C5-Azobenzene-substituted 2'-Deoxyuridine-containing Oligodeoxynucleotides for Photo-Switching Hybridization

A new photoisomeric nucleoside dUAz bearing an azobenzene group at the C5-position of 2'-deoxyuridine was designed and synthesized. Photoisomerization of dUAz in oligodeoxynucleotides can be achieved rapidly and selectively with 365 nm (forward) and 450 nm (backward) irradiation. Thermal denaturation experiments revealed that dUAz stabilized the duplex in the cis-form and destabilized it in the trans-form with mismatch discrimination ability comparable to thymidine. These results indicate that dUAz could be a powerful material for reversibly manipulating nucleic acid hybridization with spatiotemporal control.


Introduction
Regulation of nucleic acid hybridization by some external stimuli is a rewarding challenge due to its potential to control gene expression flow from DNA to protein at a predetermined place and time. This technique could allow for spatiotemporal controllable pharmacotherapy based on nucleic acid agents. The regulation of nucleic acid hybridization is also important in the field of nanotechnology, such as in the construction of DNA-origami [1][2][3]. Modified oligonucleotides (ONs) that can reversibly alter the hybridization ability by noninvasive external stimuli are therefore necessary. The most promising OPEN ACCESS external stimulus is light, due to the possibility of accurately controlling the location, dosage and time of the irradiation. For example, Asanuma et al. have reported reversible photoregulation of DNA duplex formation via installation of azobenzene moieties on ONs [4,5]. Azobenzene and its derivatives are commonly adopted due to their rapid photoisomerization and drastic changes in geometry and dipole moment [6,7].
In this study, we describe a new type of azobenzene-modified nucleoside that reversibly changes its properties upon photoisomerization by ultraviolet (365 nm) or visible light (450 nm). There are several positions to attach a photochromic moiety to a nucleoside, and we have selected the C5 position of 2'-deoxyuridine (dU Az , Figure 1) [8]. It is predicted that the azobenzene moiety of dU Az is projected into the major groove of the double helix via a rigid ethynyl linker. We assumed that the duplexes containing trans-dU Az would be destabilized because the hydrophobic azobenzene moiety extends to the outside of the groove [9] which surrounded by a highly polar aqueous phase, and interferes with hydration and the formation of interstrand cation bridges to stabilize the duplexes [10,11]. Meanwhile, cis-dU Az -modification would not affect the duplex stability due to compact conformation of the azobenzene moiety. In other words, the affinity of ONs containing dU Az for complementary single-stranded DNA or RNA may be reversibly changed, triggered by light.

Synthesis of dU Az Phosphoramidite and dU Az -Modified Oligodeoxynucleotides
The synthetic route of dU Az phosphoramidite is outlined in Scheme 1. dU Az nucleoside 1 was synthesized from the corresponding 2'-deoxy-5-iodouridine (2) through a palladium-catalyzed cross-coupling reaction [12] with 4-ethynylazobenzene 3 [13]. Tritylation at the primary hydroxyl group of 1 with DMTrCl and phosphitylation at the secondary hydroxyl group yielded phosphoramidite 5. The amidite 5 was incorporated into the oligodeoxynucleotide using conventional solid-phase phosphoramidite synthesis and purified by reverse-phase HPLC (29% yield). The ON sequences used in this study are shown in Table 1

Photoisomerization Property of dU Az
We initially investigated the efficiency of the dU Az cis-trans photoisomerization property in ON by UV spectra and HPLC analysis. UV spectra of trans/cis ON 7, showed that photoisomerization of trans-dU Az to cis-dU Az decreased absorbance at 365 nm and increased absorbance at 310 nm and 450 nm (Figure 2a). The λ max of cis-form (340 nm) was blue-shifted compared to that of the trans-form (365 nm), as was the case with previous reports [6,7,14]. The trans-form dU Az was photoisomerized to the cis-form by a 10-second irradiation of 365 nm monochromic light with 60% conversion, as determined by the HPLC peak areas (Figure 2b). In addition, subsequent 10-second irradiation of 450 nm yielded the trans form isomer with 80%. The HPLC analysis showed no side products from the reactions.
Even when the photoirradiation was repeated three times, the efficiency of the dU Az cis-trans photoisomerization was not attenuated (Figure 2c). It can therefore be concluded that dU Az has a rapid and highly efficient cis-trans photoisomerization property and the potential to work as a photo-switch for various biomolecules. We investigated the differences in the thermal stability of 12-bp duplexes containing dU Az in the trans-and cis-forms by monitoring the melting temperature (T m ) following the way of azobenzenemodified nucleoside containing ONs (Table 2) [15,16]. DNA duplex 7/8 showed a modest T m difference (ΔT m ) between the trans-and cis-forms, namely, the T m value of the cis-form was 2 °C higher than that of the trans-form. On the other hand, the ON 7/RNA 12 duplex showed a larger T m difference. The T m value of the cis-form was 5 °C higher than that of the trans-form. It is noteworthy that the cis-ON 7/RNA 12 duplex showed a T m value comparable to that of natural DNA 6/RNA 12 duplex. According to past studies, the cis-form photochromic moieties generically destabilize the duplex because of its interference with the vicinity bases stacking interaction [4,5,[17][18][19].In this study, ON containing dU Az showed a higher hybridization ability when dU Az is cis-form rather than transform, unlike ONs containing the exiting photochromic nucleoside. Brown et al. have reported that hydrophobic buta-1,3-diynyl anthracene in ON leads to significant destabilization of the duplex, probably because the aromatic moiety is exposed to the aqueous environment [9]. The azobenzene moiety of trans-dU Az also would extend to the outside of the major groove, a highly polar aqueous phase. This may have an impact on the groove hydration and the formation of interstrand cation bridges, and lead to destabilization of the duplex containing trans-dU Az . Finally, we investigated the mismatch discrimination ability of ON containing dU Az . The T m values of mismatched DNA duplexes containing dU Az were found to be 14 or 15 °C lower than that of ON7/DNA8 in both trans-and cis-form (Table 3). Toward complementary ssRNA, ON containing dU Az could also discriminate mismatched bases comparable to ON7 (Table S1 in Supplementary Material). These results indicate that the mismatch discrimination ability of ON containing trans-/cis-dU Az is not spoiled by the C5-substituted-azobenzene moiety of dU Az . We achieved synthesis of the photoisomeric nucleoside, dU Az , for which the hybridization can be controlled by using different wavelengths of light. The ΔT m value between the trans-and cis-form is more remarkable in the DNA/RNA duplex than the DNA duplex. Although dU Az photoisomerization induced modest T m differences, the modification of ONs with multiple dU Az units or the introduction of substituents to the azobenzene moiety [20] could enhance the ΔT m value between the trans-and cis-forms. Our strategy indicated the possibility of photo-switches based on dU Az -modified ONs for the development of unique molecular machines and the control of various biological phenomena.

General
Reagents and solvents were purchased from commercial suppliers and were used without purification unless otherwise specified. All experiments involving air and/or moisture-sensitive compounds were carried out under N 2 or Ar atmosphere. All reactions were monitored with analytical TLC (Merck Kieselgel 60 F254). Column chromatography was carried out with a Fuji Silysia FL-100D. Physical data were measured as follows: NMR spectra were recorded on a JEOL JNM-ECS-500 spectrometer in CDCl 3 or DMSO-d 6 as the solvent with tetramethylsilane as an internal standard. IR spectra were recorded on a JASCO FT/IR-4200 spectrometer. Optical rotations were recorded on a JASCO P-2200 instrument. FAB mass spectra were measured on a JEOL JMS-700 mass spectrometer. (1) Under an argon atmosphere, 4-ethynylazobenzene (3 [13], 1.06 g, 5.12 mmol), Pd(PPh 3 ) 4 (592 mg, 0.512 mmol), and CuI (113 mg,0.512 mmol) was dissolved in dry DMF (50 mL). Then, Et 3 N (3.6 mL) and 2'-deoxy-5-iodouridine (2, 1.81 g, 5.12 mmol) were added. The reaction mixture was stirred at 60 °C for 4 h. The resultant mixture was filtered over Celite. The filtrate was concentrated in vacuo.

Synthesis of dU Az -Modified Oligodeoxynucleotides
Solid-phase oligonucleotide synthesis was performed on an nS-8 Oligonucleotides Synthesizer (GeneDesign, Inc., Osaka, Japan) using commercially available reagents and phosphoramidites with 5-(bis-3, 5-trifluoromethylphenyl)-1H-tetrazole (0.25 M concentration in acetonitrile) as the activator. dU Az phosphoramidite was chemically synthesized as described above. All of the reagents were assembled, and the oligonucleotides were synthesized according to the standard synthesis cycle (trityl on mode). Cleavage from the solid support and deprotection were accomplished with concentrated ammonium hydroxide solution at 55 °C for 12 h. The crude oligonucleotides were purified with Sep-Pak Plus C18 cartridges (Waters) followed by RP-HPLC on a XBridge TM OST C18 Column, 2.5 μm, 10 × 50 mm (Waters) using MeCN in 0.1 M triethylammonium acetate buffer (pH 7.0). The purified oligonucleotides were quantified by UV absorbance at 260 nm and confirmed by MALDI-TOF mass spectrometry (Table 4).

UV Melting Experiments
Melting temperatures (T m ) were determined by measuring the change in absorbance at 260 nm as a function of temperature using a Shimadzu UV-Vis Spectrophotometer UV-1650PC equipped with a T m analysis accessory TMSPC-8. Equimolecular amounts of the target DNA/RNA and oligonucleotides were dissolved in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl to give a final strand concentration of 4.0 µM. The melting samples were denatured at 100 °C and annealed slowly to room temperature. Absorbance was recorded in the forward and reverse directions at temperatures of 5 to 90 °C at a rate of 0.5 °C/min.

Conclusions
We have synthesized a new photoisomeric nucleoside, C5-azobenzene-modified 2'-deoxyuridine dU Az using Sonogashira-type cross-coupling as a key step. dU Az showed very rapid reversible cis-trans photoisomerization with monochromic light at the appropriate wavelength in oligodeoxynucleotide. dU Az -modified oligodeoxynucleotide showed an interesting duplex-forming property, namely, the T m values of both the dU Az -modified ON/DNA and dU Az -modified ON/RNA were higher for the cis-form than for the trans-form, unlike conventional azobenzene-modified ONs. Additionally, it was revealed that installation of dU Az into oligodeoxynucleotide had little influence on the mismatch recognition ability.