Noninvasive and Spatiotemporal Control of DNAzyme-Based Imaging of Metal Ions In Vivo Using High-Intensity Focused Ultrasound

Noninvasive and Spatiotemporal Control of DNAzyme-Based Imaging of Metal Ions In Vivo Using High-Intensity Focused Ultrasound

Xiaojing Wang,

^◆

Gun Kim,

^◆

James L. Chu,

^◆

Tingjie Song, Zhenglin Yang, Weijie Guo, Xiangli Shao, Michael L. Oelze, King C. Li,* and Yi Lu*

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^sı Supporting Information

ABSTRACT: Detecting metal ionsin vivowith a high spatiotemporal resolution is critical to understanding the roles of the metal ions in both healthy and disease states. Although spatiotemporal controls of metal-ion sensors using light have been demonstrated, the lack of penetration depth in tissue andin vivo has limited their application.

To overcome this limitation, we herein report the use of high- intensity focused ultrasound (HIFU) to remotely deliver on-demand, spatiotemporally resolved thermal energy to activate the DNAzyme sensors at the targeted region both in vitro and in vivo. A Zn²⁺- selective DNAzyme probe is inactivated by a protector strand to block the formation of catalytic enzyme structure, which can then be activated by an HIFU-induced increase in the local temperature. With this design, Zn²⁺-specific fluorescent resonance energy transfer (FRET) imaging has been demonstrated by the new DNAzyme-

HIFU probes in both HeLa cells and mice. The current method can be applied to monitor many other metal ions forin vivoimaging and medical diagnosis using metal-specific DNAzymes that have either been obtained or can be selected usingin vitroselection.

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INTRODUCTION

Metal ions are essential for many biological processes because of their many roles, including stabilizing biomolecules, regulating molecular interactions, and catalyzing chemical and biological reactions. Abnormally low or high levels of metal ions can lead to diseases, such as anemia and growth retardation.^1,2 Therefore, site-specific monitoring of the distribution and concentration fluctuation of metal ions in situ can provide critical information for human health and diseases. To achieve this goal, instrumental analyses, such as atomic absorption/emission spectroscopy,³ inductively coupled plasma mass spectrometry,^4,5 and X-ray fluorescence microtomography,⁶are used to detect and measure metal ions.

However, these techniques do not permit real-time monitoring of metal ions in living cells and tissues. To overcome this limitation, sensors based on small molecules,⁷⁻⁹ peptides,¹⁰ and proteins¹¹⁻¹³ are extensively explored for metal ion detection. Despite progress made by many investigators, sensors are still limited to a small number of metal ions.

More importantly, since metal ions are distributed in different locations of biological systems with time-dependent fluctua- tion, it is important to develop sensors with high spatiotemporal resolution. Toward this objective, optical control of metal-ion sensors has been reported.¹⁴ However, it is difficult to employ light to monitor metal ions in animal

and human bodies for such control because of the low tissue penetration depth.

DNAzymes, which are DNA molecules with enzymatic activities, have emerged as an important class of sensors for metal ions.¹⁵⁻¹⁷ Compared with other metal-ion probes,⁷⁻⁹ DNAzymes with high metal ion selectivity can be obtained through in vitro selection from a large DNA library of up to 10¹⁵ sequences and can be converted to fluorescence, colorimetric,¹⁸ and other types of sensors¹⁹ by conjugating with a variety of reporters.²⁰ During the past decades, DNAzyme-based sensors have been reported for the detection of many metal ions (e.g., Na⁺, Mg²⁺, Ca²⁺, Cu⁺/Cu²⁺, Pb²⁺, UO2²⁺, Hg²⁺, Tl⁺, and Cd²⁺), and the metal-ion-specific DNAzyme toolbox is still expanding.^21,22 In addition, DNAzymes have the merits of easy synthesis, and high programmability and sensitivity for targeted metal ions in vitro.²³⁻²⁶ Benefitting from the biocompatibility of DNA,

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DNAzymes have also been used in metal ion detection in living cells and zebrafish. The first intracellular application of DNAzymes as a metal-ion sensor was reported for the detection of uranyl, but the application lacked spatiotemporal control.¹² Later reports introduce spatiotemporal control in either living cells or zebrafish using a photocaging group at the putative cleavage site and the detection can be controlled by removing the caging group using UV light (365 nm) directly,^27,28 or by upconverting near-infrared (IR) light to UV light by conjugating the DNAzyme to lanthanide-doped upconversion nanoparticles (UCNPs).²⁹ Taking advantage of the light-responsive nanomaterials, such as the nanoshell,³⁰ that can increase the local temperature, spatiotemporal control of the DNAzyme-based metal ion detection was achieved without using a caging group. Although the spatiotemporal control of DNAzyme-based metal ion detection using an external light source has been achieved in living cells and zebrafish, it would be difficult to apply the method in animals

and human bodies because of the penetration depth limitation (<12 mm).³¹

To address the above issues of the spatiotemporal control of DNAzyme-based metal ion detection, we herein report a remote and noninvasive control to modulate the activation of a DNAzyme-based metal-ion probe with high-intensity focused ultrasound (HIFU). Ultrasound is a minimally invasive modality that is capable of penetrating deep into intervening tissues and overcoming the weakness of light-based con- trol.³²⁻³⁶Acoustic energy from the ultrasound system can be focused onto a focal spot determined by the excitation frequency, making it possible to activate the DNAzyme in a specific target area. Because of these advantages, HIFU has found success in applications including tumor ablation,^35,37 neurosurgery,^33,38,39pain management,⁴⁰and drug delivery.⁴¹ Importantly, by focusing an ultrasonic wave onto the target location, the high intensity of irradiation can also cause localized heating from energy dissipation and can be further used to control the gene expression of bacteria⁴²or the release Figure 1.Schematic of HIFU-activated noninvasive and spatiotemporal control of DNAzyme-based sensor for Zn²⁺detectionin vivo.

of drugs.^34,43Since lower thermal dose at mild temperature has less effect on the survivability of cells and tissues,^44,45 we integrate the heat control of HIFU with DNAzyme for metal ion detectionin vivo.

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RESULTS AND DISCUSSION

The design of a HIFU-activated DNAzyme sensor is shown in Figure 1. We choose Zn²⁺-specific 8−17 DNAzyme⁴⁶ as the proof of concept for method development because it is the most well studied and broadly used DNAzyme for cellular and in vivoapplications.^29,30In addition, Zn²⁺plays important roles in biological systems, including protein function regula- tions.^47,48 Dysregulation of Zn²⁺ has also been linked to prostate cancer.⁴⁹ In our design, DNAzyme is inactivated by hybridization between the protector strand (P) with the enzyme strand (E) so that DNAzyme is inactive until the introduction of HIFU. Upon HIFU treatment, the heat is generated to increase the local temperature from 37 to 43°C, which can induce the dehybridization of P−E strands (Figures S1 and S2).⁴³We choose to increase the local temperature to 43 °C because it is the upper temperature limit of mild hyperthermia in mice and has been used in drug delivery.⁵⁰As a result, the E strand can then hybridize to the substrate strand (S) to form the active DNAzyme conformation. At the same time, a helper strand (H) is also added into the system to eliminate the free P strand after HIFU stimulation. In the presence of target Zn²⁺, the S strand is cleaved by the E strand.

Because of the low melting temperature of the cleaved product (27 °C), it is released from the E strand at the physiological temperature to generate detectable signals.

At present, most DNAzyme-based sensors are designed with a one-step “off−on” signal change for metal ion detection.

They have difficulty in distinguishing the false-positive signal from background activation or knowing the detailed procedures of HIFU-triggered multiple-step DNAzyme-cata- lyzed reactions. To monitor the step-by-step reactions and produce optical signals from the HIFU-activated DNAzyme sensor, we conjugated Cy3 to the 3′of the E strand, IABkFQ to the 5′of P strand, and Cy5 to the 5′of the S strand (Figure 1). Since the IABkFQ quencher was close to Cy3 upon hybridization between the E and P strands, the initial three- stranded construct would display minimal fluorescence signal from Cy3. This prediction from the design was indeed observed when the three DNA strands were hybridized using a protocol described in theSupporting Information. As shown in Figure 2a, upon excitation at 514 nm, the three-strand DNA probe and H construct displayed minimal signal at 565 nm where Cy3 is expected to befluorescent; only a weak signal at 666 nm was observed, which could be attributed to fluorescence from Cy5. Once P was released from the complex structure upon HIFU heating, the quencher IABkFQ moved away from Cy3, while Cy5 on the substrate strand moved close to Cy3 due to the formation of active DNAzyme configuration.

As a result, a fluorescent resonance energy transfer (FRET) signal between Cy3 and Cy5 was expected. To simulate such a case of an increase in temperature due to HIFU, we incubated the above DNAzyme probe at 43 °C and observed increased fluorescence signals at both 565 and 666 nm (Figure 2b), indicating that the FRET between Cy3 and Cy5 indeed occurred. Upon addition of Zn²⁺, the Cy3fluorescence signal at 565 nm increased, while the Cy5fluorescence signal at 666 nm decreased. This result was consistent with the design shown inFigure 1that, upon Zn²⁺-dependent cleavage of the S

strand, the cleaved product containing Cy5 was released from Cy3, causing the FRET between Cy3 and Cy5 to disappear and thus resulting in an increasedfluorescent signal of Cy3 and a decreasedfluorescent signal of Cy5. Importantly, without the two-dye design, it is difficult to know if the resulting signal is triggered by HIFU and then Zn²⁺in a stepwise manner.

To investigate whether the helper strand (H) was necessary for the design shown in Figure 1, we removed H from the above mixture and found that, although the P strand involved three-strand probe was stable before the thermal treatment; a lowerfluorescent signal was observed after the activation than that of the three-strand construct (see Figures S3 and S4).

Therefore, P and H strands play important roles in ensuring the performance of our sensors. First, P prevents the hybridization between the S and E chains and stabilizes the probe before the HIFU treatment. In addition, since P and H have sequences complementary to each other; they form a double-stranded complex after the thermal treatment to avoid any potential inhibition effect from the protectors to the S or E strand (Figure S5).

After validating the design of the DNAzyme-HIFU probe, its performance was evaluated with the kinetic study. As shown in Figure 3a, at 37 °C, no obvious Cy5 fluorescence change at Figure 2.Fluorescence response of the DNA probe and helper strand (a) before heat treatment, (b) after heat treatment at 43°C for 30 min, and (c) when the heat-treated probe is added with zinc ions at 37°C for 30 min.

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666 nm was observed, even after the addition of 20μM of Zn²⁺

at the 30 min time point (Figure 3a), demonstrating that the E strand is well protected by the P strand, preventing it from cleaving. Interestingly, the Cy3 fluorescence intensity at 565 nm increased slightly after the addition of Zn²⁺. This result suggests that Zn²⁺ may promote the formation of the active DNAzyme configuration³⁰(see the red region of the E strand in Figure 1) and weaken the hybridization between P and E strands. On the other hand, if the DNAzyme-HIFU probe is incubated at 43 °C for 30 min, thefluorescence intensity at 565 nm keeps constant, while that at 666 nm gradually increases up to 3.3-fold compared to that when it is incubated at 37°C for the same time (Figure 3b). These results indicate that the protector P is released under higher temperature and active DNAzyme is formed in 30 min. Then, we removed the heat treatment and added 20 μM zinc ions. The fluorescence intensity at 565 nm increased and the signals at 666 nm decreased, consistent with the cleavage of the S strand and Cy5 moving away from Cy3.

Given that the Cy5/Cy3 FRET signal of the DNAzyme- HIFU probe is correlated to Zn²⁺-dependent DNAzyme activity (Figures 2c and 3b), we investigate the quantitative relationship between the FRET signal and Zn²⁺concentrations.

As shown in Figure 3c−e, the FRET ratio (F_A/F_D) shows a linear relationship with Zn²⁺concentrations ranging from 0.5 to 10μM. Based on the linearfit of this relationship, a limit of detection (LOD) of 345 nM Zn²⁺was obtained, based on the common LOD definition of 3 times the standard deviation of the blank samples’ FRET ratios divided by the slope (3σ/ slope).

After establishing that the DNAzyme-HIFU probe works when heated at 43°C, we evaluated its performance using the proposed HIFU setup to temporally control and elevate the temperature for 30 min (Figure S6). As shown in Figure 3f, without Zn²⁺and HIFU, a minimalfluorescence signal at 565 nm was observed, indicating that the Cy3 fluorescence was

mostly quenched by the IABkFQ quencher. After HIFU sonication 30 min, but in the absence of Zn²⁺, fluorescence signals at 565 and 666 nm increased (Figure 3f), indicating the formation of the active DNAzyme, consistent with the thermal treatment experiments (cf. Figures 2b and 3b). Finally, after the HIFU treatment, and upon the addition of 20 μM Zn²⁺, the Cy3 fluorescence at 565 nm increased and Cy5 signal at 666 nm decreased, which was consistent with the thermal treatment experiments (cf.Figures 2c and3b), suggesting that the S strand was cleaved and Cy5-conjugated cleaved strand moved away from the Cy3-conjugated E strand.

Having demonstrated the DNAzyme-HIFU probe in test tubes, we evaluated its performance in cells. First, we tested the stability and feasibility of the probe in cell lysates. As shown in Figure S7, after incubation of the probe in cell lysate at 37°C for 2 h, a weak increase in thefluorescent signals at 565 and 666 nm was observed. This increase would be expected because of the robust nuclease activity found in HeLa cell lysates. We observed that thefluorescence at 565 nm increased by 2.46-folds upon heat treatment and Zn²⁺ addition (Figure S7) compared with without the heat treatment, suggesting that the probe was suitable for application in living cells.

To test the performance of the probe for sensing Zn²⁺ in HeLa cells, the probe with the TurboFect transfection reagent was first incubated with the cells for 3 h to allow sufficient uptake. Tofind out if the HIFU treatment to increase the local temperature to 43°C would cause any damage to the cells, we checked the cell morphology before and after HIFU treatment for 30 min and found minimal changes (Figure S8). To quantify the effect, we carried out the MTT assay of HeLa cells after incubating the cells at 43°C in a water bath for 1 and 2 h, and the cell viability remained 98.96 and 101.36%, respectively (Figure S9). In addition, the HIFU treatment of the cells at 43

°C for 30 min caused minimal effects on the viability of HeLa cells (Figure S9). Our results are consistent with previous studies that showed minimal effects of HIFU treatment on cell Figure 3. (a) Kinetics study of the stability of the probe before and after heat treatment at (a) 37°C and (b) 43°C. Kinetic study of the fluorescence signal change caused by the addition of zinc ions after heat treatment. (c) Zn²⁺-concentration-dependent response of probe after the thermal treatment. (d) FRET response of probe to different Zn²⁺concentrations. (e) Calibration curve betweenF_A/F_D(=FI_666nm/FI_565nm) to Zn²⁺

concentration. (f) Fluorescence response of probe with HIFU activation.λex= 514 nm.

viability.⁵¹ Therefore, a 30 min HIFU treatment of the cells was performed in the following cells studies. The HeLa cells were incubated with 40 μM Zn²⁺ for 30 min and confocal imaging was collected. To find out if 40 μM Zn²⁺ could be toxic to the cells, we performed the MTT assay on HeLa cells treated with 40 μM ZnCl₂ and 40 μM zinc ionophore pyrithione (PT). As shown inFigure S10, the cells remained viable up to 1 h under this condition. Afterward, the stability of different probe compositions was tested in HeLa cells. As shown inFigure S11, negligible Cy3fluorescence was observed in the ESPH system, indicating good stability of the probe in living cells. As shown in Figures 4a and S12, without the addition of Zn²⁺, minimalfluorescent signals were observed in the HeLa cells before the HIFU treatment (Figure 4a). After

the HIFU sonication, the Cy3 signal increased (Figure 4a,b);

the FRET ratio between Cy5/Cy3 was enhanced (Figure 4c).

The enhanced FRET ratio demonstrated the formation of active DNAzyme inside the cells, which was consistent with the in vitrostudies (Figure 3f). The newly formed DNAzyme could react with endogenous mobile Zn²⁺ and produce the Cy3 signal. These results indicated that the DNAzyme system could be used to monitor cellular Zn²⁺ions under HIFU control. To further corroborate thisfinding, we added 40μM Zn²⁺into the cells after the HIFU sonication and observed an even further increase in the Cy3 fluorescent intensity (Figure 4d,e). The HeLa cells with the HIFU treatment showed 2.32 times higher Cy3 signal than the cells without the HIFU treatment.

Figure 4.(a) Endogenous Zn²⁺detection in HeLa cells. Scale bars: 20μm. Thefluorescence intensities from the CLSM images were quantified to calculate the (b) Cy3 signal and (c) FRET ratio (Cy5/Cy3) from (a). (d) Cellular images with additional exogenous 40μM Zn²⁺and (e) the corresponding intensity analysis of Cy3 signal (two-tailed Mann−Whitneyttest,p< 0.001).

Journal of the American Chemical Society pubs.acs.org/JACS Article

Finally, to demonstrate the noninvasive and spatiotemporal control of our DNAzyme-HIFU probe to detect metal ionsin vivo(Figure S13), we performed a subcutaneous test in BALB/

c mice (Figures 5a, S14, and S15). Mice were anesthetized with 2% isoflurane and maintained on a surgical stage of anesthesia throughout the procedure (Supporting Information, Figures S16−S18). Thirty microliters of the reaction buffer (12.5 mM Tris, 150 mM NaCl, pH 7.4) containing 3.3μM of the probe and 40 μM of Zn²⁺ was subcutaneously injected bilaterally near the fourth mammary fat pads (Supporting Information, Figure S18b). As shown in Figure 5b, the mice demonstrated intrinsically weak background signals after the initial injection, which was attributed to the quenching effect from IAbRQ. We then applied HIFU heating to the left side of the mice while leaving the right side without HIFU as a negative control. The signal on the control right flank increased over time, probably due to the enzyme-triggered DNA digestion or global temperature increase by HIFU. After 25 min of HIFU sonication (Figure S18d), the fluorescence signal on the heated leftflank showed less difference compared to the rightflank without the HIFU treatment (Figure 5c,e). It was possible that the thermal conduction and DNAzyme reaction were slower inside the mice compared toin vitroand cellular experiments. Thirty minutes after sonication, the left flanks of the mice showed 1.91 times higher signal compared with the untreated right flanks (Figure 5d,e), indicating the

disassembly of the DNAzyme via activation through HIFU sonication. Furthermore, after 60 min, a 2.42-fold increase in fluorescence was observed on the HIFU-treated flanks (Supporting Information, Figure S14). To further validate that the sensor can be used to detect the endogenous metal ions in mice, a blank control was performed with probe only and without additional metal ions (Supporting Information, Figure S15). Under the same conditions, theflank sonicated by the spatiotemporally released acoustic energy from HIFU showed an enhanced fluorescent signal, demonstrating the robustness of our sensor in monitoring the microenvironment metal ionsin vivo.

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CONCLUSIONS

We have demonstrated remote spatiotemporal control of DNAzyme probes to detect and image Zn²⁺in both HeLa cells and mice using HIFU. This objective was achieved by careful designing a three-stranded DNA probe and a helper strand consisting of not only E and S strands of the DNAzymes but also a protector strand to the above E and S strands so that, upon HIFU sonication, the protector strands would dehy- bridize from the E and S strands to allow the formation of active DNAzyme configuration and thus the Zn²⁺-dependent cleavage of the S strand. The design was verified by conjugating fluorophores and quencher to the ends of S, E, Figure 5.HIFU-activated metal-ion sensingin vivo. Whole-bodyfluorescence imaging of mice after the injection of the probe and 40μM Zn²⁺. (a) From the leftflank and the rightflank (probe and 40μM Zn²⁺injection), respectively. Whole-bodyfluorescence imaging of mice after the injection of the probe and Zn²⁺. (e) Quantification offluorescence signal in mice before sonication (b), 25 min after sonication, (c) and 25 min after sonication and resting for 30 min (d) (p= 0.0223 calculated by an unpaired, two-tailedttest).

and one of the protector strands and monitoring each step to show that the probe displays minimal activity before HIFU activation and much higher activity after HIFU sonication.

This demonstration of the noninvasive and high spatiotempo- ral control of DNAzyme-based probes has broadened the applications of DNAzymes for metal ion detection, making it a powerful tool for imaging metal ions in vivo, including monitoring metal ions in metabolic processes and providing pivotal information for metal ions involved in medicine.

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ASSOCIATED CONTENT

*^sı Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.1c11543.

Experimental details and supplementary data, including optimization and kinetic studies of DNA sequences, cell culture, mice, cell viability assay, construction of HIFU- based temperature control platform, in vivo HIFU activation of subcutaneously injected DNAzyme, and in vivoimaging. Tables of DNA sequences (PDF)

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AUTHOR INFORMATION Corresponding Authors

King C. Li−Beckman Institute for Advanced Science and Technology, Carle Illinois College of Medicine, and Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States;

Email:kingli@illinois.edu

Yi Lu−Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States;

Department of Biochemistry, University of Illinois at Urbana- Champaign, Urbana, Illinois 61801, United States;

Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States; orcid.org/0000- 0003-1221-6709; Email:yi.lu@utexas.edu

Authors

Xiaojing Wang−Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States; orcid.org/0000-0003-0631-5727 Gun Kim− Beckman Institute for Advanced Science and

Technology and Carle Illinois College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States; Department of Urban and Environmental Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea James L. Chu−Beckman Institute for Advanced Science and

Technology and Carle Illinois College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States; orcid.org/0000-0003-2429-6478 Tingjie Song−Department of Chemistry, University of Illinois

at Urbana-Champaign, Urbana, Illinois 61801, United States; orcid.org/0000-0002-5696-5763

Zhenglin Yang−Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States; Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States; orcid.org/

0000-0003-2743-5779

Weijie Guo− Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States; Department of Chemistry, University of Texas

at Austin, Austin, Texas 78712, United States; orcid.org/

0000-0001-5694-2142

Xiangli Shao−Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States

Michael L. Oelze−Beckman Institute for Advanced Science and Technology, Carle Illinois College of Medicine, and Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States

Complete contact information is available at:

https://pubs.acs.org/10.1021/jacs.1c11543 Author Contributions

◆X.W., G.K., and J.L.C. contributed equally to this work.

Notes

The authors declare no competingfinancial interest.

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ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health (R35GM141931 to Y.L. and Grant R01CA184091 to K.C.L.) and the Robert A. Welch Foundation (Grant F-0020 to Y.L.).

G.K. acknowledges the research fund (1.200116.01) from the Ulsan National Institute of Science & Technology (UNIST).

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ABBREVIATIONS S a substrate strand E an enzyme strand P a protector strand H a helper strand

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