Influence of a GC Base Pair on Excitation Energy Transfer in DNA

Article pubs.acs.org/bc

Influence of a GC Base Pair on Excitation Energy Transfer in DNAAssembled Phenanthrene π‑Stacks Florian Garo and Robert Han̈ er* Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland S Supporting Information *

ABSTRACT: The effect of a GC base pair on the excitation energy transfer in a DNA-based, light harvesting assembly of phenanthrene and pyrene chromophores is described. After absorption of light at 320 nm by the stacked phenanthrene building blocks, the excitation energy is transferred to the pyrene and leads to the formation of a phenanthrene-pyrene exciplex. The fluorescence intensity depends on the number of light absorbing phenanthrenes, as well as on the type of DNA base pair flanking the phenanthrene stack. In comparison to an AT base pair, a GC base pair located next to the stacked aromatic residues results in a reduction of fluorescence. The degree of quenching is dependent on the length of the phenanthrene stack that separates the GC base pair from the exciplex. Overall, a large number of stacked phenanthrenes positively affects exciplex fluorescence by increasing the quantity of absorbed light and, at the same time, reducing the effect of quenching by GC base pairs.

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area of chemical research finds applications in the fields of molecular diagnostics, electronics, and material sciences.39−46 The DNA-guided assembly of chromophores for the construction of arrays with distinct properties, including energy transfer, has gained considerable attraction over the past decade.47−57 The use of DNA as a molecular scaffold allows for the construction of stable and well-defined π-stacks.58−64 In addition, the arrangement of non-nucleosidic, polyaromatic chromophores leads to constructs with interesting properties.65−75 We recently reported on the light harvesting properties of DNA-organized, multichromophoric arrays.76 In these arrays, light is absorbed by multiple, π-stacked phenanthrenes (P) and efficiently transferred to an exciplexforming pyrene (S) unit. The design of the assembly results in PS-exciplex fluorescence at one end of the arene π-stack. The excitation energy is transferred and concentrated to a specific location of the construct before it is reemitted. This type of array is reminiscent of the natural chlorosome and can be viewed as a mimic of one small segment. Thus, it serves as a prototype for the design of synthetic light harvesting systems. The DNA sequences used in the previous study76 excluded the presence of guanine entirely to minimize fluorescence quenching by charge separation.77−79 In the course of exploring and investigating the light harvesting phenanthrene arrays in more detail, the question about the effect of a GC base pair on the energy transport within the π-stack arose. In the hitherto

INTRODUCTION The use of sunlight as a source of energy is one of the top challenges in today’s research.1−4 Photosynthetic organisms are using sunlight in an impressively elegant way to satisfy their energy needs.5 In this process, sunlight is collected through absorption by chromophores arranged in an antenna complex, transferred to a reaction center and converted to chemical energy by charge separation across a bilayer membrane.6−8 The first two steps of the photosynthetic cascade, light harvesting and energy transfer, are essential elements for the effectiveness of the whole process. In plants and purple bacteria, a protein scaffold places and organizes the antenna chromophores in the proper distance and orientation to ensure efficient light harvesting and fast energy transfer.9−13 In the chlorosome, the light harvesting antenna complex of green sulfur bacteria, thousands of chromophores are self-assembled, π-stacked and highly organized without the aid of a protein scaffold.14−17 The vast number of antenna chromophores and their packing within the arrays allows for energy production under very low light intensities rendering chlorosomes probably the most efficient antenna complexes found in nature. Inspired by nature, scientists are aiming at the development of mimics of the light harvesting complexes (LHCs). Over the last two decades, different approaches have been pursued in the design of artificial photosynthetic systems.18−25 The preparation of synthetic light harvesting and energy transferring devices is therefore a topic of ongoing interest.26−32 DNA is increasingly used as a scaffold for the placement and assembly of functional components including proteins, chromophores, metal ligands, and nanoparticles.33−38 This © XXXX American Chemical Society

Received: June 7, 2012 Revised: September 18, 2012

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dx.doi.org/10.1021/bc300302v | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry

Article

Figure 1. Schematic representation of the DNA-assembled chromophoric systems studied in this paper. Phenanthrene units (P, blue discs) and one pyrene unit (S, green discs) built the core segment. The influence of the DNA base pair (Y and R, red and gray square plates) next to the core segment was investigated. Gray cylinders on both sides of the core segment represent the DNA sequences composed entirely of AT base pairs. The used artificial building blocks are displayed on the right side.

square plates, R and Y), which may also be a GC base pair. Guanine, which has the lowest oxidation potential of the four canonical DNA nucleobases,80−82 acts as a fluorescence quencher. In the four different sets, S and G are either in direct contact (control set) or separated by an intermediate phenanthrene stack (sets 1, 2, and 3).

studied systems containing only AT base pairs, the PS-exciplex acted as the major “energy collecting center”. The presence of a GC base pair at the opposite end of the π-stack will quench some of the excitation energy. The degree of quenching can be directly measured by a reduction of the exciplex fluorescence. Here, we describe the effect of a GC base pair on the excitation energy transfer in a DNA-based light harvesting antenna composed of phenanthrene and pyrene chromophores. Sixteen different oligomers (1−16) were synthesized (Figure 1 and Table 1) and are grouped into four sets of hybrids for this

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EXPERIMENTAL PROCEDURES Synthesis. Syntheses of the pyrene (S) and phenanthrene (P) phosphoramidite building blocks were carried out as previously described.83−85 The abasic site analogue (φ, Glen Research) and the natural nucleoside phosphoramidite building blocks were purchased from SAFC Proligo. Oligonucleotides 1−16 were synthesized on an Applied Biosystems 394-DNA/ RNA synthesizer (“trityl-off” mode) using standard phosphoramidite chemistry on a 1 μmol synthesis scale. The holding time in the coupling step was set at two minutes for the artificial building blocks. The oligonucleotides were cleaved from the solid support and deprotected by treatment with 30% ammonium hydroxide solution (55 °C, 2 h) and purified by reverse-phase HPLC (LiChrospher 100 RP-18 5 μm column; eluents: acetonitrile and 0.1 M triethylammonium acetate at pH 7.0). The oligonucleotides were characterized on a Shimadzu LCMS-2010EV (Waters XTerra MS C-18 3.5 μm column; eluents: acetonitrile and 50 mM ammonium formate) and quantified by absorbance measurement at 260 nm (phenanthrene P: ε260 = 26 900 L mol−1 cm−1, pyrene S: ε260 = 8600 L mol−1 cm−1). Spectroscopy. All experiments were carried out at a 2 μM oligonucleotide concentration (each strand), 100 mM sodium chloride, and 10 mM sodium phosphate buffer at pH 7.0. Absorbance and thermal denaturation experiments were carried out on a Varian Cary-100 Bio-UV/vis spectrophotometer (optical path length 10 mm). Melting temperature (Tm) values were determined from the first derivative of the second cooling ramp of a cooling−heating−cooling cycle in the temperature range 10−90 °C using a temperature gradient of 0.5 °C/min. Fluorescence data were collected on a Varian Cary Eclipse fluorescence spectrophotometer [2.5 nm ex./em. slit width; detector sensitivity: 750 V (fluorescence), 700 V (excitation); 15 °C probe temperature]. DNA hybrids: a solution of a 1:1 mixture of the two complementary oligomers was heated to 90 °C and allowed to cool to room temperature. Fluorescence spectra were recorded after an equilibration time of 15 min.

Table 1. Oligomers 1−16 and Melting Temperature (Tm) Values of the Hybrids no. control set

set 1

set 2

set 3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

hybrid 5′-TAA 3′-ATT 5′-TAA 3′-ATT 5′-TAA 3′-ATT 5′-TAA 3′-ATT 5′-TAA 3′-ATT 5′-TAA 3′-ATT 5′-TAA 3′-ATT 5′-TAA 3′-ATT

TAA ATT S TTA AAT AAT ATT TAA φ AAT TTA TTA TAA ATC S TTA AAT AAT ATT TAG φ AAT TTA TTA TAA ATT P S TTA AAT AAT ATT TAA Pφ AAT TTA TTA TAA ATC P S TTA AAT AAT ATT TAG Pφ AAT TTA TTA TAA ATT P P S TTA AAT AAT ATT TAA P Pφ AAT TTA TTA TAA ATC P P S TTA AAT AAT ATT TAG P Pφ AAT TTA TTA TAA ATT P P P P S TTA AAT AAT ATT TAA P P P Pφ AAT TTA TTA TAA ATC P P P P S TTA AAT AAT ATT TAG P P P Pφ AAT TTA TTA

Tma [°C] 27.0 31.0 34.5 40.0 36.5 41.5 37.0 41.0

Conditions: oligomer concentration 2 μM (each strand), 100 mM sodium chloride, and 10 mM sodium phosphate buffer at pH 7.0; Tm values ±1 °C. a

study. Phenanthrene (P, blue disks) and pyrene (S, green disk) units constitute the central segment (core) of the system, as illustrated in Figure 1. The core of each hybrid includes different numbers of P units. An S unit, which is placed opposite an abasic site analogue (orange disk, φ) to eliminate the possibility of positional isomers, was located at one end of the π-stack. The resulting hybrids contain, thus, one S unit and zero, two, four, or eight P units. The two outer segments (gray cylinders) of the hybrids are exclusively composed of AT base pairs, except for one base pair next to the π-stack (red and gray B



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Figure 2. UV/vis absorption profiles of (a) selected single strands (5 black, 6 gray, 9 blue, 10 light blue, 13 red, 14 orange) and (b) hybrids (1*2 black, 3*4 gray, 5*6 light blue, 7*8 blue, 9*10 red, 11*12 orange, 13*14 green, and 15*16 light green).

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RESULTS The melting temperature (Tm) values of the hybrids obtained by thermal denaturation experiments varied between 27.0 and 41.5 °C (Table 1 and SI). The relatively low Tm values in the control set (1*2 and 3*4) are due to the high AT content as well as to the abasic site analogue φ, which is only partly compensated by the presence of the intercalating S residue.85−87 A growing number of interstrand stacking residues in sets 1, 2, and 3 lead to an increase in hybrid stability. In all four sets, the presence of a GC base pair next to the core segment had a stabilizing effect. To ensure duplex stability for all hybrids, all UV/vis and fluorescence experiments were performed at 15 °C. Figure 2 shows the UV/vis absorption profiles of selected single strands and all hybrids. The spectra can be divided into two regions: below 300 nm, where the DNA nucleobases absorb and above 300 nm, where only P and S absorb. The long wavelength absorbance bands of P (λmax ∼320 nm) and S (λmax ∼360 nm) are well separated to allow selective excitation of either type of building block (P: λex = 320 nm; S: λex = 365 nm). The oligomers of the control set were next investigated by fluorescence spectroscopy. The emission spectra of single strands 1 and 3, possessing one S but no P units, are given in Figure 3. The fluorescence intensities of single strand 1 with a 5′-T next to S (Figure 3a, black line) and of single strand 3 with a C in this position (Figure 3b, black line) were compared. The curves of 1 and 3 with maximum intensity around 400 nm (pyrene monomer emission) show no significant differences in shape and intensity after excitation of S at 355 nm. Thus, the two different nucleobases, T or C, do not lead to significant changes in the emission behavior of S. The fluorescence emission spectrum of hybrid 1*2 showed an emission spectrum (Figure 3a, red line) that was nearly identical in emission intensity and curve shape as that of single strand 1 alone. In contrast, the intensity changed significantly upon hybrid formation between 3 and 4 (Figure 3b, red line). The fluorescence at 400 nm of 3*4 was strongly quenched compared to 3 alone. No pyrene-guanine exciplex emission was detected.88,89

Figure 3. Fluorescence emission spectra of single strands and hybrids from the control set after excitation at 355 nm (black lines = single strands, red lines = hybrids) with (a) an AT base pair next to the core segment (1*2) and (b) a GC base pair in the same position (3*4).

Oligomers of sets 1, 2, or 3 that contain both types of chromophores (P and S) can be excited separately at either of these units (see above, Figure 2). Excitation of P at 320 nm in single strands having one S and one, two, or four P units in the same sequence results in a broad emission with a maximum at around 450 nm (strands 5, 7, 9, 11, 13, and 15, Figure 4a and b, black lines). This band has been previously assigned to phenanthrene/pyrene-exciplex fluorescence.90,91 As in the control set, no significant differences in the emission behavior was observed if a C or T pyrimidine nucleobase was adjacent to the phenanthrenes. Intensities and shapes of the emission curves of all mentioned single strands were essentially identical. Excitation of single strands containing only P units (strands 6, 8, 10, 12, 14, and 16, Figure 4a,b, blue lines) at 320 nm showed relatively weak fluorescence with maximum intensities between 380 and 410 nm depending on the number of P units. The presence of G in the 3′-position of P (Figure 4b, strands 8, 12, C



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Figure 4. Fluorescence spectra of single strands and hybrids containing a variable number (2n) of phenanthrenes in each strand. (a) Excitation at 320 nm of hybrids 5*6 (n = 1), 9*10 (n = 2), and 13*14 (n = 4). Single strands with only P units (6, 10, 14) are shown in blue, single strands with P and S units (5, 9, 13) in black and DNA hybrids in red. (b) Excitation at 320 nm of hybrid 7*8 (n = 1), 11*12 (n = 2), and 15*16 (n = 4). Single strands with only P units (8, 12, 16) are shown in blue, single strands with P and S units (7, 11, 15) in black, and DNA hybrids in green. (c) Excitation of all hybrids at 365 nm. Hybrids with AT next to the core segment (5*6, 9*10, and 13*14) are shown in red, hybrids with GC next to the core (7*8, 11*12, and 15*16) are shown in green.

and 16) resulted in significant fluorescence quenching compared to adenine (A) in the same position (Figure 4a, strands 6, 10, and 14). Hybrid 5*6 showed a higher exciplex emission intensity at 450 nm than the single strand 5 alone (Figure 4a, n = 1, red line versus black line). Hybrid 7*8, on the other hand, had a less intense emission curve than single strand 7 alone (Figure 4b, n = 1, green line versus black line). This is in agreement with the control set where addition of a G in the hybrid next to the chromophore resulted in strong quenching of the S monomer fluorescence. The presence of G also resulted in the quenching of exciplex fluorescence in hybrid 7*8 compared to hybrid 5*6. The hybrids in set 2 behaved in analogy to those in set 1. Hybrid 9*10 with an AT base pair next to the core segment showed stronger fluorescence at 450 nm compared to hybrid 11*12 with a GC base pair at this site. As expected, excitation of hybrid 13*14 at 320 nm in set 3 resulted in the highest exciplex fluorescence emission intensity at 450 nm. This hybrid contains the largest number of P residues and no GC base pair next to the chromophore segment. Again, in the GC-containing hybrid 15*16, the emission was quenched in comparison to 13*14. In contrast to the observations made with sets 1 and 2, however, the emission intensity of 15*16 was higher than that of single strand 15. As mentioned above, selective excitation of S at 365 nm is possible in all hybrids. In this case, the resulting emission curves of hybrids 5*6, 9*10, and 13*14 showed equal exciplex fluorescence intensities at 450 nm (Figure 4c, red lines). One S unit is present in all these hybrids, and the varying number of P units has no influence on the PS-exciplex emission intensities. Furthermore, no significant variation in the maximum emission wavelength was observed compared to excitation at 320 nm. On the other hand, emission intensities of hybrids 7*8, 11*12,

and 15*16, which have the GC base pair next to the chromophore stack, were different (Figure 4c, green lines) from those of hybrids with an AT base pair in this position. Hybrid 7*8 had the least intense exciplex emission and was considerably quenched compared to 5*6. This quenching effect by GC was less pronounced in set 2; in set 3, hybrid 15*16 showed almost the same emission intensity as hybrid 13*14. Thus, the quenching effect was gradually reduced with an increasing number of P units located in the stack between the site of excitation (pyrene, S) and the GC base pair. An overview on the emission intensities of the various hybrids is given in Table 2 and the values are displayed Table 2. Fluorescence Intensities (Ifl, auca) of Phenanthrene/Pyrene-Containing Hybrids λex = 320 nm

a

λex = 365 nm

hybrid

Ifl [auc]

ΔIfl [auc]

relative [%]

Ifl [auc]

5*6 7*8 9*10 11*12 13*14 15*16

20 877 12 506 30 612 22 262 49 038 39 424

8371

100 60 100 73 100 80

13 504 9228 13 375 11 351 13 699 12 810

8350 9614

ΔIfl [auc]

relative [%]

4276

100 68 100 85 100 94

2024 889

Area under the emission curve.

graphically in Figure 5. The chart is grouped into emission intensities observed after excitation at 320 nm (excitation of P, blue and black bars) or 365 nm (excitation of S, red and green bars). Black and green bars represent hybrids with an AT base pair next to the core segment (5*6, 9*10, and 13*14; n = 1, 2, and 4), whereas red and blue bars stand for hybrids with GC D



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Figure 5. Dependence of the fluorescence intensity (area under curve, auc) of different hybrids on the number of P units, the DNA sequence, and the excitation wavelength. Excitation (λex): 320 nm (blue and black bars) or 365 nm (red and green bars). Black and green bars: hybrids with an AT base pair adjacent to the arene segment. Blue and red bars: hybrids with a GC base pair adjacent to arene segment.

Figure 6. Excitation spectra of different hybrids observed at 450 nm: (a) uncorrected and (b) normalized at 365 nm (5*6 black, 7*8 gray, 9*10 blue, 11*12 light blue, 13*14 red, and 15*16 orange).

next to the core segment (7*8, 11*12, and 15*16). Fluorescence intensities [Ifl, given as area under the curve (auc) values] after excitation at 320 nm were reduced by the presence of a GC base pair in each set by approximately the same fraction compared to the corresponding hybrids with an AT base pair (black vs blue bars, see also SI). The differences in intensity (ΔIfl values) were relatively constant, i.e., to a large part independent of the number of P units present in the chromophore stack. A look at the relative intensities, however, reveals that the quenching effect of the GC base pair after excitation at 320 nm becomes less important when the number of P units increases. Hybrid 7*8 shows an exciplex fluorescence which is quenched by 40% compared to hybrid 5*6, while emission of hybrid 15*16 is only quenched by 20% compared to 13*14. Fluorescence intensities after excitation at 365 nm (S unit) reveal a clear length dependence (green bars versus red bars): the more P residues present, the less important the quenching effect of the GC base pair on the PS-exciplex fluorescence becomes. A doubling of the number of P units between each set results in a reduction of the quenching effect

(ΔIfl) by approximately a factor of 2. In all hybrids that are lacking the GC base pair, the fluorescence intensity is equal after excitation at 365 nm (green bars). Thus, it can be concluded that a GC base pair reduces exciplex fluorescence by constant (absolute) value if excitation occurs at 320 nm (phenanthrenes), but after excitation at 365 nm (pyrene), the quenching effect is gradually diminished by increasing numbers of phenanthrenes. Excitation spectra between 230 and 420 nm of all hybrids are shown in Figure 6a (λem = 450 nm, maximum intensity of the PS-exciplex). To better visualize the quenching effect of the GC base pair, the spectra are normalized at 365 nm, the maximum of pyrene excitation (Figure 6b). The obtained values are also summarized in Table 3. The intensity at 320 nm (phenanthrene) increases steadily with increasing number of P units, whereas the values at 365 nm are relatively constant. Within each set, the hybrid containing the GC base pair shows a less intense excitation band at 320 nm compared to the hybrid with the AT base in this position. The difference between the values (ΔIF) within one set is almost constant at 320 nm (Table 3) E



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increase of exciplex emission upon increasing the number of P units (see also SI, Figure S17). The picture changes when a GC base pair is introduced into the DNA-part, directly adjacent to the chromophore stack (hybrids 7*8, 11*12, and 15*16). The introduction of this GC base pair results in the presence of a second “trap” for the excitation energy. Fluorescence quenching by the formation of charge-separated states between excited chromophores and guanine is a well-known and extensively studied mechanism.81,96,97 This effect is clearly present, as shown, e.g., by the control set (1*2 and 3*4) exhibiting a strong reduction of S fluorescence by the GC base pair (Figure 3). This quenching effect is also present, albeit to a lesser extent, when both chromophores, S and P, are present in the hybrid. Thus, hybrid 7*8 shows reduced exciplex fluorescence compared to 5*6 (Figure 4b, green lines). The emission spectrum of hybrid 7*8 is not superimposable with the sum of the spectra reported for single strands 7 and 8 alone (see SI, Figure S14). Despite this quenching effect, also GC-containing hybrids (7*8, 11*12, and 15*16) reveal an increase of the exciplex emission intensity with a growing number of P units. After irradiation at 320 nm, the absolute amount of fluorescence quenching compared to hybrids lacking the GC base pair is rather constant, irrespective of the number of involved P units (see also Figure 5, black bars versus blue bars). This is best illustrated by a fairly constant ΔIfl-value over the three sets after excitation at 320 nm (see Table 2). Analysis of the excitation spectra recorded at the maximum emission wavelength of the exciplex (λem = 450 nm) confirms this observation. Hybrids containing a GC base pair showed a signal of lower intensity at 320 nm compared to hybrids lacking the GC base pair. The P-excitation band was reduced by a rather constant amount. Thus, less excitation energy from the P-units leads to exciplex emission in the presence of a GC base pair, and a relatively constant (absolute) fraction of excitation energy is absorbed by guanine from the Pstack irrespective of the number of P units. Consequently, less excitation energy is transferred to the S unit resulting in a reduction of exciplex fluorescence. It can be concluded that longer P-arrays partly “compensate” the energy trapping effect of the GC base pair. The relative quenching effect is getting smaller as the length of the P-stack increases (Table 2). Even when quenching by a GC base pair was observed, it is important to note that in all cases studied the main direction of energy transfer in the P-stack remained toward the PS-exciplex, i.e., the quenching effect never exceeded 40%.

Table 3. Intensities (IF) in Excitation Spectra of the Different Hybrids (λem = 450 nm; see Figure 6) excitation spectra hybrid

# of P units

IF (320 nm)

5*6 7*8 9*10 11*12 13*14 15*16

2 2 4 4 8 8

138 84 196 151 324 264

ΔIF 54 45 60

IF (365 nm) 90 62 87 76 90 84

ratio ΔIF

(320/365)

[%]

28

1.51 1.36 2.25 1.95 3.61 3.16

100 88.5 100 87.0 100 86.3

11 6

but decreases at 365 nm when the length of the phenanthrene stack is extended. Normalization to the 365 nm band reveals that the 320 nm band is reduced by a relatively constant fraction (∼12−14%).

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DISCUSSION The present system constitutes a light harvesting assembly of phenanthrene (P) and pyrene (S) chromophores. The arenes are arranged in a π-stacked assembly within a double-stranded DNA framework. The P building blocks absorb light at 320 nm. On excitation, a PS-exciplex is formed which results in intensive exciplex fluorescence around 450 nm. A linear increase of exciplex fluorescence after excitation at 320 nm of hybrids 5*6, 9*10, and 13*14 with an AT base pair next to the arene segment (Figure 5, black bars) is attributed to efficient excitation energy transfer from P-units to the PS-exciplex. This energy transfer becomes also apparent when the fluorescence emission curves of hybrid 5*6, 9*10, and 13*14 are compared to the emission curves of the single strands alone. The fluorescence curve resulting from a hybrid is not corresponding to the sum of the two spectra of the single strands (see also SI, Figures S14−S16). The hybrid exhibits increased fluorescence intensity and a more pronounced signal (Figure 4a, red versus black and blue lines) than the single strands. No significant S or P monomer emission was detected in the emission spectra of hybrids containing phenanthrenes indicating that loss of excitation energy due to inefficient transfer to the exciplex is minimal. Formation of P excimers to a minor degree cannot be completely ruled out based on the emission spectra of single strands 10, 12, 14, and 16.92−95 Even if these kinds of excimers were formed after excitation, they are, obviously, not affecting the overall efficiency of the excitation energy transfer process, which is best illustrated by a linear

Figure 7. Amber-minimized model100 of hybrid 15*16. The pyrene unit (S) is shown in green, phenanthrene units (P) in blue, and guanine (G) in brown. Excitation at 320 nm (step 1) is followed by excitation energy transfer (step 2) from P units to the PS-exciplex, which results in fluorescence at 450 nm (step 3). The presence of a GC base pair next to the core segment leads to a partial quenching of the excitation energy. The degree of quenching depends inversely on the number of phenanthrenes in the stack. F



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After selective excitation of the S unit at 365 nm, exciplex fluorescence is constant and not dependent on the number of P residues (Figure 5, green bars), as long as a GC base pair next to the arene core segment is avoided. Introduction of a GC base pair, however, again reduces exciplex fluorescence intensity also after S excitation. The effect correlates again inversely with the number of intervening P units (Figure 5, red bars). Whereas the quenching effect was significant (∼32%, Table 2) in set 1 (only two P units), only a marginal reduction (∼6%) was observed in set 3 (eight P units). Thus, exciplex fluorescence quenching by guanine after excitation of the S unit is significant with two and four intervening P units but becomes negligible if eight P units are present in the stack. The findings are summarized and illustrated in Figure 7. A stack of up to eight P units acts as light absorbing unit. Excitation leads to formation of a PS-exciplex with an emission maximum at 450 nm. Fluorescence intensity increases with the number of phenanthrenes. A guanine located at the end of the P-stack leads to a partial quenching of exciplex fluorescence. The degree of quenching is dependent on the length of the phenanthrene stack that separates the GC base pair from the exciplex. Overall, a large number of stacked phenanthrenes positively affects exciplex fluorescence by increasing the quantity of absorbed light and, at the same time, reducing the effect of quenching by GC base pairs.

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Article

ASSOCIATED CONTENT

S Supporting Information *

Analytical data for modified oligomers, Tm value curves, UV/vis and fluorescence spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by the Swiss National Science Foundation (grant 200020-132581) is gratefully acknowledged. REFERENCES

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CONCLUSIONS

The effect of a GC base pair on the excitation energy transfer in a DNA-embedded, light harvesting assembly of phenanthrene (P) and pyrene (S) chromophores is described. After absorption of light at 320 nm by the stacked P building blocks, the excitation energy is transferred to S and results in the formation of a fluorescent PS-exciplex (Figure 7). The fluorescence intensity is dependent on the number of light absorbing P units, as well as on the nature of the DNA base pair next to the P-stack. Several double-stranded hybrids were studied that contained P-stacks of zero, two, four, and eight units. Excitation of the hybrids at 320 nm (excitation of P units) results in a steady increase of exciplex fluorescence with increasing length of the P-stack. On the other hand, it was found that a GC base pair adjacent to the P-stack results in significant fluorescence quenching in comparison to an AT base pair at this location. The reduction of fluorescence intensity (ΔIfl) was rather constant in all sets of hybrids. In relative terms, however, the quenching effect by this GC base correlates inversely with the number of P units separating it from the exciplex. The stacked P units have, thus, a length-dependent, insulating effect98,99 that prevents quenching of the exciplex by the GC base pair. The PS-exciplex can also be generated by excitation of the S unit. As expected, the degree of exciplex fluorescence is not influenced by the number of P units in the hybrids composed of AT base pairs only. If, however, a GC base pair is present, exciplex fluorescence is quenched also in this case. Again, a longer P-stack counteracts the quenching effect and becomes negligible in the hybrid containing a total of eight P units. The π-stacked arrays of aromatic hydrocarbons described here represent a valuable model system to practically and theoretically study light harvesting and energy transfer effects and may find relevance in the design and construction of related systems with other kinds of chromophores. G



Article

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Influence of a GC Base Pair on Excitation Energy Transfer in DNA

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