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Functional Nanostructured Materials (including low-D carbon)
DNA Origami Based FRET Nanoarrays and their Application as Ratiometric Sensors Youngeun Choi, Lisa Kotthoff, Lydia Olejko, Ute Resch-Genger, and Ilko Bald ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03585 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018
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ACS Applied Materials & Interfaces
DNA Origami Based FRET Nanoarrays and their Application as Ratiometric Sensors. Youngeun Choi†,‡,§, Lisa Kotthoff†,‡, Lydia Olejko†, Ute Resch-Genger‡,§, and Ilko Bald*†,‡,§ †Department of Chemistry, Physical Chemistry, University of Potsdam, 14476 Potsdam, Germany ‡BAM Federal Institute for Materials Research and Testing, 12489 Berlin, Germany §School of Analytical Sciences Adlershof, Humboldt-Universität zu Berlin, 10099 Berlin, Germany ABSTRACT: DNA origami nanostructures provide a platform where dye molecules can be arranged with nanoscale accuracy allowing to assemble multiple fluorophores without dye-dye aggregation. Aiming to develop a bright and sensitive ratiometric sensor system, we systematically studied the optical properties of nanoarrays of dyes built on DNA origami platforms using a DNA template that provides a high versatility of label choice at minimum cost. The dyes are arranged at distances, at which they efficiently interact by Förster resonance energy transfer (FRET). To optimize array brightness, the FRET efficiencies between the donor fluorescein (FAM) and the acceptor cyanine 3 (Cy 3) were determined for different sizes of the array and for different arrangements of the dye molecules within the array. By utilizing nanoarrays providing optimum FRET efficiency and brightness, we subsequently designed a ratiometric pH nanosensor using coumarin 343 (C343) as pH inert FRET donor and FAM as pH responsive acceptor. Our results indicate that the sensitivity of a ratiometric sensor can be improved simply by arranging the dyes into a well-defined array. The dyes used here can be easily replaced by other analyte responsive dyes demonstrating the huge potential of DNA nanotechnology for light harvesting, signal enhancement, and sensing schemes in the life sciences.
KEYWORDS: DNA origami, nanoarray, FRET, ratiometric sensing, pH sensing █ INTRODUCTION Multiparametric fluorescence techniques utilizing molecular and nanoscale fluorescent reporters are widely used in the life and material sciences due to their ease of use, suitability for in vitro and in vivo imaging, sensitivity down to the single molecule level, and nanoscale resolution1,2. A common strategy for signal enhancement are multichromophore systems. The efficiency of such systems is, however, largely controlled by dyedye interactions. Depending on the orientation of the dye dipole moments this can lead to the formation of H-type dimers and aggregates, which are commonly not or barely emissive and can act as energy sink via Förster resonance energy transfer (FRET) between chemically identical, yet spectroscopically distinguishable fluorophores3,4. FRET is a nonradiative energy transfer process that occurs between an excited donor molecule and an acceptor molecule through dipoledipole interactions. Its distance dependent nature has prompted development of many analytical tools for sensing including biologically relevant molecules5. However, for multichromophore systems it is necessary to develop strategies to avoid fluorescence-diminishing dye-dye interactions. A powerful tool that allows for controlled arrangement of molecules with different functionalities is the DNA origami technology.6,7 As each oligonucleotide is addressable and can be easily modified chemically, a DNA origami structure can be folded to incorporate functionalities at predetermined positions. DNA origami structures have been meanwhile employed for the self-assembly of molecular and nanocrystal moieties8,9 including organic dye molecules10–13, nanoparticles14–16, quan-
tum dots17–19, and proteins20–22. Due to their unique features, DNA origami nanostructures also present very interesting platforms for all applications utilizing FRET such as creating photonic wires23,24, light harvesting constructs25–27, and sensing schemes28. These FRET based structures provide yet another crucial advantage for developing sensors as they can be designed as self-referenced dual wavelength probes. This eliminates analyte-independent signal distortions often encountered for single color fluorescence intensity measurements29–32. The broad applicability of such nanostructures requires not only the design of DNA origami nanoarrays with optimum brightness and spectroscopic performance, but also with minimum cost. This can be achieved by a DNA template design, where dyes can be easily replaced to allow for simple adaptation to different tasks. Aiming to explore the potential of the DNA origami technology for light harvesting, signal amplification, and sensing, we assemble different donor and acceptor fluorophores on DNA origami structures and systematically assess the influence of nanoarray size and dye pattern on their optical properties and FRET efficiency using steady state and time-resolved fluorometry. The optimum design concept was then expanded to the design of ratiometric pH nanosensors utilizing pH inert and pH responsive dyes as FRET donors and acceptors. █ METHODS DNA origami synthesis The triangular DNA origami nanostructures were fabricated as previously described28 using M13mp18 (tilibit nanosystems) viral strand with 208 short oligonucleotides in TAE buffer (10 x concentrated) containing 150 mM MgCl2 and
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Figure 1. (A) Folding process into DNA origami based dye array. A triangular DNA origami is self-assembled first with extended staple strands to act as handles. The dye molecules are then positioned on the DNA origami by hybridization of the dye labeled ssDNA with the overhanging ssDNA from the DNA origami. (B) FRET arrays and nomenclature used. 12 positions from the DNA origami were chosen to act as dye labeling sites, allowing to construct arrays of different size ranging from (2x1) to (3x4). The donor (orange) and acceptor molecules (red) were arranged in a checkerboard pattern while keeping the ratio of donor to acceptor fixed at 1:1. (C), (D) Normalized excitation (dotted line) and emission spectra (solid line) of donor and acceptor molecule with the spectral overlap of donor emission and acceptor excitation colored-in in grey, with (C) of FAM as donor and Cy3 as acceptor used for systematic study of the nanoarray and (D) of coumarin 343 (C343) as donor and FAM as acceptor for its application as pH ratiometric sensor.
ultrapure water (Merck Millipore). The solution was subsequently annealed from 80 °C and slowly cooled down to 8 °C in 2 hours using a thermal cycler (PEQLAB/VWR). The solution was purified using centrifugal filters (100 kDa MWCO, Merck Millipore) by washing 3 times (6000 rpm, 7 mins) with 1 x TAE with 15 mM MgCl2 to remove excess staple strands. Subsequent hybridization was performed by mixing the DNA origami solution with dye modified oligonucleotides (Metabion) in 1 x TAE, 15 mM MgCl2 solution, heating up to 45 °C and cooling down to 25 °C over 1 hour using again the thermal cycler. The final DNA origami solution was purified again using centrifugal filters 6 times. Final concentration of DNA origami in solution was set to approximately 5 nM for fluorescence measurements, and checked using UV-Vis absorption spectroscopy (NanoDrop 2000, Thermo Scientific). For pH ratiometric sensing each pH buffer (1x TAE, 15 mM MgCl2) was prepared by adding appropriate amounts of HCl and NaOH, and the pH measured using a pH meter (Orion 3 Star, Thermo Scientific). Buffers were exchanged at second washing step of DNA origami folding to have the desired pH. For more detailed description of folding scheme see SI (Figure S1). AFM imaging AFM imaging was performed for each DNA origami sample to confirm their structure. 2 µL of the purified sample was adsorbed on freshly cleaved mica (Plano) with 28 µL of buffer (1 x TAE with 15 mM MgCl2) and incubated for 30 s. After incubation, the sample was washed with ultrapure water and dried with compressed air. The imaging was performed under dry conditions (Flex AFM, Nanosurf) using cantilevers (Tap150 Al-G, Budget Sensors) with a resonance frequency of 150 kHz and a spring constant of 5 Nm-1. An AFM image of a typical triangular DNA origami structure is shown in Figure S2. Steady-state fluorescence spectroscopy Steady-state spectroscopy measurements were performed with a spectrophotometer (Fluoromax P, HORIBA Jobin Yvon) with 3 mm quartz cuvettes (Hellma Analytics). Fluo-
rescence emission was collected at a right angle to the excitation beam using an internal quantum correction system. For FRET nanoarray analysis using FAM and Cy3, an excitation wavelength of 450 nm was chosen, and for ratiometric pH sensing with C343 and FAM an excitation wavelength of 400 nm, respectively. For collecting excitation spectra, the emission wavelengths were set to 600 nm and 550 nm, respectively. For all spectra, acquisition at an increment of 1 nm, integration time of 0.2 s, and bandpass of 5 nm were used. Time-correlated single photon counting The FRET efficiency is strongly distance-dependent according to eq. 1, "#$% &''()(&*)+ ,
-./ / -./ 0123
.
(1)
In eq. 1, #4 is the Förster distance, at which the FRET efficiency is 50 %, and 567 is the distance between donor and acceptor molecule. In order to avoid inaccuracies due to direct excitation of the acceptor, FRET efficiencies have been determined from the fluorescence lifetimes of the donor: "#$% &''()(&*)+ , 1 9
:;23 :2
.
(2)
In eq. 2,
