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Directed Energy Transfer through DNA-templated J-aggregates Sarthak Mandal, Xu Zhou, Su Lin, Hao Yan, and Neal W. Woodbury Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00043 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019
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Bioconjugate Chemistry
Directed Energy Transfer through DNA-templated J-aggregates Sarthak Mandal,1,4, ‡, * Xu Zhou,2,3, ‡ Su Lin,1, 3 Hao Yan2, 3* and Neal Woodbury1, 3* 1Center for Innovations in Medicine at the Biodesign Institute, Arizona State University, Tempe, Arizona, 85287, USA 2Center for Molecular Design and Biomimetics at the Biodesign Institute, Arizona State University, Tempe, Arizona, 85287, USA 3School of Molecular Sciences, Arizona State University, Tempe, Arizona, 85287, USA 4Department of Chemistry, National Institute of Technology Tiruchirappalli, 620015, Tamil Nadu, Indian
ABSTRACT: Strongly coupled molecular dye aggregates have unique optoelectronic properties that often resemble those of light harvesting complexes found in nature. The exciton dynamics in coupled dye aggregates could enhance the longrange transfer of optical excitation energy with high efficiency. In principle, dye aggregates could serve as important components in molecular-scale photonic devices, however, rational design of these coupled dye aggregates with precise control over their organization, interactions and dynamics remains a challenge. DNA nanotechnology has recently been used to build an excitonic circuit by organizing pseudoisocyanine (PIC) dyes forming J-aggregates on the templates of poly(dA)-poly(dT) DNA duplexes. Here, the excitonic properties of the PIC J-aggregates on DNA are characterized spectroscopically in detail using poly(dA)-poly(dT) tract lengths of 24 and 48 base pairs. The excitonic properties of these DNA templated dye assemblies depend on the length and sequence of the DNA template. The incorporation of a gap of two GC base pairs between two segments of poly(dA)-poly(dT) DNA markedly reduces the delocalization of excitation in the Jaggregates. Using a quantum dot (QD) as the light absorber and energy donor and using Alexa Fluor 647 (AF647) as the energy acceptor, with a DNA-templated J-aggregate in between, significant energy transfer from QD to AF647 is observed over a distance far longer than possible without the aggregate bridge. By comparing the efficiency of energy transfer through a continuous J-aggregate with the efficiency when the aggregate has a discontinuity in the middle, the effects of energy transfer within the aggregate bridge between the donor and acceptor are evaluated.
Introduction Understanding energy transfer in rationally designed dyeaggregate structures is of interest in the development of artificial light harvesting and biophotoelectronic devices.13 Nature makes use of ordered pigment aggregates within specific protein scaffolds to achieve highly efficient and rapid exciton transport involving localized quantum coherence.4-5 The excitonic properties of strongly coupled dye aggregates, particularly J or J-like aggregates, resemble those of natural pigment aggregates.6-10 Excitons in dye Jaggregates are delocalized over several monomers and are often characterized in terms of their coherence length. This coherent delocalization is thought to enhance exciton transport efficiency compared with incoherent hopping of localized excitons.9-13 The packing arrangement of the dyes in a J-aggregate largely dictates the extent of exciton delocalization and thus its performance in exciton transport.14-18 Fluorescence resonance energy transfer (FRET)-based multichromophoric assemblies have been explored previously using different templates including supramolecular polymer/lipid assemblies, biomacromolecular assemblies (proteins/peptides) and metal-organic frameworks.19-23 Recently, DNA nanostructures have been used as scaffolds to control and optimize the number and spatial arrangements of dyes through covalent or noncovalent attachment for
programmed FRET studies.24-28 A variety of fluorescent dye intercalators and quantum dots have been used to develop energy transfer relays on simple DNA duplexes for demonstrating long range energy transfer.29-36 In most of these systems, the chromophores are spatially separated by distances that result in weak electronic coupling interactions between localized excitons. Simply moving multiple chromophores close to each other without specific structural constraints generally results in selfquenching which competes with energy transfer and limits its efficiency. This comes about when chromophores are close enough so that their electronic states are modulated by intermolecular movement, providing a route for nonradiative decay of the excited state. The design of strongly coupled biomimetic dye-aggregates with appropriate structural constraints that limit energy dissipation via self-quenching and tailored excitonic properties holds promise in overcoming this issue. Recently, programmed assemblies of DNA templated pseudoisocyanine (PIC) dye aggregates have been introduced for mediating nanoscale energy transfer through strongly coupled, densely packed dyes.37-38 Such excitonic systems can potentially be used as building blocks in the design of long distance energy transfer systems (Figure 1a). In these aggregates, the coupling between adjacent molecules is strong enough so that the exciton is delocalized over a small number of neighboring molecules. That strong coupling allows rapid energy
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transfer, probably via a mixed mechanism of short range coherent transfer and longer range incoherent transfer.37-38 However, the extent of exciton delocalization and its dependence on PIC dye aggregate structure (upon varying length and sequence of DNA templates) is not well characterized. The present work focuses on two issues in this regard. First, the excitonic properties of PIC Jaggregates on long poly(dA)-poly(dT) DNA duplexes (referred to as J on AT duplexes; Figure 1b, left panel) with poly-A tract lengths of 24 and 48 base pairs are investigated. In addition, to explore how those properties change with the perturbation of the DNA-templated Jaggregate structure, a gap of two GC base pairs is introduced in the middle of the AT segments of the poly(dA)-poly(dT) DNA (referred to as J(gap) on AT:GC:AT duplexes; Figure 1b, middle panel). The excitonic properties of the J–aggregates and J(gap)– aggregates are then compared with that of nonspecifically bound PIC monomer molecules on control DNA duplexes with sequences of alternating dA and dT base pairs (referred to as PIC (monomer) assemblies on ATA duplexes; Figure 1b, right panel). In the second part of this work, the influence of exciton delocalization and its perturbation in J-aggregates on energy transfer properties is investigated using a quantum dot (QD) as the donor (D) and Alexa Fluor 647 (AF647) as the terminal acceptor (A), with several different versions of DNA-templated PIC aggregates (J/J(gap)/PIC (monomer)) serving as a bridge between them. QDs are often used as FRET donors due to their tunable and spectrally narrow fluorescence, their stability with regard to photobleaching, and their high quantum yield. The broad absorption cross-section of the quantum dots with high molar extinction coefficients at short wavelengths (e.g., 400 nm) where the absorption of PIC and AF647 are minimal is advantageous in the studies described here, in comparison with most molecular dyes. For example, ATTO-390 was used earlier to demonstrate energy transfer through J-aggregates but its spectral characteristics made analysis of light absorbed by the donor and light absorbed by the aggregate difficult.38 Moreover, the narrow emission spectrum of QD donors will help suppress the degree of reabsorption of donor emission by the necessarily high concentrations of free PIC that must be used to ensure aggregate formation on the DNA.
Results and Discussion DNA-Templated PIC Aggregates. PIC dye spontaneously forms J-aggregates with the right-handed charily on poly(dA)-poly(dT) DNA duplexes (referred to as AT duplexes) through head-to-tail molecular dipole arrangement.38 The melting temperature of 24 bp AT duplex increase ~7 °C after J-aggregates formed (Figure S16). A red-shifted, narrow absorption band characteristic of the J-aggregates appears at 550 nm in the absorbance spectrum (Figure 1c, left panel). The line-width of the
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excitonic J-aggregate band is known to exhibit an N–1/2 dependence where N relates to the length of exciton delocalization.39 The amount of strongly coupled dye aggregate increases as the length of the template (poly-A and poly-T tract length) is increased from 24 to 48 base pairs as indicated by the increased intensity of the sharp Jaggregate absorption band at 550 nm.37-38 The intensity of the J-aggregate absorption band decreases significantly with incorporation of two GC base pairs in the middle of the AT duplex (Figure 1c, middle panel). PIC does not assemble into J-aggregates very effectively on G/C duplex regions of DNA, so the two GC base pairs interrupt the Jaggregate structure (referred to as J(gap)-aggregates), perturbing the number of strongly coupled dye molecules and their excitonic structure. Similarly, poly-AT/poly-TA duplexes (referred to as ATA duplexes) do not exhibit the characteristic J-band absorption at 550 nm, consistent with the previous reports (Figure 1c, right panel).38
Figure 1. (a) Sequence of an AT duplex and chemical structure of PIC dye (left panel) and schematic of the structural organization of PIC J-aggregates on AT duplex (right panel). (b) Schematic representations of PIC dye aggregates on DNA duplexes of different lengths and sequences. (c) Absorption spectra of PIC in aqueous buffer (gray) and assembled in different DNA duplexes (green and orange).
PIC J-aggregate formation on AT duplexes results in a strong narrow emission band centered at 565 nm and a broad shoulder peaking at 600 nm in the fluorescence emission spectra collected at 490 nm excitation wavelength (Figure 2a). Similar spectral properties are also observed for the samples using 400 and 525 nm excitation wavelengths (Figure S1). The broad shoulder presumably corresponds to emission from localized excitons in free/non-specifically bound PIC molecules or from the self-trapped excitons in J-aggregates as earlier suggested by Malyukin et al.16 The spectral features of Jaggregates become more intense with increasing length of AT duplexes (Figure 2a) consistent with the increase in the J-band absorption intensity (Figure 1c). A perturbation of
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Bioconjugate Chemistry
the aggregate structure with the formation of J(gap)aggregates on AT:GC:AT duplexes results in a significant decrease in J-band emission (Figure 2b). The broad emission from non-specifically bound monomer assemblies on ATA duplexes exhibits a spectral profile similar to PIC monomers in aqueous buffer and lacks the characteristics of J aggregates (Figure 2c). The intensity of the PIC emission is, however, almost two orders of magnitude higher in ATA duplexes than in aqueous buffer, presumably because of the interaction of PIC with ATA duplexes. The decay pathway to the ground state through free rotation of the two quinonlinic rings of PIC is reduced upon binding to DNA and hence the fluorescence intensity increases relative to that in water.40 Thus, there are really three forms of PIC in these samples: PIC in DNA-templated aggregates, PIC nonspecifically bound to DNA and PIC free in solution. The fluorescence excitation spectra monitored at 600 nm were compared for all samples (Figure S2a-c). The excitation spectra of the J-aggregates and J(gap)aggregates formed on AT and AT:GC:AT duplexes, respectively, are similar (Figure S2a,b) and have a sharp band at 550 nm that matches the J-aggregate band’s absorbance spectrum, suggesting that the emission is primarily contributed by the J-aggregates. The excitation
spectra of the PIC on ATA duplexes is similar to the profiles of the absorption spectra of PIC monomers (Figure S2c). Further, the relative amount of PIC in the monomer and Jaggregate forms can be estimated by comparing the 1Transmitance spectrum with that the fluorescence excitation spectrum illustrated in Figure S2d as an example. Excitation at 400 and 500-565 nm predominantly excites the PIC in the J-aggregate, while excitation at 490 nm excites both PIC in all three forms (aggregate, nonspecifically bound and free). The relative fluorescence yield (fluorescence relative to the number of photons absorbed) drops by ~2-fold at 490 relative to 565 nm, thus at least half the molecules excited at 490 nm either do not fluoresce at 600 nm or are less fluorescent than molecules excited at 565 nm, indicating there is a significant population of PIC molecules that are not in the strongly coupled aggregate. Based on the previous estimate of an effective dissociation constant of 25 µM for PIC binding to AT dsDNA38 and given 90 µM PIC and roughly an equal number of potential binding sites on the DNA (4 µM DNA with ~23 potential sites per molecule), one would calculate that the concentration of free PIC would be a little more than half of the total, consistent with the excitation spectra.
Figure 2. Steady-state fluorescence emission spectra of PIC dye J-aggregates or monomer on (a) AT, (b) AT:GC:AT, and (c)ATA duplexes of tract length of 24 (green) and 48 (orange) base pairs. The spectra were recorded with excitation wavelength of 490 nm (see Figure S1 for comparing the fluorescence spectra of the samples with 400- and 525-nm excitations). Average fluorescence lifetime values of PIC on AT, AT:GC:AT and ATA duplexes with tract length of (d) 24 base pairs and (e) 48 base pairs. The fluorescence decay traces of all samples were recorded at 565 nm and fitted to a bi-exponential function. (f) Decrease of coherence length of the PIC J-aggregates with the incorporation of a gap of GC base pairs on the AT segments of 24AT and 48AT DNA duplexes.
The superradiance and exciton delocalization properties of the DNA-templated PIC J-aggregates were estimated from the relative fluorescence yield and lifetime measurements.17 The quantum yield of fluorescence for free PIC monomers in aqueous buffer solution in the absence of DNA is extremely low (
