Novel Ratiometric Fluorescent Nanothermometers Based on

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Novel ratiometric fluorescent nanothermometers based on fluorophores-labeled short single-stranded DNA Youshen Wu, Jiajun Liu, Ya Wang, Ke Li, Lei Li, Jianhua Xu, and Daocheng Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01554 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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ACS Applied Materials & Interfaces

Novel ratiometric fluorescent nanothermometers based on fluorophores-labeled short single-stranded DNA Youshen Wu†, Jiajun Liu‡, Ya Wang‡, Ke Li‡, Lei Li§, Jianhua Xu§ and Daocheng Wu‡* † Department of Chemistry, School of Science, Xi’an Jiaotong University, Xi’an, 710049, China ‡ Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and Technology, Xi'an Jiaotong University, Xi’an, 710049, China § State Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai, 200062, China KEYWORDS: fluorescent nanothermometer, ratiometric thermometer, single-stranded DNA, MD simulation, FRET.

Corresponding Author

* E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT:

Novel

ratiometric

fluorescent

short

single-stranded

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DNA

(ssDNA)

nanothermometers (ssDNA FT) were developed using the fluorescence resonance energy transfer (FRET) effect of the ssDNA’s end labeled fluorophores. An optimal ssDNA sequence and associated ssDNA FT were determined through combined MD simulation and temperaturerelated FRET analysis. Their fluorescence properties and thermo-responsivities were analyzed using fluorescence spectra. The influences of ssDNA’ sequence length, sequence composition and fluorescent labels for temperature sensing were investigated. Results revealed the prepared, optimized ssDNA FT showed a high average temperature sensitivity of 7.04% °C-1, wide linear response range of 0-100 °C, and excellent stability with various environmental factors. Furthermore, this ssDNA FT was successfully used for intracellular temperature sensing in cancer cells and was used for in vivo thermos-imaging during microwave hyperthermia of tumor tissue. Advantages in size, sensitivity, and stability proved the feasibility of ssDNA FT in nanoscale thermometry applications, and this novel fluorescent thermometry mechanism is of large potential in the development of FTs. This investigation of ssDNA’s molecular thermosensitivity could give rise to a new prospective in the nanothermometry field.


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1. Introduction Nanothermometry is concern with the analysis of temperature variation and distribution at the submicron scale; this field has attracted increasing attention in the research of nanotechnology and nanomedicine1, 2. Fluorescent nanoprobes with temperature-sensitive emissions have been employed as fluorescent nanothermometers (FTs), and high-resolution real-time temperature mapping could be achieved through measurement and analysis of fluorescence spectra of the used FTs3-6. In recent years, various FTs have been developed with fluorescent materials, including quantum dots (QDs)7, lanthanide (Ln)-doped materials8, conjugated polymer nanoparticles (NPs)7, fluorescent dye-incorporated NPs9,

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, luminescent metal clusters11,

thermosensitive nanogels12, 13, and DNA-based nanostructures14-18. These developed FTs can be used for thermal mapping in the liquid or solid phase, and some of which have been successfully applied for nanothermometry in diverse heterogeneous environments, such as in vivo and intracellular temperature sensing. Among them, DNA-based nanothermometers have attracted increasing attentions. The shape of DNA-based nanostructure is sensitive to temperature changes, and this property is utilized for DNA FT fabrication19. Most of the reported DNA FTs are fluorescent-labeled hairpin-shaped DNA molecular beacons (MBs)14-17, which comprise a single-stranded loop and a double-stranded stem with end-labeled fluorophores. The shape or conformation of MBs can reversibly change with temperature between the hairpin and random coil states, which change the relative position and fluorescence emissions of the labeled fluorophores20. Compared with FTs prepared with fluorescent NPs, DNA FTs present advantages of defined molecular structure, small size, tunable sensing property, and good biocompatibility14, 16. However, these materials also exhibit some drawbacks: 1) Given that the thermosensitivity of MBs is usually attributed to


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temperature-related hydrogen bond formation and breaking between nucleobases, they can only act at the melting temperature (Tm) of the double-stranded stem; moreover, the sensing ranges of the prepared MB FTs are usually restricted in Tm ± 10 °C14-16, 18; 2) Fluorescence signals of MB FTs change with temperature in a S-shaped curve with limited linear range, and most of them are non-ratiometric14-16; and 3) Base pairing is also influenced by factors, such as temperature, viscosity, salt concentration, and pH; thus, the stability and accuracy of MB FTs in complexed environments (such as in tissues and cells) are limited14. The above mentioned FTs only utilize DNA as polymer materials with temperature sensitive hydrogen bonding properties, the highly complicated, sequence related conformational behavior of DNA is seldom investigated21, 22. Notably, the conformation of certain single-stranded DNA (ssDNA) changed with temperature, which has been confirmed by small-angle neutron scattering, ultraviolet optical density measurement, and differential scanning calorimetry23-25. Zhou et al. found that the radius of gyration (Rg) of a 10-mer ssDNA (5’-ATGCTGATGC-3’) random coil increases from 0.8 to 2.8 nm when the temperature increases from 25 °C to 80 °C, indicating a significant expansion in the ssDNA molecular dimension23. Ramprakasha et al. analyzed the thermodynamics of the conformation transition of 24 10 to 12-mer ssDNA sequences and revealed that temperature-related ssDNA conformational behaviors are highly sequence-dependent24. Therefore, conformation properties (such as Rg, a typical index for molecular dimension) of ssDNA random coil without complimentary segments are temperaturesensitive in certain conditions. Furthermore, when fluorescence resonance energy transfer (FRET) donor and acceptor fluorophores are labeled at the ends of the ssDNA, these properties could be revealed with FRET. Hence, ssDNA can be applied for the design and preparation of DNA FTs, which possess the above-mentioned advantages as MB-based FTs. DNA FTs can also


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be designed for specific requirements because molecular dynamic (MD) simulation has been successfully applied tor conformational behaviors of ssDNA26-28. Moreover, investigation of ssDNA’s molecular thermosensitivity may give rise to a new prospective in the field of nanothermometry. Nevertheless, to date, only a few temperature-related conformational behaviors of ssDNA sequences have been reported, little fluorescent label strategy of the ssDNA random coil is observed, and the principle of the relationship among the sequence length, composition, and random coil’s thermosensitivity of ssDNA remains unclear.

Scheme 1. Ratiometric fluorescent nanothermometry of the ssDNA FT. Therefore, to develop a sensitive ssDNA-based ratiometric FTs, temperature-related conformations of series of ssDNA sequences with varied sequence length and composition were initially studied through MD simulation. On the basis of the simulation results, several ssDNA sequences with large temperature-related conformational changes were selected, and pairs of


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FRET donor and acceptor fluorophores were labeled at the ends of these ssDNA to prepare ratiometric fluorescent short ssDNA nanothermometer (ssDNA FT, as showed in Scheme 1). Results showed that the obtained ssDNA FT can provide fluorescent signal that ratiometrically changes when temperature varies. This ssDNA FT can also act in the wide temperature range of 0-100°C and show a high average temperature sensitivity of 7.04% °C-1. In addition, it exhibits excellent stability and accuracy in complex environments, such as in cells and tissues, and has been successfully applied in intracellular thermometry and thermoimaging during tumor hyperthermia process. This study not only presents a series of biocompatible, stable, highly sensitive FTs, but also reveals the great potential of ssDNA materials in the development of FTs and other thermal response functional materials with advanced performance. 2. Experimental Methods 2.1 MD simulation MD simulations were performed using the GROMACS code. The all-atom CHARMM force field and potential parameters of nucleic acid were used for ssDNA. The TIP3P model was utilized as a water source. The initial coordinates of the ssDNA were obtained by unzipping chain A (removing the complementary chain B) of the B-form helical DNA built using the UCSF Chimera software. The overall charge of the system was neutralized by adding Na+ ions. The systems were initially minimized using the gradient energy minimization method, and the temperature of the systems was gradually increased to a set temperature in a short MD run of 100 ps and a constant pressure of 1 atm under isothermal-isobaric ensemble (NPT) conditions. The systems were processed with NPT and constant volume and temperature ensemble (NVT) equilibration run of 5 and 5 ns, respectively. Afterward, long micro canonical ensemble (NVE) production runs of 50 or 100 ns were performed using the systems.


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2.2 Synthesis and labeling of the ssDNA FTs The fluorophores labeled ssDNA FTs were synthesized with an ABI 3900 DNA Synthesizer (Applied Biosystems Inc, Foster City, USA). The phosphoramidite nucleotides and related solvents and reagents were purchased from the Glen Research (Sterling, USA). The phosphoramidites of 6-carboxyfluorescein (FAM), carboxy-tetramethyl-rhodamine (TAMRA) and Carboxy-X-Rhodamine (ROX) were obtained from Applied Biosystems Inc (Foster City, USA). Texas Red NHS-ester was purchased from ThermoFisher. The ATTO 647N were purchased from ATTO-TEC GmbH (Siegen, Germany). All of the ssDNA sequences are indicated from 5’ to 3’, and were labeled as follows: FAM-AAAAAA-Texas Red (FAM-6A-TR) FAM-AAAAAAAAAAAA-Texas Red (FAM-12A-TR) FAM-CCCCCCCCCCCC-TAMRA (FAM-12C-TAMRA) FAM-TTTTTTTTTTTT-TAMRA (FAM-12T-TAMRA) FAM-ATCCTGAATGCG-TAMRA (FAM-12R1-TAMRA) L-FAM-ATCCTGAATGCG-TAMRA (FAM-12R1-TAMRA) L-ROX-ATCCTGAATGCG-ATTO 647N (L-ROX-12R1-ATTO)

The synthesized and labeled ssDNA were purified using a Waters Prep 100q SFC HPLC system equipped with a XBridge Oligonucleotide BEH C18 Column (Milford, USA). 2.3 Cubosome encapsulation of the ssDNA FTs Cubosome nanoparticles with bicontinuous cubic and hexagonal phases were used to encapsulate the ssDNA FTs. The Pluronic F127, 1-(cis-9-Octadecenoyl)-rac-glycerol (monoolein, glyceryl monooleate, GMO content >99%), and didodecyldimethylammonium bromide (DDAB) were purchased from Sigma-Aldrich (St. Louis, USA). The synthesized


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ssDNA FTs were encapsulated with the GMO cubosomes as previously reported. In a typical preparation, the GMO (25 mg), DDAB (0.5 mg) and F127 (10 mg) were solubilized in 5 mL of chloroform and mixed on a votex mixer, and solution was placed in a vacuum drying oven at 50°C for 4 h to remove chloroform. The mixture was heated to 50°C to melt the lipids, and 3.5 mL of ssDNA FT solution (10 µg/mL, heated to 50°C) was added to the mixture. The mixture was then dispersed by ultrasonication with a JY92-IIDN ultrasonic processor (Scientz, China) for 3 min in pulse mode (5 s pulses and 5s breaks) at 30% power. The obtained homogeneous transparent dispersion was rotated mixed at 37°C for 24 h for the equilibration of the cubosomes. 2.4 Measurements of temperature-related fluorescence changes The temperature related steady-state fluorescence measurements of ssDNA FTs were performed using a FluoroMax-4 fluorescence spectrophotometer (HORIBA Jobin Yvon, Kyoto, Japan) with a temperature controller (Wavelength Electronics Inc., Bozeman, USA). In a typical measurement, ssDNA FT was diluted to 25 nM in 10 mM phosphate buffer. Fluorescence lifetimes were measured using a time-correlated single-photon counting instrument (44MXs-B; LeCroy, USA). The solution was excited using a fiber laser (SC400-4-PP; Fianium Ltd., Southampton, UK), and the temperature was controlled with a temperature controller (Wavelength Electronics, Inc., Bozeman, USA). The results were recorded using a PicoHarp300 single-photon counting apparatus (PicoQuant, Berlin, Germany). Data were analyzed with multiple exponential models. 2.5 Cell culture and imaging PC-2 cancer cells were grown in RPMI 1640 medium containing 10% fetal bovine serum. The cells were cultured at 37 °C in 95% air and 5% CO2 for 24 h. The culture was conducted in a 10 cm diameter plate transplanted to 24-well-plates with poly-D-Lysine coated coverslips


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(CITOGLAS Co., Ltd., Nanjing, China). The cells were first cultured for 24 h before sequentially incubating with the ssDNA FTs. 5 µL of the ssDNA FTs encapsulated cubosome soluition (10 mg/mL) was diluted in 50 µL of RPMI 1640 medium without serum, and the mixture was added to culture wells containing cells and 0.5 mL of the medium without serum or antibiotics. The cells were then grown at 37 °C in 95% air and 5% CO2 for 4 h. The obtained cell culture coverslips were then washed with PBS solution and placed in lidded microcuvettes for fluorescence measurements. The calibration curve was obtained by averaging of the temperaturerelated emission intensity ratios data of 5 samples. Laser scanning confocal microscopy images were captured using a Carl Zeiss LSM 700 laser scanning confocal microscope (Oberkochen, Germany) equipped with a heating stage to control temperature. 2.6 In vivo imaging Animal experiments were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals of the Medical Research Council of Xi'an Jiaotong University. Tumor transplants were established in BALB/C nude mice by subcutaneously injecting 5 × 106 PC-3 cells in the left and right leg of each mouse. Imaging experiments were performed 24 d after injection. Images were captured with the IVIS Lumina XRMS Series III system (PerkinElmer, USA). An excitation filter of 580 ±10 nm and two emission filters of 620 ±20 nm (for ROX) and 670 ±20nm (for ATTO 647N) were used for the tow-channel fluorescence imaging. 50 µL of the ssDNA FTs encapsulated cubosome soluition (10 mg/mL) were subcutaneously injected into the tumor tissue on the right leg of the mouse, and in vivo fluorescence images were captured 5 min after the injection. To observe the temperature related ratiometric fluorescence emission changes during the intra-tumor hyperthermia process, the injeceted tumor tissue was then heated by microwave radiation (40w, 2450MHz) with a Shengpu (Xuzhou, China) SPW-1A microwave


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therapy apparatus for 5 min, and a planar spiral antenna was used for directional heating of the tumor zone. 3. Results and Discussion 3.1 Thermosensitivity analysis with MD simulation MD simulation is widely applied in conformational behavior studies of biomacromolecules, such as protein and nucleic acids28. This method is also used to investigate the molecular mechanism of thermosresponsivity of poly(N-isopropylacrylamide)29, 30. Recently, Chakraborty et al. reported their MD simulation study of a 12-mer ssDNA (5'-CGCGAATTCGCG-3') in aqueous medium, and their simulation results revealed that ssDNA presents a fluctuating collapsed coil-like conformation in solution27. Using a similar approach, temperature-related conformations of a series of ssDNA sequences were analyzed. The considerable changes in the molecular dimension of ssDNA result in remarkable changes in the FRET signal, and high thermo-sensitivity of the obtained FT is observed. Therefore, to select ssDNA sequences for highly sensitive FTs, thermosensitivities of the ssDNA sequences were compared by their Rg (molecular dimension) variation magnitude between 293 K and 353 K temperatures. First, 3-mer to 18-mer ssDNA sequences that were consisted by a single kind of base (polyT, polyC, polyA, and, polyG) were analyzed through MD simulations (random coil of ssDNA length at more than 18 mers is complicated). Simulation results showed that these ssDNA possess random coil conformations (Figure S1), and their Rg and Rg’s fluctuation ranges increase with their sequence length (Figure 1A). Their Rg values are also related with sequence composition, where ssDNA consisting of pyrimidines bases (such as polyT and polyC) present smaller Rg than that of ssDNA consisting of purine bases (polyA and polyG) (Figure 1A). Thus, the molecular


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dimensional properties of ssDNA are mainly dependent on their sequence length and composition, and these two factors are the key parameters for the design of ssDNA FTs.

Figure 1. Average molecular dimension (indexed by Rg) of 3-18 mer PolyT, PolyC, PolyA and PolyG ssDNA sequences at 293 K and 353 K (A1 and A2). Sequence dependent thermosensitivities of the ssDNA of varied lengths and compositions (B and C). As indicated by the simulation results, polyC, polyT, and polyA sequences with sequence length larger than 6-mer show remarkably higher thermosensitivity than those of polyG sequences (Figure 1B). Furthermore, the Rg variation trends of the ssDNA sequences also differ from one another; for polyC and polyT ssDNA, their Rg increases with temperature, which indicated their expanded conformations at high temperature. On the contrary, the Rg of polyA decreases at high temperature because of the shrinkage of random coils. In comparison, 12-mer ssDNA sequences show high average thermosensitivity, and 12-mer polyT ssDNA shows the highest Rg variation of 31%. The molecular dimensions of 12-mer sequences usually vary in a


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range of 3-6 nm, which are also suitable for FRET analysis. Thus, 12-mer was selected as the sequence length for further study. Furthermore, the thermosensitivities of ssDNA sequences consisting of multiple kinds of bases were investigated. Given the large number of combinations of four kinds of bases with sequence length of 12 (412 = 16,777,216), evaluating all of these sequences is difficult in practice. Therefore, dozens of 12-mer random ssDNA sequences were selected as representatives, and their temperature-related conformations were analyzed with MD simulations. Table 1. Thermosensitivity of some 12-mer ssDNA Sequences Nos 1 2 3 4 5 6 7 8 9 10

Abbreviations 12A 12T 12C 12G 12AC 12TG 12TC 12AG 12R1 12R2

Sequence 5’-AAAAAAAAAAAA-3' 5’-TTTTTTTTTTTT-3' 5’-CCCCCCCCCCCC-3’ 5’-GGGGGGGGGGG-3’ 5’-ACCACACACCAA-3’ 5’-TTGGTGTGTGGT-3’ 5’-TCCTCTCTCCTT-3’ 5’-AAGGAGAGAGGA-3’ 5’-ATCCTGAATGCG-3’ 5’-CGCATTCAGGAT-3’

Thermosensitivity (%) -15.9 30.6 22.7 -3.0 44.8 58.8 47.9 36.4 90.3 65.2

Given the hydrogen bonding between complimentary base pairs (A-T, C-G), ssDNA sequences with high complimentary base contents may exhibit complex conformations, and their dynamic behaviors are also highly complex. By contrast, 12-mer ssDNA sequences with two kinds of noncomplimentary bases or equivalent amount of four kinds of bases (in which hydrogen bonding is hindered by the steric hindrance) present coil-like conformations and significant thermosensitivities. As shown in Figure 1C, Rg of 6 kinds of ssDNA sequences (Sequence information corresponding to these abbreviations is shown in Table 1) largely increases at high


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temperature. These sequences also show higher thermosensitivities than those of sequences consisted of a single kind of base. The Rg of 12R1 ssDNA increases to 90% at 353 K than that in 293 K and shows the highest thermosensitivity among all the simulated 12-mer ssDNA sequences. 3.2 Preparation and characterization of series of ssDNA FTs To verify the design principle, a series of ssDNA random coil FTs was prepared as the representative ssDNA sequences with properly selected FRET fluorophores. The relationship between FRET efficiency (E) and the fluorophore interdistance (r) is given by the formula: E = (R06)/(R06+r6), where R0 is the Förster distance of the fluorophores31. This equation shows that FRET efficiency drastically changes when the fluorophore interdistance varies in the vicinity of R0. Thus, to prepare ssDNA FTs with high sensitivity, the used FRET fluorophores should exhibit a R0 that is close to the molecular dimension variation range of the selected ssDNA (Figure S2). As indicated by the MD simulation results, molecular dimensions of SsDNA strands are proportional to their sequence length, so do their variation ranges. For ssDNA strands with longer sequence length, they may have larger molecular dimension, and interdistance of the terminally tagged fluorophores may vary in a range of ten to tens of nanometers. However, R0 of most FRET fluorophore pairs are in the range of 2 to 8 nm, thus short ssDNA with sequence length of 12-mer were used for the preparation of ssDNA FTs. According to the simulation results, the molecular dimension of 12-mer polyA ssDNA decreases with temperature in a range of 3.2-4.1 nm, and FAM (as donor) and Texas Red (as acceptor) fluorophores (with R0 of approximately 3.6 nm) were labeled at the ends of both 12-mer and 6-mer polyA ssDNAs for the preparation of polyA ssDNA FTs (noted as FAM-12A-TR and FAM-6A-TR FTs). The fluorescence emission of FAM-12A-TR FT significantly changes with temperature (Figure 2A).


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The donor/acceptor emission intensity ratio (D/A ratio) of FAM-12A-TR FT decreases with increasing temperature, thereby indicating an increased FRET efficiency between the FAM and TR fluorophores (Figure S3A1). For FAM-6A-TR FT, the emission intensity of both fluorophores only slightly decreases at high temperature (Figure 2B), and its D/A ratio remains unchanged in the temperature range of 0-100 °C （Figure S3B1). These results indicated the good thermostability of the used fluorophores and the unchanged FRET efficiency. The fluorescent lifetime of FAM fluorophore (as the FRET donor) in FAM-12A-TR FT decreases with increasing temperature (Figure S3A2) and that of FAM in FAM-6A-TR FT exhibits little change with temperature (Figure S3B2), which coincides with the analysis of the steady-state emissions.


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Figure 2. Temperature related emissions of the FAM-12A-TR FT (A), FAM-6A-TR FT (B), FAM-12T-TAMRA FT (C) and FAM-12R1-TAMRA FT (D). Ratiometric working curves of some ssDNA FTs (E) and their D/A ratio variations in repeated heating-cooling cycles (F). As shown by the MD simulation results, the molecular dimensions of most of the simulated 12-mer ssDNA sequences increase with temperature. The molecular dimensions of 12-mer polyC, polyT, polyTC, and 12R1 ssDNA significantly increase in a range of 3.2-5.2 nm with temperature. FRET labels of FAM (as FRET donor) and TAMRA (as FRET acceptor) fluorophores (with R0 of 4.8 nm) were used, and ratiometric ssDNA random coil FTs were prepared using four sequences, which are noted as FAM-12C-TAMRA, FAM-12T-TAMRA, and FAM-12R1-TAMRA FTs. Steady-state spectrum analysis showed that the emission spectra of these ssDNA random coil FTs ratiometrically change with temperature (Figure S4A and Figure 2C and D), and their related D/A ratios all increase with temperature (Figure 2E, Figure S4B) This result indicated the decreased FRET efficiencies between the FAM and TAMRA fluorophores at high temperatures. These temperature-dependent FRET efficiency variation trends were also confirmed by the fluorescence lifetime analysis of the donor fluorophore of FAM (Figure S4D). Consequently, temperature-related fluorescence properties of the prepared ssDNA FTs are all consistent with the thermosensitivity analysis of the related ssDNA sequences obtained by MD simulations. Such properties also proved the effectiveness of the design principles of ssDNA random coil FT. Thermometry applications require that FTs should have reversible responses with no hysteresis1, 3, 4. Thermostability of the prepared series of ssDNA random coil FTs was evaluated by repeated heating and cooling cycles in the temperature range of 10-90 °C, and their D/A ratio variations were recorded. As shown in Figure 2F, the D/A ratios of the ssDNA random coil FTs reversibility change with temperature and show good


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reproducibility. The ratiometric working curves of the FTs were obtained by measuring the D/A ratio versus temperature curves of the prepared ssDNA random coil FTs. Comparisons showed that FAM-12R1-TAMRA FT possesses the largest D/A ratio variation amplitude, which increases from 0.29 to 2.03 in the temperature range of 0-100 °C. This FT also shows high average temperature sensing sensitivity of 7.04% °C-1, which is significantly higher than those of ratiometric FTs prepared with QDs or Ln-doped materials3, 4, 6. The D/A ratio of FAM-12R1TAMRA FT also increases almost linearly with temperature (R2 = 0.9953, Figure S4C) and presents unchanged sensitivity at the wide temperature range of 0-100 °C. On the contrary, thermosensitive nanogels and MB-based FTs can usually only act in a narrow temperature range of ±10 °C around their transition temperature 12-14, 18. In consideration of these advantages, FAM12R1-TAMRA FT was selected and used for following studies. 3.3 Ratiometric fluorescent thermometry in solutions and cellular environment. Compared with those FTs that only depend on emission intensities, measurements of ratiometric FTs is not affected by the inhomogeneity of probe concentration and excitation, thereby indicating improved sensing accuracy 3, 4, 6. Experimental results showed that the sensing properties of FAM-12R1-TAMRA FT remain unchanged at different probe concentrations and excitation power (Figure 3A). The thermosensitivity of ssDNA random coil FTs is independent from the hydrogen bonding of complimentary bases. Thus, ssDNA random coil FTs also exhibit better stability than that of MB-based DNA FTs in complex environments. Results showed that the sensing properties of FAM-12R1-TAMRA FT remain unchanged in solutions of different concentrations of NaCl, KCl, and phosphates (Figure 3B, C and D). The ssDNA FTs can also be applied in highly viscous media, and the sensing properties of FAM-12R1-TAMRA FT show little changes in solutions with different concentrations of glycerol (Figure 3E) and bovine serum


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albumin (BSA, Figure 3F). All of these results proved that FAM-12R1-TAMRA FT is a highly sensitive ratiometric FT with high stability in complex environments.

Figure 3. The D/A ratio versus temperature curves of FAM-12R1-TAMRA FT of various probe concentrations (A) and in solutions containing different concentrations of NaCl (B), KCl (C), phosphate (D), glycerin (E) and BSA (F). Compared with FTs prepared with QDs, Ln-doped materials, and polymer NPs, DNA FTs contain no metal ions, and they present better biocompatibility2, 4, 5, 14-16. However, previously reported MB-based DNA FTs are seldom applied in accurate intracellular temperature sensing because natural DNA (D-DNA) could be deactivated by enzymolysis or hybridization in cells. LDNA is a mirror-image of D-DNA and exhibits the same physical characteristics as the latter. As


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demonstrated in a previous study of MB-based DNA FTs, the L-DNA probes possess good biocompatibility and long-term intracellular stability14. The prepared L-DNA MB-based DNA FTs are not ratiometric, and their working curves vary in different cell lines14. Considering their highly stable ratiometric sensing properties, ssDNA random coil FTs present some advantages in intracellular temperature sensing over MB-based DNA FTs when they could be stably transfected into the target cells. To prepare ssDNA FTs with enhanced intracellular stability, 12R1 ssDNAs with L-conformation were synthesized and labeled with FAM and TAMRA fluorophores to prepare L-FAM-12R1-TAMRA FT. The obtained L-DNA FT shows the same sensing properties as its D-DNA analogue (Figure S5A); it could also act in the lysate of PC-3 cells, thereby showing good resistance to enzymolysis. The L-FAM-12R1-TAMRA FT exhibits excellent stability in repeated heating and cooling cycles, unchanged sensing properties in various solution environments as other ssDNA FTs, and shows long-term intracellular stability in cellular environment, which is of strong stability for intracellular thermometry applications. As hydrophilic negatively charged molecules, ssDNA usually exhibits a low cell uptake rate32. Cubosome NPs were used as carriers to transfect L-FAM-12R1-TAMRA FT into HeLa cells33. Laser scanning confocal microscopy images of the transfected cells showed that the L-FAM12R1-TAMRA FT probes are eventually distributed in the nucleus and cytoplasm. The signal intensities recorded by the FAM and TAMRA channels ratiometrically change with temperature and consequently produce colorimetric-turned cell images (Figure 4A, B and C). However, signal intensities ratios of the related channels are not numerically equal to the peak intensity ratios obtained from the temperature related emission spectra dates. Accuracy of the temperature controller of the used confocal system is ±1°C, which is not sufficient to establish an accurate working curve. For quantitative measurement of intracellular temperature, intracellular emission


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spectra of the ssDNA FTs should be analyzed. Furthermore, the fluorescence emissions of the LFAM-12R1-TAMRA FT-transfected cell samples were analyzed at varied temperatures using the laser micro–spectrum method. Results showed that the sensing properties of L-FAM-12R1TAMRA FT show little changes in cellular environment at the physiological temperature range of 20-50°C, which proved the accuracy of L-FAM-12R1-TAMRA FT in cellular thermometric applications (Figure S5B).

Figure 4. LSCM images of the L-FAM-12R1-TAMRA FT transfected PC-3 cells at varied temperatures (A, B and C). Fluorescence images of the L-ROX-12R1-ATTO FT injected tumor tissues before (D) and after (E) microwave irradiation. With the obtained intracellular D/A ratio versus temperature curves, cellular temperature and temperature variation could be quantitatively measured by laser micro–spectrum methods.


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Although the quantitative measurement of intracellular actual temperature is difficult, the ratiometric working curve of L-FAM-12R1-TAMRA FT show little changes as compared with its in vitro ratiometric working curve in solution which can be precisely quantitative measurement (See Figure S5), thus the accuracy of L-ssDNA FT in quantitative measurement of intracellular temperature is reliable. In consideration the difference between the internal temperature of the living cells and the set temperature of the thermostat, there might be a slight difference between the detected temperature and the actual temperature, however, accuracy of temperature variation (∆T) measurement is sufficient. 3.4 Fluorescent thermoimaging in tumor tissue Hyperthermia utilizes the increased sensitivity of the lesions of cancerous cells at increased temperature and received increasing attention in recent years due to its low side effects and improved therapeutic efficiency34, 35. The study and development of hyperthermia technology require accurate monitoring of internal temperature distribution of the tissues2, 3, 5. Given the absorption, scattering, and autofluorescence effect of biological tissue, in vivo fluorescent thermoimaging requires the use of FTs exhibiting temperature-related emissions at the near– infrared (NIR) region5. Nevertheless, the thermosensitivity of most of the developed FTs is highly related with the properties of the used fluorophores; in addition, FTs with tunable emission wavelengths are seldom reported3, 4, 6. The thermosensitivity of ssDNA FTs is mainly determined by their ssDNA sequences, and their emission characteristics can be adjusted by using different pairs of FRET fluorophores. HEX, TET, and FAM are fluorescein derivatives with different maximum emission wavelengths, and they are frequently used for the fluorescent label of DNA. HEX and TET, instead of FAM, were used as donor fluorophores in preparing Hex-12R1-TAMRA and TET-12R1-TAMRA FTs. Moreover, the temperature-related emissions


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were measured and analyzed (Figure S6A and B). Results showed that all of the three kinds of 12R1-based FTs demonstrate similar D/A ratio variation trends toward temperature changes (Figure S6C), which further confirmed the previous analysis. Furthermore, 5' and 3' ends of L-12R1 ssDNA were labeled with ROX (as the donor) and ATTO 647N (as the acceptor) fluorophores to prepare FT with emission peaks at the red to NIR region. The obtained L-ROX-12R1-ATTO FT shows significant ratiometric thermosensitivity (Figure S6D), and its D/A ratio also increases with temperature at the physiological temperature range of 20-50°C. The thermoimaging effects of the L-ROX-12R1-ATTO FT were also evaluated in vivo. L-ROX-12R1-ATTO FT solution was injected into the tumor tissue with a depth of 3.5 mm, and the tumor was heated by 40 W microwave irradiation for 5 min. The in vivo fluorescence images of the tumor bearing nude mice before and after hyperthermia treatment were captured. As shown in Figure 4D and E, the fluorescence of L-ROX-12R1-ATTO FT significantly changes during hyperthermia treatment, and the emission intensity ratios of the two channels increased from 0.75 to 0.95, which indicated the enhanced intratumor temperature distribution. These preliminary results proved the practicability of ssDNA FTs in in vivo fluorescent nanothermometry applications. 4. Conclusion In conclusion, a series of ratiometric FTs was developed using ssDNA terminally-tagged fluorophores. An optimal ssDNA sequence and associated ssDNA FT were determined through combined MD simulation and temperature-related FRET analysis. This ssDNA FT can act as ratiometric FTs and show a high average temperature sensitivity of 7.04% °C-1, wide linear response range of 0-100 °C, and excellent stability under various environmental factors.


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Additionally, this ssDNA FT was successfully used in temperature mapping in vitro and primary use in vivo. This investigation of ssDNA’s molecular thermosensitivity could give rise to a new prospective in the nanothermometry field, and it indicates that ssDNA possess considerable potential in theory and applications of nanothermometry. ASSOCIATED CONTENT Supporting Information. Additional spectroscopic analysis, these materials are available free of charge via the Internet at http://pubs.acs.org. ACKNOWLEDGMENT This work was sponsored in part by National Natural Science Foundation of China (NO 81271686, 81228011, 61178085 and 81471771), the grants of National Key Research and Development Program of China (NO.2016YFC0100701).

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Table of Contents Graphic and Synopsis


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Scheme 1 90x81mm (300 x 300 DPI)


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Figure 1 74x54mm (300 x 300 DPI)



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Figure 2 111x123mm (300 x 300 DPI)


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Figure 3 111x123mm (300 x 300 DPI)



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Figure 4 107x95mm (300 x 300 DPI)


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Figure S1 142x203mm (300 x 300 DPI)



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Novel Ratiometric Fluorescent Nanothermometers Based on

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