Perylene Attached to 2′-Amino-LNA: Synthesis, Incorporation into

Bioconjugate Chem. 2008, 19, 1995–2007

1995

Perylene Attached to 2′-Amino-LNA: Synthesis, Incorporation into Oligonucleotides, and Remarkable Fluorescence Properties in Vitro and in Cell Culture Irina V. Astakhova,†,‡ Vladimir A. Korshun,‡ Kasper Jahn,§ Jørgen Kjems,§ and Jesper Wengel*,† Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia, and Interdisciplinary Nanoscience Center, Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus, Denmark. Received May 19, 2008; Revised Manuscript Received July 8, 2008

During recent years, fluorescently labeled oligonucleotides have been extensively investigated within diagnostic approaches. Among a large variety of available fluorochromes, the polyaromatic hydrocarbon perylene is an object of increasing interest due to its high fluorescence quantum yield, long-wave emission compared to widely used pyrene, and photostability. These properties make perylene an attractive label for fluorescence-based detection in Vitro and in ViVo. Herein, the synthesis of 2′-N-(perylen-3-yl)carbonyl-2′-amino-LNA monomer X and its incorporation into oligonucleotides is described. Modification X induces high thermal stability of DNA:DNA and DNA:RNA duplexes, high Watson-Crick mismatch selectivity, red-shifted fluorescence emission compared to pyrene, and high fluorescence quantum yields. The thermal denaturation temperatures of duplexes involving two modified strands are remarkably higher than those for double-stranded DNAs containing modification X in only one strand, suggesting interstrand communication between perylene moieties in the studied ‘zipper’ motifs. Fluorescence of single-stranded oligonucleotides having three monomers X is quenched compared to modified monomer (quantum yields ΦF ) 0.03-0.04 and 0.67, respectively). However, hybridization to DNA/RNA complements leads to ΦF increase of up to 0.20-0.25. We explain it by orientation of the fluorochrome attached to the 2′-position of 2′-amino-LNA in the minor groove of the nucleic acid duplexes, thus protecting perylene fluorescence from quenching with nucleobases or from the environment. At the same time, the presence of a single mismatch in DNA or RNA targets results in up to 8-fold decreased fluorescence intensity of the duplex. Thus, distortion of the duplex geometry caused by even one mismatched nucleotide induces remarkable quenching of fluorescence. Additionally, a perylene-LNA probe is successfully applied for detection of mRNA in ViVo providing excitation wavelength, which completely eliminates cell autofluorescence.

INTRODUCTION

Chart 1. Structures of Pyrene and Perylene Fluorochromes

During the past decade, there has been remarkable growth in the use of synthetic oligonucleotide analogues in nucleic acid research. Thus, fluorescently labeled oligonucleotides have now been introduced into a variety of studies within molecular biology and biochemistry (1-3). The sensitivity of fluorescence detection, its simplicity, and relative cheapness stimulate continuing development of medicinal tests based on fluorescence. Detection of nucleic acid hybridization is an important task in this area, which includes diagnostics of genetic and infectious diseases, discovery of gene-targeted drugs, and other biomedicinal studies (1-5). To become an effective tool for hybridization analysis, the labeled oligonucleotide has to display considerable change of fluorescence upon formation of complexes with complementary strands. Development of such detection methods has led to several formats of homogeneous fluorescence assays (4). For example, fluorescence polarization detection is based on the measurement of the increase in anisotropy when labeled DNA binds to the complementary strand (5). Fluorescence resonance energy transfer (FRET), another useful process for the above purposes that occurs in the excited state, is determined by the distance between the * Corresponding author. E-mail: [email protected], Tel: +45-65502510, Fax: +45-6550-4385. † University of Southern Denmark. ‡ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry. § University of Aarhus.

donor and acceptor of fluorescence (1, 3, 4). Taqman probes (also known as 5′-nuclease probes) (7), sunrise primers (8), molecular beacons (3, 9), and duplex scorpion primers (10) utilize a distance-dependent fluorescence quenching mechanism. Labeling of oligonucleotide with a single kind of hybridizationsensitive substance is another promising paradigm for fluorescent probe design (11-22). Among a wide variety of fluorochromes used to covalently label DNA, fluorescent dyes based on polyaromatic hydrocarbons (PAHs) have been extensively introduced in biochemical studies. The most common PAH in this area is pyrene (Chart 1). A long lifetime of the excited state (23), high fluorescence quantum yield (24), and the possibility to form noncovalent interactions with another PAH molecule (25-37) or nucleic acids (for example, by intercalation 38-41) have inspired researchers to use pyrene for the design of many different detection systems. Thus, fluorescence of a single pyrene label linked to oligonucleotides was shown to be sensitive to duplex

10.1021/bc800202v CCC: $40.75  2008 American Chemical Society Published on Web 09/05/2008

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Chart 2. Chemical Structures of LNA, 2′-Amino-LNA, and Its Pyrene and Perylene Derivatives

formation (19, 21, 42-46). It has also been pointed out that changes in the pyrene excimer-to-monomer fluorescence intensity ratio can be useful for detection of nucleic acid hybridization (27). More recently, Hrdlicka et al. investigated multilabeled pyrene-functionalized 2′-amino-LNA probes which exhibited high-affinity hybridization to DNA/RNA complements, quenched fluorescence of single-stranded probe, and large increase of the quantum yield and fluorescence brightness after hybridization (22). However, pyrene and its simple derivatives absorb at relatively short wavelengths (334 nm for pyrene in ethanol), which is likely to be unsatisfactory for experiments in living cells due to irradiation of several intrinsic fluorochromes at the same wavelengths (47). This limitation of pyrene can be overcome by its chemical functionalization (for example, introduction of phenylethynyl substituents 47-49), or by choosing another fluorochrome with a long-wave emission spectrum relative to pyrene. Among PAHs, perylene is such a fluorochrome (Chart 1), which resembles pyrene in its chemical behavior, but has essentially different photochemical properties (23). Fluorescence of perylene is observed at 450-500 nm; its excited lifetime is relatively short (4-5 ns), while the fluorescence quantum yield is very high (0.5-1.0). Furthermore, perylene displays a strong fluorescence anisotropy effect (24). Unlike pyrene, perylene is not a common fluorescent dye for modification of oligonucleotides, whereas it is widely used in the field of lipids (50-55). Mostly, perylene attached to DNA was employed as an acceptor of fluorescence energy in FRET-studies (27, 37, 56-59), and only scarcely in nucleic acid hybridization assays (60-65). Besides insertion of fluorescent dyes, chemical modification of oligonucleotides allows improvement of their binding affinity and selective recognition of cDNA and/or RNA. These properties have in particular been reported for locked nucleic acids (LNA (66-70), 2′-amino-LNA (71), and its N-functionalized derivatives 22, 40, 72-74) (Chart 2), and have stimulated intensive investigations of LNA-modified oligonucleotides as diagnostic tools and therapeutic agents (75, 76). Moreover, affinity enhancing LNAs are interesting monomers within nucleic acid nanotechnology, including so-called Ångstro¨m-scale chemical engineering (32, 77). Stimulated by the high-affinity hybridization of LNAs (40, 66-74), sensitivity of pyrene-functionalized 2′-amino-LNA probes to hybridization (22), and taking into account perylene’s superiority in fluorescence quantum yield and emission wavelengths relative to pyrene, we have investigated perylene-

modified 2′-amino-LNA. In this paper, we describe synthesis of 2′-N-(perylen-3-yl)carbonyl-2′-amino-LNA monomer X, its incorporation into synthetic oligonucleotides, thermal denaturation studies, and spectral properties of the conjugates containing perylene residue either in one or in two complementary strands of the duplexes (Chart 3).

EXPERIMENTAL PROCEDURES General. Reagents obtained from commercial suppliers were used as received; 3-acetylperylene (78), diisopropylammonium tetrazolide (79), and nucleoside 1 (71, 80, 81) were synthesized as described. Perylene and 9,10-diphenylanthracene used as standards for emission quantum yield measurements were recrystallized. HPLC grade toluene and acetone were distilled and stored over activated 4 Å molecular sieves. DCM was always used freshly distilled over CaH2. Other solvents were used as received. Photochemical studies were performed using spectral grade cyclohexane. NMR spectra were recorded at 303 K on Varian Gemini 2000 300 MHz and Bruker DRX 500 MHz instruments. Chemical shifts are reported in ppm, relative to solvents peaks (DMSOd6, 2.50 ppm for 1H and 39.5 ppm for 13C; 85% aq H3PO4, 0.00 ppm for 31P). 1H NMR coupling constants are reported in Hz and refer to apparent multiplicities. High resolution mass spectra were recorded in positive ion mode using IonSpec Fourier Transform ICR mass spectrometer (MALDI) or PE SCIEX QSTAR pulsar mass spectrometer (ESI). Melting points were determined using a Boetius heating table and are uncorrected. Analytical thin-layer chromatography was performed on Kieselgel 60 F254 precoated aluminum plates (Merck). Silica gel column chromatography was performed using Merck Kieselgel 60 0.040-0.063 mm. Oligonucleotide synthesis was carried out on a PerSpective Biosystems Expedite 8909 instrument in a 200 nmol scale using manufacturer’s standard protocols. In the case of LNA phosphoramidites (monomers TL and MeCL), the coupling step time was extended to 15 min. To accomplish incorporation of monomer X, a double hand-coupling procedure with extended coupling time (2 × 30 min) was performed. In the case of phosphoramidite 3 pyridine hydrochloride solution in CH3CN was used as activator. 95-99% per step coupling yields of monomer X were obtained based on the absorbance of the dimethoxytrityl cation released after each coupling. The coupling efficiencies of standard DNA and LNA amidites varied between

Perylene Attached to 2′-Amino-LNA

Bioconjugate Chem., Vol. 19, No. 10, 2008 1997

Chart 3. Representative Structures of the Perylene Conjugates Prepared in This Studya

a

Green droplets indicate monomer X.

98% and 100%. Cleavage from solid support and removal of nucleobase protecting groups was effected using standard conditions (32% aqueous ammonia for 12 h at 55 °C). Unmodified DNA/RNA strands were obtained from commercial suppliers and used without further purification, while all the modified oligonucleotides were purified by DMT-ON RP-HPLC using the Waters Prep LC 4000 equipped with Xterra MS C18column (10 µM, 300 mm × 7.8 mm). Elution was performed starting with an isocratic hold of A-buffer for 5 min followed by a linear gradient to 55% B-buffer over 75 min at a flow rate of 1.0 mL/min (A-buffer: 95% 0.1 M NH4HCO3, 5% CH3CN; B-buffer: 25% 0.1 M NH4HCO3, 75% CH3CN). RP-purification was followed by detritylation (80% aq AcOH, 20 min), precipitation (abs EtOH, -18 °C, 12 h) and washing with abs EtOH three times. The identity of ONs was verified by MALDITOF mass spectrometry (Table S1, Supporting Information). UV-visible absorption spectra and thermal denaturation experiments were performed on a Perkin-Elmer Lambda 35 UV/vis spectrometer equipped with PTP 6 (Peltier Temperature Programmer) in a medium salt buffer (100 mM NaCl, 10 mM sodium phosphate, 0.1 mM EDTA, pH 7.0). Concentrations of ONs were calculated using the following extinction coefficients (OD260/µmol): G, 10.5; A, 13.9; T/U, 7.9; C, 6.6; perylene, 33.2. ONs (1.0 µM each strand) were thoroughly mixed, denaturated by heating, and subsequently cooled to the starting temperature of the experiment. Thermal denaturation temperatures (Tm values, °C) were determined as the maximum of the first derivative of the thermal denaturation curve (A260 vs temperature). Reported Tm values are an average of two measurements within (1.0 °C. Fluorescence spectra were obtained in a medium salt buffer using a PerkinElmer LS 55 luminescence spectrometer equipped with a Peltier temperature controller. For recording of fluorescence spectra, 0.1 µM concentrations of the single-stranded probe or corresponding duplex were used. For weakly fluorescent samples, concentration was increased to 0.5 µM. Excitation spectrum was obtained recording emission at 510 nm. The fluorescence quantum yields (ΦF) were measured by the relative method using standards of highly diluted solutions of perylene (ΦF 0.93 (82)) and 9,10-diphenylanthracene (ΦF 0.95 (83)) in cyclohexane. CD spectra were recorded on a JASCO J-815 CD Spectrometer equipped with CDF 4265/15 temperature controller. In cell studies, fluorescence was visualized with a Ziess axiovert 200m microscope using excitation

wavelengths of 360 nm (DAPI), 435 nm (perylene-LNA probe), and 570 nm (mCherry protein) and emission wavelengths of 460, 480, and 630 nm for DAPI, perylene-LNA, and mCherry protein, respectively ((15 nm). 3-Perylenecarboxylic Acid. To a precooled to 0 °C solution of sodium hydroxide (600 mg, 15 mmol) in water (12 mL), bromine (205 µL, 4 mmol) and 1,4-dioxane (12 mL) were subsequently added. The resulting mixture was stirred at 0 °C for 10 min, and 3-acetylperylene (294 mg, 1 mmol) was added in 5 portions, over 10 min. The solution was stirred at 0 °C for 3 h, then left at room temperature overnight. Saturated Na2SO3 (2 mL) was added, and the stirring was continued for 1 h; then, the reaction mixture was acidified with 1 M aq citric acid to pH 3 and extracted with CHCl3 (3 × 100 mL). The combined organic phase was dried over Na2SO4 and evaporated. The product was recrystallized from 5% DMF in nitromethane (v/ v) to give 250 mg (84%) of the desired product as a red solid. Rf 0.45 (silica gel, 30% EtOH in CHCl3, v/v). Mp 331-334 °C (5% DMF in nitromethane, v/v) (lit 78. 333-335 °C). MALDIHRMS: m/z 319.0732 ([M + Na]+, C21H12O2Na+ calcd. 319.0730). 1H NMR (300 MHz, DMSO-d6) δ 8.70 (d, J ) 8.4 Hz, 1H), 8.29-8.24 (m, 4H), 8.01 (d, J ) 8.1 Hz, 1H), 7.76-7.69 (m, 2H), 7.53 (dd, J ) 8.1 Hz, 1H), 7.45-7.40 (m, 2H). Carboxylate proton signal was not observed. 13C NMR (75.4 MHz, DMSO-d6) δ 171.3, 168.4, 134.5, 134.0, 132.4, 130.7, 130.5, 130.2, 129.5, 129.2, 128.3, 128.2, 128.0, 127.1, 127.0, 126.9, 125.6, 122.3, 131.5, 121.0, 119.7. (1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy5-(perylen-3-ylcarbonyl)-3-(thymin-1-yl)-2-oxa-5azabicyclo[2.2.1]heptane (2a). To a stirred solution of the 3-perylenecarboxylic acid (116 mg, 0.39 mmol) and HBTU (136 mg, 0.36 mmol) in DMF (2 mL), diisopropylethylamine (132 µL, 0.76 mmol) was added in one portion. The mixture was stirred for 15 min at room temperature and then added dropwise to a stirred solution of nucleoside 1 (200 mg, 0.35 mmol) in DMF (2 mL). After stirring for 40 min, the reaction mixture was diluted with DCM (100 mL) and washed with water (2 × 100 mL), 5% NaHCO3 (2 × 100 mL), and water (3 × 100 mL). The organic layer was dried over Na2SO4 and evaporated. The residue was chromatographed on silica gel using gradient elution 10f20% acetone in toluene, containing 1% NEt3 (v/v/ v) to yield nucleoside 2a (267 mg, 90%) a as an orange foam (rotameric mixture ∼ 1:2.4:1.2 by 1H NMR). Rf 0.40 (50%

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Scheme 1a

a Reagents and conditions: (i) 3-perylenecarboxylic acid, HBTU, EtN(i-Pr)2, DMF (2a, 90%); (ii) dichloroacetic acid, Et3SiH, DCM, 40 min (2b, 73%); (iii) NC(CH2)2OP(N(i-Pr)2)2, diisopropylammonium tetrazolide, DCM (3, 95%); (iv) DNA synthesizer. DMT ) 4,4′-dimethoxytrityl. T ) thymin-1-yl.

acetone, 1% NEt3 in toluene, v/v/v). ESI-HRMS: m/z ) 872.2927 [M + Na]+, calc. for C53H43N3O8Na+ 872.2943. 1H NMR (500 MHz, DMSO-d6; the signals are given for the major rotamer) δ 11.12 (br s, 1H), 8.42-8.32 (m, 4H), 7.87-7.46 (m, 10H), 7.38-7.17 (m, 7H), 6.98 (m, 4H), 6.03 (m, 1H), 5.79 (s, 1H), 4.18 (m, 1H), 4.07 (s, 1H), 3.80 (s, 6H), 3.76 (d, J ) 12.0 Hz, 1H), 3.58 (m, 2H), 3.48 (d, J ) 11.0 Hz, 1H), 1.45 (s, 3H). 13C NMR (125.7 MHz, DMSO-d6; the signals are given for the major rotamer) δ 169.5, 164.6, 159.2 (2C), 150.6, 145.6, 136.3, 136.1, 136.0, 135.8, 135.3, 135.2, 135.1, 135.0, 134.7, 132.6, 132.5, 131.9, 131.5, 131.2, 131.0, 130.8, 130.7, 130.6, 130.5, 129.8, 129.5, 129.1, 128.9 (2C), 128.7, 128.6 (2C), 128.4, 127.9, 127.8, 127.7, 126.4, 114 (4C), 109.3, 88.4, 87.5, 86.7, 69.8, 66.2, 60.2, 56.0 (2C), 52.2, 12.5. (1R,3R,4R,7S)-1,7-Dihydroxy-5-(perylen-3-ylcarbonyl)-3(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (2b). To a stirred solution of 2a (85 mg, 0.1 mmol) in DCM (0.5 mL), dichloroacetic acid (20% solution in DCM, 400 µL) and triethylsilane (200 µL, 1.0 mmol) were subsequently added. After 40 min at room temperature, TLC displayed the complete conversion of starting nucleoside. The reaction mixture was transferred on a column (silica gel, gradient elution 5f10% MeOH in CHCl3, v/v) to give nucleoside 2b (40 mg, 73%) as a yellow solid (rotameric mixture ∼ 1:2.5 by 1H NMR). Rf 0.52 (10% MeOH in CHCl3, v/v). MALDI-HRMS: m/z ) 570.1648 [M + Na]+, calc. for C32H25N3O6Na+ 570.1641. 1H NMR (300 MHz, DMSO-d6; the signals are given for the major rotamer) δ 11.03 (br s, 1H), 8.35-8.26 (m, 4H), 7.90-7.81 (m, 3H), 7.71-7.58 (m, 5H), 6.24 (br.s, 1H), 5.66 (m, 1H), 4.18 (br s, 1H), 4.00-3.43 (m, 6H, partial overlap with water), 1.45 (s, 3H). 13C NMR (75.4 MHz, DMSO-d6; the signals are given for the major rotamer) δ 169.6, 164.6, 150.7, 135.7, 135.2, 134.9, 134.2, 132.6, 132.5, 131.8, 131.6, 131.2, 130.9, 130.8, 129.5, 129.2, 128.9, 128.6, 128.4, 127.9, 127.8, 122.2, 122.0, 120.7, 109.0, 89.9, 87.2, 70.0, 66.2, 57.5, 51.8, 13.3. (1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-7-hydroxy-5(perylen-3-ylcarbonyl)-3-(thymin-1-yl)-2-oxa-5azabicyclo[2.2.1]heptane (3). The LNA nucleoside derivative 2a (170 mg, 0.2 mmol) was evaporated with dry DCM (2 × 30 mL), dissolved in anhydrous DCM (20 mL), diisopropylammonium tetrazolide (52 mg, 0.3 mmol), and bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine (96 µL, 0.3 mmol) were added under argon, and the resulting mixture was stirred for 4 h. The mixture was diluted with DCM (100 mL) and washed with saturated solution of NaHCO3 (2 × 100 mL) and brine (100 mL). The organic layer was dried over Na2SO4, evaporated, and the crude material purified by chromatography (silica gel, 30% acetone in toluene, containing 1% NEt3, v/v/v). Yield 228 mg (95%). Rf 0.53, 0.50 (50% acetone, 1% NEt3, toluene, v/v/ v). ESI-HRMS: m/z ) 1072.3985 [M + Na]+, calc. for

C62H60N5O9PNa+ 1072.4020. 31P NMR (121.4 MHz, DMSOd6) δ 149.22, 148.83 (1:1.7) (main conformer).

RESULTS AND DISCUSSION Synthesis. Modified phosphoramidite was synthesized as shown in Scheme 1. The starting DMT-protected 2′-amino-LNA monomer 1 (82, 83) was coupled with 3-perylenecarboxylic acid, synthesized from 3-acetylperylene (78) under haloform oxidation conditions (84, 85). A small portion of perylenemodified 2′-amino-LNA 2a was reacted with dichloroacetic acid in the presence of triethylsilane giving DMT-deprotected nucleoside 2b required for spectroscopic and photophysical studies. The main portion of 2a was converted to the phosphoramidite derivative 3 (in 86% overall yield), which was used in automated oligonucleotide synthesis to prepare a series of modified oligonucleotides (ONs). Sequences of ONs were similar to those designed for studies of pyrene-functionalized 2′-amino-LNA (22). Synthesis of X-labeled ONs was performed following standard protocols, except for incorporation of monomer X for which a double coupling procedure with an extended coupling time (2 × 30 min) using pyridine hydrochloride as an activator was applied. This resulted in 95-99% stepwise coupling yields of monomer X based on the absorbance of the dimethoxytrityl cation released after each coupling. The coupling efficiencies of standard amidites varied between 98% and 100%. All synthesized conjugates were purified by RP-HPLC and their identity confirmed by MALDI-TOF mass spectrometry (Table S1, Supporting Information). Spectral Properties of 2′-N-(Perylen-3-yl)carbonyl-2′amino-LNA Monomer (2b). To get true solutions of nucleoside 2b for recording of UV-vis absorption, steady-state fluorescence emission, and excitation spectra in aqueous media (Figure 1), it was dissolved in a medium salt buffer with 1% DMSO (v/v). Perylene absorbs with a maximum at 445 nm, an additional band at 419 nm, and a shoulder at 396 nm. Spectral broadening of the spectrum of 2b compared to that of perylene (23) indicates increased ground-state interactions for the perylene attached to 2′-amino-LNA. These interactions are most likely caused by the nearby thymine, and less probably, by the sugar ring. Emission spectra of 2b displays a broad, featureless peak near 480 nm corresponding to a Stokes shift of 35 nm. Compared to perylene, emission of 2b is 30 nm red-shifted and significantly broadened. Fluorescence emission quantum yield (ΦF) of nucleoside 2b was determined by the relative method using highly diluted solutions of perylene (ΦF ) 0.93 (82)) and 9,10-diphenylanthracene (ΦF ) 0.95 (83)) in cyclohexane as the reference standards. Optical densities of the solutions used for quantum yield measurements were kept between 0.1 and 0.01 to avoid

Perylene Attached to 2′-Amino-LNA

Bioconjugate Chem., Vol. 19, No. 10, 2008 1999

Figure 1. Normalized absorption, emission, and excitation spectra of nucleoside 2b in a medium salt buffer containing 1% DMSO (v/v) recorded at 19 °C using an excitation wavelength of 425 nm, emission wavelength of 510 nm for excitation, 2 µM concentration of 2b for the absorption spectrum, and 0.1 µM concentration for the excitation and emission fluorescence spectra. Table 1. Thermal Denaturation Temperatures for Duplexes of Modified ONs and DNA/RNA Complementsa duplex # ON1 ON2 ON3 ON4 ON5 ON6 ON7 ON8 ON9 ON10

sequence, 5′f3′ GTG GCA GCA GTG GCA GTG GXG GXG TTX TTLT

AXA XAT TAX AXA XAX XXT AXA XTX AXA AXA

TGC CAC CAC XGC CAC TGC XGC TGC XAX CAMeCLG XAX CAMeCLG

with DNA Tm with RNA Tm (∆Tm/mod)/°C (∆Tm/mod)/°C 31.0 (+3.0) 35.0 (+7.0) 34.5 (+6.0) 43.5 (+7.8) 42.0 (+7.0) 32.5 (+0.5) 43.0 (+5.0) 38.5 (+2.3) 37.5 (+0.5)b 42.5 (+2.3)

32.5 (+6.5) 32.0 (+7.5) 31.0 (+6.5) 57.0 (+15.5) 38.0 (+6.8) 37.5 (+4.0) 44.0 (+6.0) 46.0 (+5.5) 42.0 (+2.0) 47.0 (+4.3)

a Thermal denaturation temperatures Tm (°C) (change in Tm per modification relative to corresponding reference duplex, ∆Tm/mod (°C)). The melting temperatures of the unmodified duplexes or LNA-modified duplex (in the case of ON9–ON10) used as references were as follows: 28.0 °C for ON1–ON5,ON7:DNA, 26.0 °C for ON1,ON4,ON7:RNA, 24.5 °C for ON2,ON3,ON5:RNA, 31.5 °C for ON6,ON8:DNA, 29.5 °C for ON6,ON8:RNA, 35.5 °C for ON9–ON10:DNA, and 34.0 °C for ON9–ON10:RNA. Tm values measured as the maximum of the first derivatives of the melting curves (A260 vs temperature). Reported expected to be hybridized to its RNA target. Tm values are averages of at least two measurements. X ) 2′-N-(perylen-3-yl)carbonyl-2′-aminoLNA monomer; TL and MeCL ) thymin-1-yl and 5-methylcytosin-1-yl LNA monomers, respectively. All Tm values were recorded in medium salt buffer. b Tm value for ON9:DNA measured using perylene absorbance at 425 nm.

uncertainties. The ΦF values were corrected with the refractive index of the solvents. Quantum yield of 2b in aerated buffer solution containing 1% DMSO measured using these conditions was ΦF ) 0.67, while ΦF for the 2′-amino-LNA nucleoside functionalized with pyrene was ΦF ) 0.34 (22). Superiority of perylene relative to pyrene with respect to quantum yield becomes clear if fluorescence rate constants are compared (Kf ) 0.25 · 10-7 s-1 for pyrene and 14.62 · 10-7 s-1 for perylene in cyclohexane, respectively) (86). Labeled Oligonucleotides and Duplexes Having Monomer X in One Strand. (a) Thermal Denaturation Studies. Thermal denaturation temperatures were determined in medium salt buffer using 1.0 µM concentration of the two complementary strands, and were compared to the denaturation temperatures of the corresponding unmodified duplexes (Table 1). The perylene-modified ONs displayed higher affinity toward complementary RNA while exhibiting lower affinity toward DNA. Previously, it had been reported that conjugates containing functionalized 2′-amino-LNA monomers form stable complexes

to both DNA and RNA complements, and that N2′-acylated 2′-amino-LNA derivatives exhibit higher thermal stabilities than N2′-alkylated (22, 72). In agreement with these results, the thermal stabilities of duplexes involving singly labeled ON1–ON3 were much higher than those of the corresponding unmodified duplexes and of duplexes involving a recently reported perylenemethyl-2′-amino-LNA monomer (37), but similar to those for N2′-pyrenecarbonyl-functionalized 2′-aminoLNAs (22). This implies that neither a perylene nor a pyrene moiety attached to LNA essentially distorts the structure of nucleic acid helixes. Incorporation of two monomers X separated by one base pair induces even larger increases in thermal stabilities, especially in the case of duplex ON4:RNA (∆Tm/ mod ) +15.5 °C). On the contrary, insertion of modifications in the neighboring positions, as well as incorporation of an additional monomer X, results in lower melting temperatures compared to doubly labeled conjugates ON4–ON5:DNA/RNA (Table 1, Tm values for ON6–ON8:DNA/RNA), which again parallels the behavior of pyrene attached via an amide linkage to 2′-amino-LNA (22). The thermal denaturation curves of all the above-mentioned duplexes displayed S-shaped monophasic transitions similar to those of the unmodified reference duplexes. Surprisingly, 13mer ON9 having four perylenes showed a sigmoidal melting curve only against its RNA complement, but not its DNA complement. Therefore, a thermal denaturation experiment was performed at perylene’s absorbance at 425 nm (Supporting Information, Figure S1). The resulting melting curve displayed clear S-shaped perylene hypochromism similar to that previously reported for pyrene (19). In the case of duplex ON9:RNA, the melting temperature values determined using absorbance at 260 and 425 nm coincided precisely. The observed hypochromism can be explained by the increase of the distance between perylene residues in the more rigid duplex compared to the flexible single-stranded oligonucleotide. Upon incorporation of an additional number of fluorochromes, the total absorbance of perylene residues at 260 nm may become rather significant and, therefore, may affect the ‘nucleotide’ melting curve. The Watson-Crick selectivity of the perylene-functionalized conjugate ON7 was determined also for the duplexes containing single mismatched nucleotides at positions 4-6 (Table 2). The presence of mismatches in the DNA/RNA target resulted in significantly decreased thermostability of the complexes compared to the corresponding fully matched duplexes ON7:DNA/ RNA (data presented in Table 2 compared to Tm values of ON7: DNA/RNA). It was previously reported that mismatches opposite of LNA monomers are generally discriminated more strongly than opposite of unmodified nucleotides (67). However, in the case of monomer X, mismatches at position 6 of target DNA resulted in more remarkable destabilization of the complexes than at position 5. (b) Spectral Properies. The UV-visible absorption and steady-state fluorescence emission spectra were obtained in medium salt buffer using 0.1-1.0 µM concentration of the single-stranded probe or the two complementary strands. For the recording of fluorescence spectra, an excitation wavelength of 425 nm was used. Table 3 lists the resulting spectral and photophysical properties of single-stranded ONs (SSP) and their duplexes with DNA and RNA complements. The UV-visible spectra recorded of all the modified ONs and duplexes, as well as the spectrum of monomer 2b, contain two main absorption bands in the visible region with λmax ≈ 448-456 nm and λmax ≈ 422-429 nm. Noteworthy, perylene longwave absorption maxima of the X-modified ONs and duplexes are red-shifted compared to those of the nucleoside 2b. As a result of perylene-ON interactions, the UV-visible absorption spectrum of the perylene unit has lost many of its

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Table 2. Thermal Denaturation Temperatures for Duplexes of ON7 and DNA/RNA Complements Containing Single Mismatchesa Tm/°C DNA target ON7:TARGET

B:

A

5′-GXG AXA XGCa 3′-CAC TBT ACG

43.0

5′-GXG AXA XGC 3′-CAC BAT ACG

25.5