water (10 ml) was then added and the solution concentrated to approximately 10 ml. Saturated methanolic ammonia (10 ml) was added, the solution maintained overnight at room temperature and then concentrated to 10 ml. After extraction with CH2C12 (10 ml), the aqueous phase was further concentrated to 5 ml and the product (DMT-on) purified by preparative reversed phase HPLC using a DuPont 8800 instrument equipped with a UV detector, preparative pump heads (40 ml) and a 8 ml injection loop connected to a column (21 cm x 30.5 cm) packed with LiChrospher 100 RP-18 (Merck, Darmstadt). A linear gradient of 14%-56% CH3CN in 50 mM triethyl ammonium bicarbonate (pH 7) in 20 min at a flow rate of 15 ml/min was used. Productcontaining fractions were collected and evaporated to dryness. The DMT-group was removed then by treatment of the residue with 80% acetic acid (10 ml) and by extraction with ethyl acetate. The dinucleotide was purified by reversed phase chromatography, this time with a linear gradient of 3.5%-28% CH3CN in 50 mM triethylammonium-bicarbonate (pH 7) in 20 min at a flow rate of 15 ml/min. Product-containing fractions were evaporated, coevaporated three times with methanol and dried over night in vacuum. Yield: 98 mg (72%). Proton-decoupled 31P NMR (H2O), -0.53; 13C NMR (H2O, adjusted to pH 9), 92.71 (C r > • To whom correspondence should be addressed + Thermal stabilities of ollgonucleotldes containing 2'-amino-2'-deoxycytldines were determined and compared to those of the unmodified ollgonucleotldes. The presence of the 2'-amlnonucleoside destabilised duplexes in a RNA as well as a DNA context at pH 7 as well as at pH 5. The pK, of the 2'-amlno group was determined by 13C-NMR spectroscopy to be 6.2. The reactivity of an oligonucleotide containing a 2'-amlnonucleoslde was exploited for the Incorporation of rhodamlne by Its Isothlocyanate derivative. Present address: Ribonetics, Rudolf-Wissel-StraBe 28, D-37079 Gdoingen, Germany Nucleic Acids Research, 1994, Vol. 22, No. 1 21 NH2U, s), 89.71 (C r , thym., s), 80.44 (C3., NH2U, d; JC4., 5.6 Hz), 75.08 (C3., thym., s), 70.02 (C5., thym., d, JC.P, 5.4 Hz), 61.11 (C r , NH2U, d; JC.P, 5.8 Hz), 65.82 (C5., NH2U, s), 42.94 (Cr, thym., s). Attachment of rhodamine to oligonucleotides After gel purification, 4.8 Ajgo units (54 nmol) of the oligoribonucleotide containing one 2'-amino-2'-deoxycytidine was dissolved in 100 /tl of 100 mM sodium borate buffer (pH 8.5) followed by the addition of 100 /tl of 23 mM rhodamine-5-isothiocyanate (Molecular Probes Inc., Eugene USA) in N,N-dimethylformamide. The reaction solution was incubated for 15 h at 25 °C in the dark. The oligoribonucleotide was recovered by precipitation at -20°C by the addition of 100 /tl of 1.2 M NaOAc (pH 5.8) and 800 /tl of absolute ethanol. After centrifugation, the pellet was redissolved in 120 /d of water and the oligonucleotide precipitated at -20°C by the addition of 80 /tl of 3 M NaO Ac (pH 5.8) and 800 /tl of absolute ethanol. Precipitation was repeated twice. The dye-conjugated oligoribonucleotide was dissolved in 200 /tl of water, mixed with 200 /tl of 7 M urea stopmix and loaded on denaturing, preparative 20% acrylamide gel. The rhodamine conjugated oligoribonucleotide migrated as a single red coloured band that was retarded by more than the equivalent of one nucleotide relative to the unreacted species. It was excised, and the oligonucleotide recovered by electroelution followed by Sep-pak purification (14). Yield: 3.3 A260 units (corrected for the rhodamine contribution at 260 nm), 69 %. X ^ (H2O), 258 and 553 nm; A ^ / A j ^ , 1.24. The rhodamine-labeled oligonucleotide was digested with snake venom phosphodiesterase and alkaline phosphatase, and the nucleoside mixture was analysed by reverse phase HPLC as described except that a gradient of acetonitrile from 3.5 to 70% in 22 min was used (14). The retention time for the rhodaminelabeled 2'-amino-2'-deoxycytidine in this mixture was 11.50 min which was identical with that of the separately prepared nucleoside. For comparison the retention time for 2'-amino-2'deoxycytidine was 1.69 min. Native gel purification of rhodamine labelled RNA duplex Neither precipitation, gel electrophoresis, reverse phase HPLC nor size ex (...truncated)