Bioconjugate Chem. 2003, 14, 919−926
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Aminomodified Nucleobases: Functionalized Nucleoside Triphosphates Applicable for SELEX Thomas Schoetzau,† Josmar Langner,† Elisabeth Moyroud, Ingo Roehl, Stefan Vonhoff, and Sven Klussmann* NOXXON Pharma AG, Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany. Received December 13, 2002; Revised Manuscript Received March 24, 2003
5-Aminoallyl-2′-fluoro-dUTP, 5-aminoallyl-UTP, and N6-([6-aminohexyl]carbamoylmethyl)-ATP were systematically tested for their suitability for the systematic evolution of ligands by exponential enrichment (SELEX) process with the aim of introducing additional functionalities to RNA libraries. All three aminomodified nucleoside triphosphates proved to be compatible with the enzymatic steps required for SELEX and maintained strict Watson-Crick basepairing. Complementary RNA molecules modified with the two uridine analogues show a significantly increased melting temperature, whereas the introduction of N6-([6-aminohexyl]carbamoylmethyl)-ATP leads to a decreased Tm and thus less stable basepairing. The chemical synthesis of 5-aminoallyl-2′-fluoro-dUTP is reported in detail.
INTRODUCTION
In vitro selection of oligonucleotides, also known as systematic evolution of ligands by exponential enrichment (SELEX), was developed independently by Joyce (1), Szostak (2), and Gold (3) in the late 1980s and early 1990s. SELEX has been successfully used to obtain aptamers that bind to various molecular targets derived from many different substance classes from combinatorial nucleic acid libraries. In addition to mere binding, new ribozymes and even deoxyribozymes were selected to catalyze various reactions such as cleavage of DNA (4), ligation of RNA (5), Diels-Alder reactions (6), peptide bond formation (7), or the synthesis of pyrimidine nucleotides (8). While the sequence and structural complexity of nucleic acid libraries is high, their chemical and biophysical diversity is limited since they comprise only four different nucleotide monomers. This stands in contrast to peptides and libraries thereof, which consist of 20 different amino acids with side chains, ranging from lipophilic to hydrophilic, charged and reactive, like carboxyl, sulfhydryl, hydroxyl, guanidinium, and ammonium groups. These amino acid side chains mediate the functional variety and diversity of peptides and proteins. In analogy, modifying their constituent nucleobases is expected to enhance the binding and functional repertoire of nucleic acids. The enzymatic generation of modified aptamers is expected to be less demanding for DNA than for RNA, where reverse transcription is required. Thus, only one enzyme, a thermostable DNA polymerase, is involved, that has to accept the modified dNTPs. In the context of DNA SELEX a series of modifications such as C5aminomodified dUTP analogues, Lee et al. (9), 5-(1pentynyl)-dUTP, Latham et al. (10), several highly functionalized dUTP derivatives, Sakthivel and Barbas (11), and C7-aminomodified 7-deaza-dATP, Gourlain et al. (12), are closely related to our work. Perrin et al. (13) reported the simultaneous use of 5-(3-aminoallyl)-dUTP * To whom correspondence should be addressed: sklussmann@ noxxon.net. † Both authors contributed equally.
and 8-(2-(4-imidazolyl)ethylamino)-dATP, Battersby et al. (14) employed 5-(3-aminopropynyl)-dUTP, and Santoro et al. (15) described C5-imidazole-functionalized dUTP. An overview of more modifications with expanded chemical functionalities is given by Bittker et al. (16). For RNA SELEX modified NTPs have to be accepted and efficiently incorporated by RNA polymerases, commonly derived from phages, that seem to be more substrate-specific than thermostable DNA polymerases. In addition, RNA transcripts have to be converted to cDNA by reverse transcriptases. In both reactions, strict Watson-Crick basepairing is essential to ensure fidelity and to keep the selected information. Apart from various publications on 2′-modified NTPs, Vaish et al. (17) successfully tested 5-(3-aminopropyl)-UTP and 5-(2-mercaptoethyl)-UTP in RNA SELEX. Dewey et al. (18) used several 5-modified UTP derivatives, Ito (19) and Ito et al. (20) employed N6-modified ATP and N4-biotin-linkedCTP, and Zinnen et al. (21) introduced 2′-F-5-[(N-imidazole-4-acetyl) propylamine]-dUTP. Here we report in detail about the synthesis of 5-aminoallyl-2′-fluoro-2′-deoxyuridine 5′-triphosphate 7. We compare RNA transcription yields in the presence of 7 with 5-aminoallyl-UTP 8 and N6-([6-aminohexyl] carbamoylmethyl)-ATP 9. Moreover, we show the compatibility of all three aminomodified triphosphates with the general SELEX process and discuss the thermal melting behavior of such modified RNA molecules. EXPERIMENTAL PROCEDURES
General. Aminomodified nucleoside triphosphates 8 and 9 were purchased from Sigma. Anion exchange chromatography was performed on Sephadex DEAE-A25 (400 × 26 mm), Amersham Biosciences. Analytical reversed phase HPLC was performed on Waters 2690 systems with PDA-UV detection from 200 to 300 nm and a 250 × 4 mm stainless steel column packed with Hypersil ODS (C18, 5 µm). A flow rate of 1 mL/min was used with 100 mM aq. TEAAc (pH 7) and a linear gradient of 0-25% acetonitrile. 1 H-NMR spectra were obtained on a 300 MHz Varian spectrometer using the respective solvent as internal
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standard. For 31P- and 19F-NMR spectra phosphoric acid and fluorotrichlormethane were used as external standards. NMR samples of nucleotide triphosphates were dissolved in D2O (99.9%), Aldrich. Mass spectral analysis of nucleoside triphosphates was obtained in negative mode on a Hewlett-Packard G2025A MALDI TOF with 3-hydroxy picolinic acid as matrix. Crotalus adamanteus venom phosphodiesterase I and calf intestine alkaline phosphatase were purchased from USB and Roche, respectively. Syntheses. 2′-Deoxy-5′-(4,4′-dimethoxytrityl)-2′-fluoroD-uridine 3. 2,2′-O-Anhydro-D-uridine 2 (100 mmol) was coevaporated with anhydrous pyridine (2 × 150 mL). Pixyl chloride (220 mmol) was added to a solution of 2 (100 mmol) in anhydrous pyridine (120 mL) and the solution was stirred at room temperature overnight. The reaction was quenched with water (50 mL) and was concentrated by evaporation. The residue was dissolved in DCM (200 mL) and washed with diluted NaHCO3 solution (2 × 100 mL). The organic phase was evaporated to dryness, suspended in methanol (1200 mL) and heated to reflux for 2 h with 2 M NaOH (250 mL). After 16 h at 4 °C, the mixture was evaporated to dryness. The residue was dissolved in DCM (300 mL) and washed with dil. NaHCO3 solution (2 × 200 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo to give 3′,5′di-O-pixyl-D-arabinofuranosyluridine as a yellowish foam (purity by TLC: >80%, Rf 0.41 (DCM/MeOH 95/5)). After drying of the sample overnight, the intermediate product (40 mmol) was suspended in a mixture of DMF and CH3CN (1:4, v:v, 250 mL) in a Teflon bottle. The suspension was cooled to 0 °C and (diethylamino) sulfur trifluoride (DAST) (0.2 mol) was slowly added under nitrogen. The clear brownish mixture was stirred for 10 min at 0 °C followed by 1 h at room temperature. NaF (0.8-1.6 mol) was added and the suspension was stirred at room temperature overnight. The reaction was cooled to 0 °C and TEA (20 mL) and methanol (60 mL) were added. After filtration and washing of the solid with methanol (3 × 60 mL), the filtrate was evaporated in vacuo, dissolved in DCM (200 mL), and extracted with water (2 × 50 mL). The organic phase was dried over Na2SO4 and evaporated to give 3′,5′-di-O-pixyl-2′-deoxy-2′-fluoro-Duridine as a brownish oil (Rf 0.66, DCM/MeOH 95/5). The crude compound (0.12 mol) was dissolved in a mixture of methanol and DCM (5:1, v:v, 1.2 L) and cooled to 0 °C. 1 M HCl (150 mL) was added and the mixture was stirred for 30 min at room temperature. The solution was neutralized with aq. 1 M NaOH solution and evaporated in vacuo. The residue was dissolved in water (400 mL) and extracted with DCM (3 × 200 mL). The aqueous layer was evaporated to dryness, and coevaporated with methanol (2 × 50 mL). The residue was dissolved in methanol (150 mL), and the precipitate (NaCl) was removed by filtration and washed with methanol (2 × 150 mL). After evaporation to dryness 2′-deoxy-2′-fluoroD-uridine (Rf 0.10, DCM/MeOH 9/1) was yielded. The crude uridine analogue (60 mmol) was coevaporated with dry pyridine (3 × 50 mL) and dissolved in dry pyridine (125 mL). DMAP (30 mmol) was added and DMTCl was added in portions (3 × 66 mmol). After 2 days at room temperature, methanol (20 mL) was added and the mixture was stirred for 30 min. The solvent was evaporated and the residue was dissolved in DCM (300 mL) and washed with 5% aq. NaHCO3 solution (3 × 100 mL). The organic layer was dried over Na2SO4 and evaporated. Silica gel chromatography using a gradient of 0 to 3% of MeOH in DCM containing 2% TEA gave 3 as a brownish foam (overall yield 20-30%, Rf 0.38, DCM/MeOH 95/5).
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3′-Acetyl-2′-deoxy-2′-fluoro-D-uridine 4. The nucleoside 4 was prepared according to a published procedure by 3′-acylation and 5′-detritylation of 3 in 81% overall yield (22). 2′-Deoxy-2′-fluoro-D-uridine 5′-triphosphate 5. The protected nucleoside 4 (158 mg, 550 µmol) was dried in vacuo overnight and dissolved in a mixture of anhydrous pyridine (550 µL) and dioxane (1650 µL) under argon. A freshly prepared 1 M solution of 2-chloro-4H-1,2,3dioxaphosphorin-4-one (167 mg, 825 µmol) in anhydrous dioxane (825 µL) was injected into the well-stirred solution. After 20 min at room temperature, a solution of 0.5 M tri-n-butylammonium pyrophosphate (439 mg, 963 µmol) in anhydrous DMF (1926 µL) and tri-nbutylamine (550 µL) was added to the reaction mixture. After 20 min, a solution of 1% iodine in pyridine/water (22 mL, 98:2, v:v) was added. After stirring of the sample for 20 min, excess iodine was quenched by adding a 5% aq. NaHSO3 solution (2 mL) and the reaction solution was evaporated to dryness. After treatment with concentrated ammonia (10 mL), the product was purified using a Sephadex DEAE A-25 column with a linear gradient of 0.05 to 1 M LiCl (triphosphate eluted at ∼0.7 M LiCl). This purification was followed by reversed phase HPLC on a 15RPC column (100 × 26 mm, Amersham Biosciences, buffer A: 100 mM TEAAc, pH 7; buffer B: 100 mM TEAAc, pH 7, 95% acetonitrile; gradient: t ) 0-8 min 1% B, t ) 8-55 min 1% f 50% B). The product containing fractions were lyophilized and transformed into lithium salt for NMR recording. Triphosphate 5 was obtained in 49% yield. 1H-NMR (D2O, 300 MHz): 7.97 (1 H, d, H-6), 6.10 (1 H, d, H-1′), 5.96 (1 H, d, H-5), 5.20 (1 H, d, H-2′), 4.64-4.29 (4 H, m, H-3′, H-4′, H-5′, H-5′′). 31 P-NMR (D2O, 122 MHz): -4.53 (d, γP), -10.27 (d, RP), -19.90 (t, βP). 19F-NMR (D2O, 284 MHz): -203.41 (ddd). MALDI-MS: calcd for C9H13FN2O14P3 (M-H)-: 485.1, found: 483.1. RP HPLC: tR ) 8.6 min. 5-Aminoallyl-2′-deoxy-2′-fluoro-D-uridine 5′-triphosphate 7. The nucleotide 5 (11 mg, 22 µmol) and mercuric acetate (38 mg, 119 µmol) were dissolved in 0.1 M sodium acetate buffer (2.1 mL, pH 6) and the solution was stirred for 4 h at 50 °C. LiCl (9 mg, 220 µmol) was added, and the solution was extracted with EtOAc (7 × 3 mL) and DCM (1 × 3 mL). The aqueous layer was cooled to 4 °C and ice-cold ethanol (30 mL) was added. After 30 min at -18 °C, the precipitate was collected by centrifugation for 30 min. The pellet was suspended in 10 mL of ethanol and the centrifugation was repeated to give nucleotide 6, which was used without further purification in the next step. A total of 165 µL of allylamine solution prepared by neutralizing allylamine (1.5 mL) with ice-cold 4 M acetic acid (8.5 mL) was added to a 20 mM solution of compound 6 (22 µmol) in 0.1 M sodium acetate (1.1 mL, pH 5). After addition of K2PdCl4 catalyst (7 mg, 22 µmol) in 220 µL of water, the mixture was stirred at room temperature for 1 day. The precipitate was removed by filtration through a 0.8 µm membrane filter (Nalge International). The product was purified using a Sephadex DEAE A-25 column with a linear gradient of 0.05 to 1 M LiCl (triphosphates eluted at ∼0.6 M LiCl). The purification was followed by reversed phase HPLC on a 15RPC column (100 × 26 mm, Amersham Biosciences, buffer A: 100 mM TEAAc, pH 7; buffer B: 100 mM TEAAc, pH 7, 95% acetonitrile; gradient: t ) 0-8 min 1% B, t ) 8-55 min 1% f 50% B). The product containing fractions were lyophilized and transformed into lithium salt for NMR recording. The triphosphate 7 was obtained in 29% yield. 1 H-NMR (D2O, 300 MHz): 8.17 (1 H, s, H-6), 6.60 (1 H,
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Figure 1. (A) Synthesis of nucleotide 7: (i) cyanamide, MeOH; (ii) methylpropiolate, EtOH aq; (iii) PxCl, Pyr; (iv) 1 M NaOH, MeOH; (v) DAST, NaF, DMF, ACN; (vi) 1 M HCl, MeOH; (vii) DMTCl, DMAP, Pyr; (viii) acetic anhydride, DMAP, Pyr; (ix) acetic acid; (x) 2-chloro-4H-1,2,3-dioxaphosphorin-4-one, dioxane, rt, 15 min, tri-n-butylammonium pyrophosphate, tributyl-amine, DMF, rt, 20 min; (xi) mercuric acetate, 0.1 M sodium acetate buffer (pH 6.0), 50 °C, 4 h; (xii) allylamine, K2PdCl4, 0.1 M sodium acetate buffer (pH 5), rt, 22 h; (B) Commercially available aminomodified NTPs used: 5-AA-UTP 8 and N6-([6-aminohexyl]-carbamoylmethyl)ATP 9.
d, CH), 6.48 (1 H, dt, CH), 6.19 (1 H, d, H-1′), 5.17 (1 H, dd, H-2′), 4.75-4.33 (4 H, m, H-3′, H-4′, H-5′, H-5′′), 3.74 (2 H, d, CH2). 31P-NMR (D2O, 122 MHz): -5.08 (d, γP), -10.29 (d, RP), -20.38 (t, βP). 19F-NMR (D2O, 284 MHz): -203.54 (ddd). MALDI-MS: calcd for C12H18FN3O14P3 [M-H]-: 540.2, found: 539.8. reversed phase HPLC: tR ) 9.4 min. Base Composition of RNA Transcripts. 0.1 OD260 of RNA transcript were incubated at 37 °C in 55 µL of buffer containing 90 mM Tris-HCl (pH 7.5), 110 mM NaCl, and 15 mM MgCl2 with 1.6 U alkaline phosphatase and 0.2 U snake venom phosphodiesterase for 16 h to achieve complete degradation. After precipitation with 2.5 vol of ethanol in the presence of 0.3 M sodium acetate, the pellet was discarded, the supernatant was vacuumdried, the residue was dissolved in 120 µL of H2O and subjected to reversed phase HPLC analysis. In Vitro Transcription of RNA. 17-mer/79-mer RNA transcripts were in vitro transcribed from chemically synthesized 36/98 base-pair dsDNA templates consisting of a minimal T7 promoter (positions -1 to -19) followed by the sequence of the transcript itself (positions 1-17/ 1-79). The 36-base-pair dsDNA T7 template for the sense 17-mer was annealed from the ssDNA molecules 5′-TCT AAT ACG ACT CAC TAT AGG ACT GAC TGA CTG ACC-3′ and 5′-GOMeGOMeT CAG TCA GTC AGT CCT ATA GTG AGT CGT ATT AGA-3′. The corresponding template to generate the antisense 17-mer transcript was annealed from the ssDNA molecules 5′-TCT AAT ACG ACT CAC TAT AGG TCA GTC AGT CAG TCC-3′ and 5′-GOMeGOMeA CTG ACT GAC TGA CCT ATA GTG AGT CGT ATT AGA-3′. The dsDNA template (Figure 2) to transcribe the
79-mer transcript was annealed from the ssDNA molecules 5′-TCT AAT ACG ACT CAC TAT AGG AGC TCA GCC TTC ACT GCG AGA GAG AGA GAG AGA GAG ACT CTC TCT CTC TCT CTC TCT GGC ACC ACG GTC GGA TCC AC-3′ and 5′-GTG GAT CCG ACC GTG GTG CCA GAG AGA GAG AGA GAG AGA GTC TCT CTC TCT CTC TCT CTC GCA GTG AAG GCT GAG CTC CTA TAG TGA GTC GTA TTA GA-3′. All ssDNA molecules were annealed to dsDNA in 10 mM Tris-HCl, pH 8. Typical 100 µL T7 transcription reactions with 2′fluoro-2′-deoxypyrimidine triphosphates contained 0.5 µM template DNA, 1 mM of each NTP, 10 mM DTT, 150 U of T7 RNA/DNA polymerase (Epicentre), 10 U RNaseOut ribonuclease inhibitor (Invitrogen), 0.1 U inorganic pyrophosphatase (Sigma) in 1× Epicentre reaction buffer (40 mM Tris-HCl, pH 7.5, 6 mM MgCl2, 10 mM NaCl, 2 mM spermidine) supplemented with MgCl2 to a final concentration of 12 mM. Typical 100 µL transcription reactions with all other modified NTPs contained 0.5 µM template DNA, 4 mM of each NTP, 10 mM DTT, 100 U of T7 RNA polymerase (Stratagene), 10 U RNaseOut ribonuclease inhibitor (Invitrogen), 0.1 U inorganic pyrophosphatase (Sigma) in standard T7 reaction buffer (80 mM HEPES-KOH, pH 7.5, 22 mM MgCl2, 1 mM spermidine). For radioactive body-labeling and subsequent quantification of transcript amounts on a Biorad FX Phosphorimager, 180.000-720.000 Bq [R-32P]GTP were added. Reactions were incubated for 14-16 h at 37 °C and treated with ∼30 U DNaseI per 100 µL for 10 min at 37 °C to degrade dsDNA template. Full-length transcripts were purified on 7 M urea, 10-20% polyacrylamide gels. Bands were visualized by 254 nm UV-
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Figure 2. (A) 98-mer dsDNA template for the 79-mer RNA test molecule with T7 forward and reverse primers. (B) Mfold (27) secondary structure prediction for the 79-mer RNA transcript.
shadowing on ALUGRAM SIL G/UV254 sheets (Macherey & Nagel), excised, and eluted with 30 mM sodium acetate, pH 5.5. Eluates were vacuum-concentrated to 10% of the initial volume and precipitated with 2.5 vol of an ethanol/2-propanol mixture (1:1, v:v) in the presence of 200 µg/mL Glycogen (Roche). RNA pellets were dissolved in water and quantified UV-spectroscopically. Reverse Transcription and PCR. A total of 5 pmol of 79-mer RNA was reverse transcribed at a concentration of 250 nM in the presence of 5 µM reverse primer (5′-GTG GAT CCG ACC GTG GTG CC-3′), 500 µM dNTPs, 10 mM DTT, 0.5 M betaine and 200 U Superscript II reverse transcriptase (Stratagene) in 1× first strand buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2). Reactions were incubated for 20 min at 51 °C, diluted 1:4 (v:v) in PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl, 2.5 mM MgCl2) containing 1 µM T7forward primer 5′-TCT AAT ACG ACT CAC TAT AGG AGC TCA GCC TTC ACT GC-3′, 1 µM reverse primer (see above), 200 µM dNTPs, 0.5 M betaine, and 5 U Taq polymerase. Ten steps of temperature cycling between 55, 72, and 94 °C were performed in a PTC-200 thermocycler (MJ research). DNA Sequencing. PCR reactions were TA-cloned and sequenced by Sanger methods at GATC-Biotech, Konstanz, Germany. Melting Curves. Melting curves were measured on a CARY 100 Bio instrument in 10 mM sodium phosphate buffer pH 7.0 and 100 mM NaCl. 0.7 OD260 of the corresponding sense and antisense 17-mers were separately dissolved in buffer to 5 µM concentration. Equal volumes were transferred into a new cuvette and heated to 95 °C. OD260 values were recorded every 0.1 °C as the temperature was decreased to 15 °C at a rate of 0.3 °C per min, held for 5 min without recording data points, and increased again to 95 °C. This procedure was performed twice. As negative controls, melting curves for each of the 17-mers alone were recorded. Four Tm values were calculated for each measuring cycle and averaged. Differences in the melting temperatures of modified duplex molecules compared to unmodified molecules were calculated by subtracting Tm values of the latter from those of the former and dividing the result by the number of modified bases present in the duplex molecule. RESULTS AND DISCUSSION
Design and Synthesis of Aminomodified Nucleotides. By introducing the functional group in the nucleoside triphosphate, we expect improved affinity of such modified RNA toward certain targets. Assuming a pKa value between 9 and 10, the amino groups of the modified nucleoside triphosphates are expected to be protonated at neutral pH. As a consequence, the interaction between
the aminomodified RNA and acidic targets should be increased. The allyl-modification at the C5-position was chosen as various modified nucleotides have proven themselves as tolerated substrates for DNA and RNA polymerases. For the synthesis of the aminomodified uridine analogue 7 the starting material D-arabinose 1 was converted to 2,2′-O-anhydro-D-uridine 2 according to a published procedure (23). After protection of the hydroxyl-groups with pixyl chloride and ring opening with 2 M NaOH, the crude product was fluorinated using DAST (24). To increase the concentration of fluoride, NaF was added in large excess. After treatment with 1 M HCl and neutralization, the 5′-OH group was tritylated to give nucleoside 3. The 5′-DMT protected 2′-fluoro nucleoside 3 was obtained in five steps from 2,2′-O-anhydro-Duridine 2 at 30-50 g scale without purification of the intermediates and in 20-30% overall yield. After protection of the 3′-OH group by acetylation and subsequent detritylation under acidic conditions, nucleoside triphosphate 5 was obtained in 49% yield according to the method of Ludwig and Eckstein (25). Transformation of 5 into the mercurated uridine analogue 6 was achieved using mercury acetate. Analytical C18-HPLC showed an almost quantitative formation of 6. Finally, 5-aminoallyl 2′-F-dUTP 7 was prepared according to the Heck reaction (26) by treating the uridine derivative 6 with allylamine in the presence of a palladium(II) catalyst. After preparative ion-exchange HPLC followed by C18-HPLC nucleotide 7 was obtained in 29% yield starting from 5. Formation of triphosphates 5 and 7 was confirmed by 1H-, 19F-, 31P-NMR and MALDI-MS. The UV spectrum of 7 shows a shift of λmax from 260 nm in uridine analogue 5 to 289 nm indicating the presence of an exocyclic double bond. Feasibility of Modified Nucleobases for SELEX. To be feasible for RNA SELEX, modified nucleotides have to meet certain criteria: First of all, the corresponding modified nucleoside triphosphates must be accepted as a substrate by RNA polymerases. To ensure primary sequence continuity, the modified nucleotides must form strict Watson-Crick base-pairs. In reverse transcription, modified RNA molecules must be efficient templates that do not inhibit the activity of the reverse transcriptase and finally the synthesis procedures for the corresponding triphosphates should be reasonably facile. Feasibility tests were performed with one single transcript of a defined sequence that was originally designed to test the ability of T7 RNA polymerases to transcribe hairpin structures. This 79-mer, shown in Figure 2, is a derivative of an RNA pool frequently used at NOXXON. The original N40 random region has been changed to an iteration of 10 GA purine duplets followed by 10 CU pyrimidine duplets, which presumably fold into an 18
Aminomodified Nucleoside Triphosphates for SELEX
Figure 3. 79-mer RNA transcript modified with 7, 8, and 9. Lane 1: reference transcript with unmodified nucleotides. Lane 2-4: transcripts generated with wt RNA-Pol. Lane 5-8: transcripts generated with Y639F RNA-Pol (relative yield in % to the reference). ssDNA-Marker (M) is ranging from 20 to 110 bases, 10% PAGE.
base-pair stem structure. RNA transcripts were generated from a dsDNA template, annealed from two ssDNA 98-mers, encoding the 79-mer and driven by a minimal T7 promoter. The unmodified and three differently aminomodified RNAs were transcribed using wt T7 RNA polymerase and the Y639F mutant thereof, respectively. The commercially available Y639F mutant (28) efficiently utilizes 2′-modified nucleoside triphosphates. Figure 3 shows the varying yields for the modified transcripts generated with the two different enzymes. Relative yields of radioactively body-labeled transcripts with aminomodified nucleotides compared to unmodified NTPs were visualized by 254 nm UV-shadowing and quantified on a phosphorimager. Yields obtained from wt RNA polymerase, containing 8 and 9, are within the same range and comparable to unmodified reference transcript, whereas hardly any transcript can be detected with 7, since the enzyme does not tolerate the 2′-F-modification. The transcription yield of mutant Y639F polymerase is
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generally reduced to approximately one-third, and again the yields of transcripts containing unmodified NTPs, 8 or 9, are within comparable ranges. With Y639F polymerase being able to tolerate 2′-modified NTPs as substrates, the transcript containing 7 is obtained at about 10% yield compared to the reference. Depending on the modification, transcripts show slightly different migration behavior in denaturing PAGE and in general appear to be larger than the corresponding band of the ssDNA marker. Byproducts that migrate above and below the intended transcript are regularly observed, but have not been investigated further. Base Composition. Base composition analyses were performed to provide the first direct evidence that modified nucleotides triphosphates have been accepted and incorporated by T7 RNA polymerases. The modified 79mer transcripts were digested with a combination of snake venom phosphodiesterase and alkaline phosphatase. The degradation products were analyzed by C18 reversed phase HPLC. Figure 4B shows the base composition of the transcript containing exocyclic-modified purine 9. Compared to the peak of adenosine, the peak for the N6-derived purine nucleoside is clearly shifted to a higher retention time. Base compositions of transcripts containing the uridine analogues 7 and 8 completely lacked the peak of the corresponding uridine nucleoside (Figure 4C). These data suggest that the uridine analogues 7 and 8 were not incorporated into the transcripts. However, full-length transcripts were clearly visualized in polyacrylamide gels (Figure 3) and MALDI-TOF analyses of modified 17-mers were unambiguous (see below). The most likely explanation is that transcripts containing uridine analogues 7 or 8 cannot be completely degraded by snake venom phosphodiesterase. Three enzymatic reactions, RNA transcription, reverse transcription, and PCR, are combined in the cyclic SELEX process. Misincorporations that occur during T7 transcription or reverse transcription cannot be monitored individually for each of the three reactions. Arising mutations cannot be observed unless the PCR product derived from the reverse transcript is sequenced. Potential mutations could arise from either T7 or reverse transcription. In both cases, the modified nucleotide may not be suitable for SELEX. To evaluate whether elevated
Figure 4. HPLC analysis of 79-mer RNA transcripts after nuclease digestion. (A) Unmodified RNA transcript as reference. (B) RNA transcript using modified ATP 9. (C) RNA transcript using uridine analogue 8.
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Figure 5. UV melting curves of DNA (A), RNA (B), RNA containing 9 (C) in A and RNA containing 2′-F-dU (D) and 7 (E) in B. Table 1. Mutational Analysis of the 79-mer Transcripts Modified with the Uridine Analogues 7, 8 and Modified ATP 9 79-mer RNA transcripts
unmodified
generated with wtT7 RNA-Pol
7 553 8 1.4
generated with Y639F mutant Of T7 RNA-Pol
-
7
8 632 8 1.3
mutation rates occurred, the different transcripts were reverse transcribed, amplified by PCR, cloned, and sequenced. Furthermore, to rule out that random NTPs were utilized in place of aminomodified NTPs, T7 transcription reactions with incomplete NTP mixes, i.e., in the absence of any one of the four NTPs, were performed. The yields of these depleted transcription reactions were reduced by roughly 3 orders of magnitude and below the detection level of UV-shadowing (data not shown). As a control, the background level of sequence alterations that were observed after one mock-round of SELEX with unmodified NTPs was determined. The resultant RNA was reverse transcribed to cDNA, amplified by PCR, and sequenced in one direction. Insertions, deletions, and base exchanges were defined as phenotypic mutations. Sequence alterations could be mutations occurring during the enzymatic reactions of a SELEX round or just sequencing artifacts. Although the number of molecules that was sequenced for each modification (see Table 1) was too low for statistics, we conclude that a maximum of a 4-fold
8
9
3 237 13 5.5
7 553 15 2.7
sequenced molecules sequenced bases number of mutations percentage of mutations
7 553 15 2.7
7 553 11 2.0
sequenced molecules sequenced bases number of mutations percentage of mutations
mutation rate in the case of 8, a 2-fold rate for 9, and a nearly identical rate for 7 compared to the background mutation rate observed for unmodified NTPs is tolerable, if not favorable for the SELEX procedure. In fact, modest mutation rates, achievable by error-prone PCR protocols (29, 30), are useful to extend the sequence space, that can be covered with SELEX (31). Melting Behavior of Nucleic Acids Containing Aminomodified Bases. Melting temperatures were measured with two complementary single-stranded molecules, each 17 nucleotides in length, 5′-GGA CUG ACU GAC UGA CC-3′ and 5′-GGU CAG UCA GUC AGU CC3′. 17-mers were chosen, since under standard conditions at 100 mM NaCl, the complete transition would occur within a temperature range between 15 and 95 °C. PolyGACU and poly-GUCA sequences were chosen as they do not form any stable intramolecular interactions according to the structure prediction program Mfold (27). Two additional guanosines at the 5′-end of the 17-mers were introduced to initiate T7 transcription efficiently. According to the addition of two guanosine residues, two
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Table 2. Summary of the Melting Temperatures of Different 17-mer DNA and RNA Molecules Modified with Uridine Analogues 7, 8 and Modified ATP 9a
a Modified U and A bases are underlined and boldface. ∆T (°C) is the difference in T (°C) to the unmodified RNA (B). (n/d ) could m m not be determined).
cytidines were added to the 3′-end to achieve perfect duplex formation. DNA, RNA, and 2′-fluoro-2′-deoxyuridine modified RNA oligonucleotides were chemically synthesized. The 17-mers containing aminomodified nucleotides 7, 8, and 9 were generated by in vitro transcription. T7 RNA polymerase is known to cause end heterogeneity. This arises as a result of nonspecific addition of nucleotides to the 3′-end of transcripts that are not encoded by the complementary template strand. To reduce this effect, template strands were synthesized to contain two 2′-OMe-guanidines at the 5′-ends (32). The 17-mer transcripts were analyzed by MALDI-TOF and HPLC to verify both the successful incorporation of the modified nucleobases and the correct length of the transcripts (data not shown). All UV spectra show the typical increase of absorbance at 260 nm due to thermally induced strand separation. The thermal melting point of duplex C containing the aminomodified adenosine nucleotide 9 was significantly decreased by 17.3 °C (2.47 °C per modified base) compared to unmodified RNA and decreases by 5.3 °C compared to DNA. The steric hindrance of the bulky N6linker modification is likely to cause the decreased stability of the duplex. The duplexes E and F containing the modified nucleotides 7 and 8, respectively, were significantly more stable (Tm > 85 °C) than the control duplexes D and B. For the double substituted uridine 7, the increase of Tm is additive for the 2′-fluoro and the 5-aminoallyl substitution. The 2′-fluoro-modification confers a RNA-like 3′-endo sugar conformation to oligonucleotides. This type of the preorganization forming an A-form duplex structure improves binding affinity toward complementary RNA (33, 34). Additionally, the aminoallyl modification of the 5-position of the nucleobase leads to an increased stability of the duplex, which can be explained by improved stacking due to the double bond (35). The positively charged amino group at neutral pH seems to have a relatively marginal effect on the Tm since incorporation of an aminopropyl-modified uridine analogue into a short DNA hairpin showed similar UV melting temperatures to the sequences bearing a uridine or thymidine residue (36). The modified nucleotides used in this analysis met the requirements of the SELEX process. The introduction of amino functions to nucleobases increases their pKa values and thus should result in positive charges within the
RNA molecules under neutral conditions. Positive charges and the resulting zwitterionic molecules could facilitate the identification of oligonucleotides against acidic targets. Further experiments are ongoing to support this hypothesis. ACKNOWLEDGMENT
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BC0256547
