Control of aggregation-induced emission by DNA hybridization †

DNA is a useful and versatile scaffold for the assembly of functional groups with important applications in the fields of diagnostics, electronics, and materials science. In particular, the introduction of a wide range of chromophores into DNA has drawn considerable attention. Distinct photophysical effects are observed in such DNA-organized architectures including the formation of exciplexes, Hand/or J-aggregates or distinct helical arrangements with unique optical or chiral properties. In recent years, tetraphenylethylene (TPE) and related molecules have attracted interest because of their unusual fluorescence properties. These chromophores are weakly or non-emissive as unassociated monomers but they become strongly fluorescent upon aggregation. Aggregationinduced emission (AIE) is a result of restricted intramolecular rotation in the aggregated state (Fig. 1a). AIE features are of interest in diverse areas including fluorescence sensors and OLEDs. However, reports describing the interaction of AIE molecules (AIEs) with DNA, proteins or other biostructures are limited and only one recent publication has reported the incorporation of an AIE-active silole derivative into DNA strands using an enzymatic method. Herein, we describe the synthesis and incorporation of the two stereoisomers of a dialkynyl-tetraphenylethylene (DATPE) into DNA and the AIE properties of the obtained conjugates (Fig. 1b). The synthetic strategy of the two building blocks (ME and MZ) is shown in Scheme 1. Compound 1 was obtained according to the reported procedure as a mixture of E-/Z-isomers. The butynyl chains were introduced through Sonogashira coupling using 3-butyn-1-ol. The Eand Z-isomers (2 and 3) were separated by chromatography and obtained in nearly equal amounts. The products were recrystallized and their configurations established by X-ray crystallography (ESI†). Compounds 2 and 3 exhibited typical AIE characteristics. They are non-emissive in well-solubilizing solvents, such as THF. In THF–water mixtures, the fluorescence intensity strongly increases once the water fraction rises above 60%. In a 95/5 (v/v) water–THF solution the quantum yields (FF, ESI†) are 0.40 and 0.39 for 2 and 3, which compare well with similar compounds. The two diols were transformed into the corresponding monoDMT protected alcohols 4 and 5, which were subsequently converted into phosphoramidites 6 and 7. These building blocks were used in the automated synthesis of oligomers ON1-8 (Table 1) containing one or two DATPEs. All oligomers were purified by reversed-phase HPLC and characterized by ESI-MS (see ESI†). Fig. 1 AIE properties of tetraphenylethylene (a) as a monomer and (b) as a building block in single and double stranded DNA. Scheme 1 Preparation of phosphoramidites 6 and 7; (a) CuI, Pd(PPh3)2Cl2, NEt3, THF; 38%; (b) DMTCl, THF, pyridine; 29% for 4; 23% for 5; (c) CEPCl, Hünig’s base, DCM; DMT = 4,4 0-dimethoxytrityl; CEP = 2-cyanoethyl-N,N-diisopropyl-phosphoramidite; 61% for 6; 71% for 7.

3][34][35][36][37][38][39] In recent years, tetraphenylethylene (TPE) and related molecules have attracted interest because of their unusual fluorescence properties.These chromophores are weakly or non-emissive as unassociated monomers but they become strongly fluorescent upon aggregation.Aggregationinduced emission (AIE) is a result of restricted intramolecular rotation in the aggregated state (Fig. 1a). 40,41AIE features are of interest in diverse areas including fluorescence sensors 42 and OLEDs. 40However, reports describing the interaction of AIE molecules (AIEs) with DNA, 43 proteins 44,45 or other biostructures 46 are limited and only one recent publication has reported the incorporation of an AIE-active silole derivative into DNA strands using an enzymatic method. 47Herein, we describe the synthesis and incorporation of the two stereoisomers of a dialkynyl-tetraphenylethylene (DATPE) into DNA and the AIE properties of the obtained conjugates (Fig. 1b).
The synthetic strategy of the two building blocks (M E and M Z ) is shown in Scheme 1. Compound 1 was obtained according to the reported procedure as a mixture of E-/Z-isomers. 43he butynyl chains were introduced through Sonogashira coupling using 3-butyn-1-ol.The E-and Z-isomers (2 and 3) were separated by chromatography and obtained in nearly equal amounts.The products were recrystallized and their configurations established by X-ray crystallography (ESI †).Compounds 2 and 3 exhibited typical AIE characteristics.They are non-emissive in well-solubilizing solvents, such as THF.In THF-water mixtures, the fluorescence intensity strongly increases once the water fraction rises above 60%.In a 95/5 (v/v) water-THF solution the quantum yields (F F , ESI †) are 0.40 and 0.39 for 2 and 3, which compare well with similar compounds. 40he two diols were transformed into the corresponding mono-DMT protected alcohols 4 and 5, which were subsequently converted into phosphoramidites 6 and 7.These building blocks were used in the automated synthesis of oligomers ON1-8 (Table 1) containing one or two DATPEs.All oligomers were purified by reversed-phase HPLC and characterized by ESI-MS (see ESI †).A mixed solvent system composed of a buffered system with 2,2,2-trifluoroethanol (TFE, 70%, v/v) and water (30%, v/v, ESI †) 48 was used to analyze the spectroscopic properties of the DATPE-modified oligonucleotides. 49Measurements performed in buffered water alone or smaller fractions of TFE provided qualitatively similar results but the effects were less pronounced.Generally, single strands showed considerably weaker fluorescence in TFE-water mixtures than in water alone.We ascribe this to reduced molecular interactions between DATPE and the nucleotides and, consequently, to increased internal rotation in DATPEs 42 in the presence of the less polar TFE.
The melting temperature (T m ) values of the different hybrids obtained by thermal denaturation are summarized in Table 1.The DATPE units have a positive effect on the stability of the duplex with DT m values ranging from +2 to +5 1C, for hybrids containing two or four DATPE units, respectively.This stabilization can be attributed to favorable stacking interactions between the propeller-twisted DATPE molecules in the duplex.
The UV-vis and fluorescence spectra recorded for hybrid D1 are shown in Fig. 2 along with the spectra of the single strands.For the single strand ON1, the absorption band of the E-DATPE molecule locates around 327 nm.The maximum of this band is shifted to 335 nm during formation of the duplex D1.Aggregation of the two DATPE molecules by hybridization of the two single strands results in a considerable enhancement of the fluorescence intensity (Fig. 2, right).The fluorescence intensity of D1 at 490 nm is about 9 times higher than that of ON1 and roughly 3 times higher than that of ON2.These differences were also reflected in the fluorescence quantum yields (F F ) given in Table 2. F F rises to 0.22 upon hybridization compared to 0.05 and 0.10 for ON1 and ON2, respectively.Similar effects are observed for D2 containing two Z-isomers.Fig. 3 displays the titration of ON3 with ON4, in which F F gradually increases to 0.19 in the duplex D2.
Formation of the duplex results in a gradual shift in the emission maximum from 476 nm (ON3) to 490 nm for D2.Fluorescence intensity is significantly increased by annealing of the two strands (an increase by a factor of 5.6 compared to ON3, Fig. 3), indicating a restriction of internal rotation by aggregation of the two DATPEs. 41,42Thus, the AIE character of the building blocks is controlled by the hybridization process.Emission maxima and quantum yields are summarized in Table 2.
Fig. 4 illustrates the effect of molecular aggregation of DATPEs by DNA hybridization.The comparison between ON7 and D2 shows that the emission by two DATPE molecules is considerably higher in the duplex (F F = 0.19) than in the single strand (F F = 0.07).This is expected since the molecular aggregation should be positively influenced by the well-organized duplex structure compared to the single stranded random coil.A further extension of the DATPE stack, as in D4, results in a further significant increase in the fluorescence intensity (F F = 0.32).The F F values of D3 and D4 are three times higher than those of the corresponding single strands and close to the monomeric building blocks 2 and 3 in their aggregated states   (20 1C; l ex : 335 nm, ex.slit: 5 nm; em.slit: 5 nm; detector: 600 V; other conditions as in Table 1; for more details see ESI †).(F F B 0.40).These findings reflect a steady growth of the emission with an increasing degree of molecular aggregation, i.e. the compact arrangement of DATPEs effectively suppresses the intramolecular rotation. 42The effect of AIE is best illustrated in the example shown in Fig. 5, which represents the change in fluorescence between single strand ON7 and duplex D4.Duplex formation results in a 10-fold increased emission.
In conclusion, we have demonstrated that AIE can be controlled by DNA hybridization.Two AIE-active, DATPE building blocks were incorporated into oligonucleotides.Hybridization of complementary strands leads to molecular aggregation of the DATPE units.Quantum yields in hybrids reach values close to those of the monomers in the aggregated state.Considering the ease of their synthesis and their unique fluorescence properties, DATPEs are promising candidates for diagnostic probes or DNA-based nanostructures with special optical properties.
This work was supported by the Swiss National Foundation (Grant 200020-132581).

Fig. 1 Scheme 1
Fig. 1 AIE properties of tetraphenylethylene (a) as a monomer and (b) as a building block in single and double stranded DNA.

Fig. 4
Fig. 4 Fluorescence spectra of single strand ON7 and hybrids D2 and D4; conditions as in Fig 2.

Table 1
Oligonucleotides (ONs) containing E-or Z-DATPE building blocks (M E and M Z ) and T m values of DNA hybrids

Table 2
Emission maxima and quantum yields (F F ) of E-and Z-DATPE molecules (2 and 3) and modified single strands and hybrids (quinine sulfate as standard, for conditions see Table1 and ESI) a Values determined in a 95/5 H 2 O-THF (v/v) mixture.