Enhancing Catalytic Activity of Uranyl-Dependent DNAzyme by

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Enhancing Catalytic Activity of Uranyl-dependent DNAzyme by Flexible Linker Insertion for More Sensitive Detection of Uranyl Ion Mengli Feng, Chunmei Gu, Yanping Sun, Shuyuan Zhang, Aijun Tong, and Yu Xiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00490 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 24, 2019

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Analytical Chemistry

Enhancing Catalytic Activity of Uranyl-dependent DNAzyme by Flexible Linker Insertion for More Sensitive Detection of Uranyl Ion Mengli Feng,1 Chunmei Gu,1 Yanping Sun,2 Shuyuan Zhang,2 Aijun Tong1 and Yu Xiang1,* 1

Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, China 2 School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China Email: [email protected]

ABSTRACT: The uranyl-dependent DNAzyme 39E cleaves its nucleic acid substrate in the presence of uranyl ion (UO22+). It has been widely utilized in many sensor designs for selective and sensitive detection of UO 22+ in environment and inside live cells. In this work, by inserting a flexible linker (C3 Spacer) into one critical site (A 20) of the 39E catalytic core, we successfully enhanced the original catalytic activity of 39E up to 8.1-fold at low UO22+ concentrations. Applying such a modified DNAzyme (39E-A20-C3) in a label-free fluorescent sensor for UO22+ detection achieved more than one order of magnitude sensitivity enhancement over using native 39E, with UO22+ detection limit improved from 2.6 nM (0.63 ppb) to 0.19 nM (0.047 ppb) while reserving the high selectivity to UO22+ over other metal ions. The method was also successfully applied for the detection of UO22+-spiked environmental water samples to demonstrate its practical usefulness.

INTRODUCTION DNAzyme (also called deoxyribozyme or catalytic DNA) is a class of single-stranded DNA oligonucleotides displaying enzyme-like catalytic activities beyond the traditional genetic role of DNA.1-4 So far, quite a few DNAzymes have been identified through a combinatorial technique called in vitro selection,5-9 and they exhibited diverse catalytic functions such as nuclease,10,11 ligase,12,13 peroxidase,14 kinase/phosphatase,15-17 and base modifications.18 For most DNAzymes with nuclease activities, metal ion cofactors are generally required, including those relying on Zn2+,19,20 Mg2+,21 Pb2+,22 Cu2+,11 UO22+,23 Hg2+,24 Na+,25 Mn2+,26 Ag+,27 or lanthanide ions.28 Thanks to their cofactor specificity and fast catalytic turnovers, metal ion-dependent DNAzymes have been widely applied in analytic techniques for highly selective and sensitive detection of a broad range of metal ions,29-37 as well as for multiple-turnover signal amplifications.38,39 Among them, the uranyl (UO22+)-dependent 39E DNAzyme23,40 is one particular example, because UO22+ is a heavy metal ion with severe health issues and also as an indicator of potential nuclear pollutions.41,42 Taking advantage of its strongest cofactor binding ability (lowest dissociation constant Kd) among all the currently known metal ion-dependent DNAzymes, 39E is highly specific to UO22+ at nanomolar or even picomolar concentrations,23,40 so that a series of sensors based on 39E have been developed for ultrasensitive detection of UO22+ in water43-54 and inside live cells.55-57 Enhancing the original catalytic activity of DNAzymes can improve the performance of many analytical techniques based on DNAzymes for metal ion detection and other applications. Several strategies have been utilized to enhance the catalytic activity of Mg2+-dependent 10-23 DNAzyme by incorporating modified nucleotides or functional groups to increase either

substrate binding affinity58-60 or catalytic core functionality.61-65 Peroxidase-mimicking G-quadruplex DNAzymes have also been improved by attaching flanking/adjacent bases, 66,67 ribose modifications68 or cationic species.69,70 Unfortunately, so far, there is still no method available to enhance the catalytic activity of UO22+-dependent 39E DNAzyme. In this work, instead of the above approaches for improving 10-23 and G-quadruplex DNAzymes, we developed a new strategy to enhance the activity of 39E by simply inserting a flexible linker (C3 Spacer) to one critical site (A20) of 39E’s catalytic core sequence, and successfully improve the activity of such modified 39E-A20-C3 up to 8.1-fold of the native 39E, while fully preserving its high specificity to UO22+ over other metal ions. A label-free fluorescent sensor design using this C3modified 39E displayed over 10-fold sensitivity enhancement for UO22+ detection compared with the original 39E, and the high selectivity to UO22+ was maintained. The method was also successfully applied for the detection of some environmental water samples containing spiked UO22+ at low nanomolar concentrations.

RESULTS AND DISCUSSION Screening the site of insertion and the type of flexible linker for optimal activity enhancement of 39E DNAzyme. The UO22+-dependent 39E DNAzyme binds its RNA substrates via base-pairing of the two biding arms on each end, and catalyzes site-specific cleaving of the substrate in the presence of UO22+ (Figure 1a).23,40 This 39E contains a catalytic core sequence of about 30 nucleotides between the two biding arms, with each nucleotide indexed by number subscripts (Figure 1b). The mode of interaction between 39E and its cofactor UO22+ was found to be “lock-and-key” catalysis according to

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Figure 1. (a) Scheme of DNAzyme 39E-catalyzed cleavage of its substrate 39S in the presence of UO22+. Typical sequences of 39E and 39S are listed at the bottom. The binding arm sequences are in green and underlined, the catalytic core sequence is in blue, and the ribonucleotide rA is in red. The cleavage of 39S labeled with a fluorophore and a quencher gives fluorescence enhancement. (b) Screening the critical site for flexible linker insertion to enhance the activity of 39E. For each inserted 39E, a C3 Spacer was placed between each two nucleotides in the catalytic core. Purple, red and green arrows indicate reduction, reservation and enhancement of 39E activity when a C3 Spacer was inserted at the location. Activity of each inserted 39E is represented by the relative rate of fluorescence enhancement (ΔF/t) induced by the DNAzyme-catalyzed cleavage of labeled 39S. Condition: 67 nM DNAyme, 60 nM 39S, 10 nM UO22+, 50 mM MES, 300 mM NaNO3, pH 5.5, λex/λem = 495/520 nm, at 25℃.

the fluorescent resonance energy transfer (FRET) studies conducted by Lu’s group.71,72 For 39E to carry out efficient RNAcleaving reactions, UO22+ was required to bind with 39E in the optimal global DNA structures. This mechanism of action inspired us that, instead of improving substrate binding or catalytic functionality for potential activity enhancement, it could also be possible to enhance the original activity of 39E by simply inserting flexible linkers to a proper site of the catalytic core, for more structural flexibility of the DNAzyme to fold into optimal structures, thereby facilitating both UO22+ binding and RNA cleaving. We carried out a screening study on 39E’s catalytic core sequence by inserting one abasic site of C3 Spacer as the flexible linker between each two conserved nucleotides, aiming to identify the critical site where even one insertion could apparently enhance 39E’s activity. The loop fragment T9~A14 was reported to make little contribution to the DNAzyme’s activity,40,73 therefore we excluded this region and only focused on the other two parts of A2~G8 and A15~C29 (Figure 1b). The substrate 39S was covalent-labeled with a fluorophore (fluorescein, FAM) and a quencher (Iowa Black, IABkFQ) at its 5' and 3' ends, respectively, and it was utilized to quantify the RNA-cleaving catalytic activity of 39E with or without C3 Spacer insertion by fluorescence measurement. The labeled 39S displayed low fluorescence signal because of the proximity between FAM and IABkFQ in the same single-stranded oligonucleotide. Upon cleavage of 39S (60 nM) by 39E (67 nM) in the presence of UO22+ (10 nM), the fluorescence significantly increased because FAM was well separated from IABkFQ (Figure 1a). The rate of fluorescence enhancement (ΔF/t) should be proportional to the rate of substrate cleavage as well as the DNAzyme’s catalytic activity, so that we are looking for the inserted 39E version that can generate the highest rate increase. As depicted in Figure 1b, 39E-A20, the one with a C3 Spacer inserted between A20 and C21 showed strongest rate enhancement relative to the native 39E, while insertion at any other location gave either comparable or much lower activity compared with 39E. Therefore, A20 is the optimal site for flexible linker insertion on 39E. We further introduced different linkers at A20 of 39E to explore their potential for activity enhancement. Flexible linkers including dSpacer, C3 Spacer, Spacer 6, Spacer 9 and Spacer

12 (Figure 2a), as well as normal bases A, T, G and C, were each inserted at A20 to study the effect of linker size and length on activity enhancement by measuring the rate of substrate cleavage by the DNAzymes (Figure 2b). As shown in Figure 2c, insertion of A, G or C induced negative effect to the activity of 39E likely because of DNA structural perturbation via wrong base pairing. However, inserting T gave a mild activity enhancement to about 2-fold of native 39E. More fold of enhancement was observed for the abasic linkers over T, including dSpacer (2.5-fold) and C3 Spacer (4.2-fold), suggesting smaller linkers (size order: T > dSpacer > C3) was beneficial to the DNAzyme’s activity probably due to less steric effect. Extending C3 Spacer further to Spacer 6 (3.7-fold) and Spacer 9 (4.2fold) provided no further activity enhancement, and severe negative contribution occurred when the linker was too long (Spacer 12). Besides the multiple-turnover over condition

Figure 2. (a) Insertion of different flexible linkers to the A20 site of 39E to enhance its activity. (b) Kinetics of 39S cleavage by inserted 39E DNAzymes with different types of linkers. (c) Relative rate of fluorescence enhancement (ΔF/t) of inserted 39E DNAzymes with different types of linkers (S3, S6, S9, S12 and Sd represent C3 Spacer, Spacer 6, Spacer 9, Spacer 12 and dSpacer, respectively). Condition: 60 nM DNAyme, 250 nM labeled 39S, 25 nM UO22+, 50 mM MES, 300 mM NaNO3, pH 5.5, λex/λem = 495/520 nm, at 25℃.

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(excess 39S) in Figure 2c, we also confirmed the similar trend under the single-turnover condition (39S : DNAzyme = 1:1.1, Figure S1, Supporting Information). Therefore, we chose C3 Spacer insertion at the A20 site of 39E catalytic core as our strategy to enhance the DNAzyme’s activity in the presence of UO22+, and named it as 39E-A20-C3.

Characterizing the activity enhancement of linker-inserted 39E-A20-C3 DNAzyme. The relative activity (kobs) of both 39E-A20-C3 and 39E was measured in the presence of different concentrations of UO22+ ranging from 0.1 nM to 1000 nM under both single-turnover (ST) and multiple-turnover conditions (MT), as shown in Figure 3a. The plateau kobs for 39E at 500 nM UO22+ under the single-turnover condition was set as 1 for the relative kobs here. At UO22+ concentrations as high as 500~1000 nM, kobs of 39E-A20-C3 and 39E both reached the plateau, suggesting the DNAzymes were fully bound by UO 22+ under this condition. Based on the data in Figure 3a, the apparent dissociation constant (Kd) of UO22+ to DNAzymes was calculated to be about 32 ± 5 nM (MT) and 19 ± 6 nM (ST) for 39E-A20-C3 and 101 ±8 nM (MT) and 81 ±11 nM (ST) for 39E, respectively, under the hypothesis of a 1:1 binding mode between DNAzyme and UO22+. The Kd values of 81 and 101 nM for native 39E in this work is in accordance with the literature Kd of 97 nM for 39E obtained from fluorescence assays. 23 The Kd of 39E-A20-C3 was around 1/3~1/4 of 39E, suggesting the flexible linker insertion indeed enhanced the binding of UO 22+ to the DNAzyme, by about 3 folds. On the other hand, the plateau kobs values of 39E-A20-C3 at UO22+ concentrations > 500 nM represented the activity when DNAzyme was fully bound by UO22+, and Kd should contribute little to the activity enhancement in this range (Figure 3a). Considering the modification of 39E-A20-C3 was in the catalytic core instead of the binding arms, we thought 39E-A20-C3 and 39E should have similar KM (related to DNAzyme-substrate binding) because of their almost same binding affinity to 39S.40 In this case, the enhancement of kobs should be proportional to that of kcat, so that the flexible linker insertion provided about 2fold catalytic robustness (kcat) of 39E. The fold of kobs enhancement for 39E-A20-C3 over 39E was indeed dependent on the concentration of UO22+, with 8.1~1.6fold enhancement in the presence of 0.1~1000 nM UO 22+, respectively (Figure 3b), mildly different between single-turnover and multiple-turnover conditions. The much higher fold of enhancement at lower concentrations of UO22+ was because of the combined effects from both Kd and kcat. This feature of low concentration-preferred activity enhancement would be ideal for improving the sensitivity of UO22+ detection in samples containing low UO22+ concentrations. Enhancement of 39E’s RNA-cleaving activity in the presence of UO22+ should not compromise the DNAzyme’s selectivity on its metal ion cofactor, which is essential for 39E as useful sensors for UO22+ detection. We examined the UO22+ selectivity of 39E-A20-C3 over other metal ions and compared it with 39E under the same condition (Figure 3c). Both 39E-A20C3 and 39E showed excellent selectivity to UO22+ over all the other metal ions in the experiment. Therefore, 39E-A20-C3 enhanced the activity of 39E while maintaining high specificity to its metal ion cofactor.23 The effect of buffer pH was also investigated to see whether 39E-A20-C3 and 39E had the same pH preference (Figure S2, Supporting Information). The optimal pH was around 5.0~5.5 for both 39E-A20-C3 and 39E, which was in agreement with

Figure 3. (a) Observed rate constants (kobs) of 39E-A20-C3 under single-turnover (39E-C3-ST, blue) and multiple-turnover (39E-C3MT, red) conditions as well as 39E under single-turnover (39E-ST, green) and multiple-turnover (39E-MT, black) conditions in the presence of 0.1~1000 nM UO22+. (b) Fold of activity enhancement of 39E-A20-C3 over 39E in the presence of different UO22+ concentrations under single-turnover (ST, red) and multiple-turnover (MT, black) conditions. (c) Selectivity of 39E-A20-C3 and 39E to UO22+ over other metal ions under multiple-turnover conditions. 1~20: Ag+, Mg2+, Ca2+, Sr2+, Ba2+, VO2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+, Tb3+, Eu3+, Th4+, UO22+. Condition: 67 nM DNAyme, 250 nM (for MT) or 60 nM (for ST) 39S, 50 mM MES, 300 mM NaNO3, pH 5.5, λex/λem = 495/520 nm, at 25℃.

previous reports.23,40 Interestingly, although pH 6.0 was not the optimal pH for the two DNAzymes, the activity enhancement for 39E-A20-C3 over 39E was larger at pH 6.0 than 5.5. It was probably because the effective UO22+ concentration was reduced at higher pH and the fold of activity enhancement was known to be higher at lower UO22+ concentrations as illustrated in Figure 3b. Unfortunately, neither 39E-A20-C3 nor 39E had a good activity at pH higher than 6.5, since other forms of uranium ions such as UO2(OH)+ instead of UO22+ dominated under these conditions.40 In addition to the assessment by fluorescence kinetics measurement, we also confirmed the activity enhancement of 39EA20-C3 by polyacrylamide gel electrophoresis (PAGE). Under both multi-turnover (500 nM 39S and 30 nM DNAzyme) and single-turnover (500 nM 39S and 500 nM DNAzyme) conditions, 39E-A20-C3 showed much faster 39S cleavage than 39E (Figures S3 and S4, Supporting Information). The effect of linker type on the activity enhancement was also tested by PAGE under the multi-turnover condition for 39E-A20-6, 39EA20-9 and 39E-A20-12, and the trend (C3 ~ Spacer9 > Spacer6 > 39E > Spacer12) was in accordance with the fluorescence kinetics studies in Figure 2b.

Applying 39E-A20-C3 DNAzyme in a label-free fluorescent sensor design for more sensitive detection of UO22+ in water. With such an improved DNAzyme 39E-A20C3 in hand, we further utilized it to construct fluorescent sensors for more sensitive detection of UO22+ in water samples. Label-free74,75 fluorescent sensors based on DNAzymes were less complicated, low-cost and less intrusive than their labeled counterparts, but usually compromised sensitivity, e.g. detection limits of 3.0 nM and 0.045 nM for previously reported label-free and labeled fluorescent sensors for UO22+, respectively.23,76 Therefore, we set to make the vacant site label-free

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Figure 4. (a) Scheme of the label-free fluorescent sensor based on the vacant site approach, which was improved by using 39E-A20C3-tr3 instead of 39E-tr3. (b) Kinetics of ADMND fluorescence enhancement of the label-free sensor based on 39E-A20-C3-tr3 in the presence of different amounts of UO22+. (c) Calibration curve for UO22+ detection using the label-free sensor based on 39E-A20C3-tr3, with an inset figure illustrating the low concentration range. (d) Selectivity of the label-free sensor based on 39E-A20-C3-tr3 to UO22+ over other metal ions, 1~18: Mg2+, Ca2+, Sr2+, Ba2+, VO2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+, Tb3+, Eu3+, Th4+, UO22+. Condition: 2 μM 39S-L, 3 μM 39E-A20-C3-tr3, 1 μM ADMND, 50 mM MES, 300 mM NaNO3, pH 5.5, λex/λem = 346/403 nm, at 4℃.

sensor design76 more sensitive using 39E-A20-C3 to detect water samples containing UO22+ at low nanomolar concentrations. The truncated 39E-A20-C3-tr3 with 3 nt trimmed at its 3' end and the elongated 39S-L with a hairpin sequence addition at its 5' end formed the DNA duplex containing a vacant site, which was positioned between two flanking guanines and with a cytosine on the opposite (Figure 4a).76 This vacant site was known to strongly bind the fluorophore, 2-amino-5,7-dimethyl-1,8naphthyridine (ADMND), via hydrogen bonds, π-π stacking and electrostatic interactions.76-82 ADMND binding to the vacant site caused its fluorescence quenching. Upon the addition of UO22+, the DNA duplex as well as the vacant site were destructed because of 39S-L cleavage by 39E-A20-C3-tr3. Subsequently, ADMND was released from DNA and its fluorescence was recovered. The rate of ADMND fluorescence recovery (enhancement) was related to the DNAzyme activity and could be used for quantification of UO22+ in the solution. As illustrated in Figure S5 (Supporting Information), about 70% fluorescence of ADMND (1 μM) was quenched in the presence of 39S-L (2 μM) and 39E-A20-C3-tr3 (3 μM). After the addition of 500 nM UO22+ to the above solution, efficient cleavage of 39S-L resulted in about 100% fluorescent enhancement after 10 min reaction. In a solution containing 1 mM ethylene diamine tetraacetic acid (EDTA), no fluorescence enhancement was observed even with the addition of 500 nM UO22+, suggesting the essential role of free UO22+ for the activity of 39E-A20-C3-tr3. Replacing 39E-A20-C3-tr3 by 39E-tr3 (39E-A20-C3-tr3 without C3 insertion at A20) gave almost the same fluorescence changes

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in the presence of UO22+ after efficient cleavage of 39S-L, but its rate of fluorescence enhancement (ΔF/t) was about only 1/4 of 39E-A20-C3-tr3 (Figure S5, Supporting Information). We measured ΔF/t in the solutions containing different concentrations of UO22+ and established the calibration curves for the sensor designs using 39E-A20-C3-tr3 and 39E-tr3, which achieved detection limits of 0.19 nM and 2.6 nM for 39E-A20-C3-tr3 and 39E-tr3 respectively, on the basis of 3σ/slope, where σ is standard deviation of the blank samples (Figure 4b and 4c, and Figure S6, Supporting Information).76 A 13-fold enhancement of sensitivity was thus achieved using the improved DNAzyme 39EA20-C3 developed in this work. The calibration curves at low nanomolar range clearly showed that the signal increase of the 39E-A20-C3-tr3 design was much faster than that of 39E-tr3. Such a phenomenon was in line with the findings in Figure 3b, supporting that 39E-A20-C3 was especially advantageous over 39E in responding to low UO22+ concentrations. The selectivity of the label-free sensor design based on 39E-A20-C3-tr3 was also investigated. Micromolar concentrations of competing metal ions tested in Figure 4d gave negligible signals compared with nanomolar concentrations of UO22+. The excellent selectivity of native 39E to UO22+ was thus well preserved by 39EA20-C3-tr3 in the label-free sensor design. Table 1. Detection of environmental samples containing spiked UO22+ using the improved label-free fluorescent sensor based on 39E-A20-C3-tr3. Spiked

2 nM

10 nM

50 nM

Lake water

1.93±0.07 nM (96.5±3.5%)

9.88±0.25 nM (98.8±2.5%)

50.3±0.85 nM (100.6±1.7%)

Tap water

1.96±0.09 nM (98.0±4.5%)

9.51±0.73 nM (95.1±7.3%)

50.3±2.0 nM (100.6±4.0%)

Drinking water

1.92±0.12 nM (96.0±6.0%)

9.93±0.54 nM (99.3±5.4%)

50.6±3.2 nM (101.2±6.4%)

In the table are the detected concentrations as well as recovery (%) in the brackets. Standard deviations are based on 3 separated tests of the samples.

The method was further applied for the detection of environmental water samples containing spiked UO22+. Each concentration (2, 10 or 50 nM) of spiked UO22+ was successfully detected with satisfactory recovery (Table 1), suggesting the usefulness of the method for practical detection of UO22+ in water. The spiked samples were also confirmed by inductively coupled plasma mass spectrometry (ICP-MS) and gave similar results of UO22+ concentration and recovery (Table S1, Supporting Information). Note that the original label-free method based on 39E cannot be used for the detection of 2 nM UO22+ because its detection limit (2.6 nM) was above the 2 nM criterion.76

EXPERIMENTAL SECTION Materials and methods. The fluorophore 2-amino-5,7-dimethyl-1,8-naphthyridine (ADMND) was synthesized as described previously.77-80 Metal salts and reagents for buffers were purchased from Sigma-Aldrich Inc. (St. Louis, MO). Lake water, tap water and drinking water were all from the campus of Tsinghua University. Fluorescence spectra were taken on a JASCO FP-6500 fluorometer (Tokyo, Japan). The concentrations of DNA were determined by a JASCO V-550 UV-VIS spectrophotometer (Tokyo, Japan) at 260 nm. Inductively coupled plasma mass spectrometry (ICP-MS) measurement was

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carried out on a PerkinElmer Nexion300X ICP-MS (Waltham, USA). PAGE gel imaging was on a BIO-RAD Universal Hood II (Hercules, USA) using SYBR Gold channel with a 300 nm filter. Oligonucleotides were synthesized and purified by either Integrated DNA Technologies, Inc. (IA, USA) or Sangon Biotech Co., Ltd. (Shanghai, China), with the following sequences and modifications (from left to right, 5' to 3'): For multiple-turnover experiment: Labeled 39S: /56-FAM/ACT CAC TAT rAGG AAG AGA TG/3IABkFQ/ Native 39E: CAT CTC TTC AGT CGG GTA GTT AAA CCG ACC TTC AGA CAT AGT GAG T 39E-A20-X: CAT CTC TTC AGT CGG GTA GTT AAA CCG AXC CTT CAG ACA TAG TGA GT For single turnover experiment: Labeled 39S long: /56-FAM/ACT CAC TAT rAGG AAG AGA TGG ACG TG/3IABkFQ/ Native 39E long: CAC GTC CAT CTC TTC AGT CGG GTA GTT AAA CCG ACC TTC AGA CAT AGT GAG T/3BHQ_1/ 39E-A20-X long: CAC GTC CAT CTC TTC AGT CGG GTA GTT AAA CCG AXC CTT CAG ACA TAG TGA GT/3BHQ_1/ For label-free detection: 39S-L: GAC GAT GAA ACA TCG TCC CAC TAT rAGG AAG AGA TGG ACG TG 39E-tr3: CAC GTC CAT CTC TGC AGT CGG GTA GTT AAA CCG ACC TTC AGA CAT AGT G 39E-A20-C3-tr3: CAC GTC CAT CTC TGC AGT CGG GTA GTT AAA CCG A/iSpC3/C CTT CAG ACA TAG TG The /56-FAM/ at 5' end is a fluorescein modification. The /3IABkFQ/ and /3BHQ_1/ at 3' end are efficient quenchers for fluorescein. The ribonucleotide rA in 39S is essential for the DNAzyme-catalyzed cleavage. The X indicates C3 Spacer, Spacer 6, Spacer 9, Spacer 12 or dSpacer. The /iSpC3/ specifically represents C3 Spacer. For Polyacrylamide gel electrophoresis (PAGE) experiment, labeled 39S or 39S long without 3' quenchers were used to avoid any bias in fluorescent gel imaging.

Screening for the optimal site for linker insertion. For each measurement, 6 μL of 10 μM DNAzyme (native or C3modified, see Supporting Information for the detail list) and 5.4 μL of 10 μM labeled 39S were added to 879 μL of the reaction buffer (50 mM MES, 300 mM NaNO3, pH 5.5). The solution was heated to 75oC and allowed to cool down gradually to 25oC in 1.5 h. Then 297 μL of the above solution was mixed with 3 μL of 1 μM UO22+, giving a final solution of 67 nM DNAzyme, 60 nM 39S and 10 nM UO22+, which was immediately monitored by the fluorometer for time-dependent fluorescence intensity (λex/λem = 495/520 nm) at 25℃. Multiple-turnover Activity of native and modified 39E DNAzymes determined by kinetic fluorescence measurement. For each measurement, 1.8 μL of 10 μM DNAzyme (native or modified at A20) and 2.5 μL of 30 μM labeled 39S were added to 293 μL of the reaction buffer (50 mM MES, 300 mM NaNO3, pH 5.5). The solution was let to stand for 10 min, giving a solution containing 60 nM DNAyme and 250 nM 39S. Desired amount of UO22+ was then added to the solution via 3 μL UO22+ stock solutions of proper concentrations, and after

brief vortexing the final solution was immediately monitored by the fluorometer for time-dependent fluorescence intensity (λex/λem = 495/520 nm) at 25℃. To test the effect of buffer pH, the reaction buffers with different pH values (5.0, 5.5, 6.0, 6.5, 7.0) were prepared while keeping concentrations of MES and NaNO3 unchanged. In the selectivity experiment, other metal salts were used to replace UO22+, including AgNO3, MgCl2, CaCl2, SrCl2, Ba(ClO4)2, VOSO4, MnSO4, Fe(NH4)2(SO4)2, Fe(NO3)3, Co(OAc)2, Ni(NO3)2, CuSO4, ZnCl2, CdCl2, Hg(ClO4)2, Pb(NO3)2, TbCl3, EuCl3, and Th(NO3)4.

Single-turnover Activity of native and modified 39E DNAzymes determined by kinetic fluorescence measurement. A large volume (1 mL in each tube, 25 mL in total) of a solution containing 67 nM DNAzyme (native 39E long or C3modified) and 60 nM labeled 39S long in the reaction buffer (50 mM MES, 300 mM NaNO3, pH 5.5) was heated to 75oC and allowed to cool down gradually to 25oC in 1.5 h. For each measurement, 297 μL of the above solution was mixed with 3 μL of UO22+ stock solution at proper concentrations, and after brief vortex the solution was immediately monitored by the fluorometer for time-dependent fluorescence intensity (λex/λem = 495/520 nm) at 25℃. PAGE Activity Assay. The reaction solution containing 30 nM DNAzyme and 500 nM labeled 39S as a multi-turnover system, or 500 nM DNAzyme long and 500 nM 39S long as a single-turnover system, was prepared in the reaction buffer (50 mM MES, 300 mM NaNO3, pH 5.5). Then UO22+ was added to the solution at a final concentration of 100 nM. After brief vortex, each 10 μL aliquot was taken from the solution at every designated time point and quenched by 2 mM EDTA. A sample containing only 39S and 100 nM UO22+ was prepared as the negative control (NC). Each of the 10 μL aliquot was mixed with 10 μL loading buffer containing 1 M sucrose and 6 M urea, and then loaded to 20% denatured or native polyacrylamide gels for electrophoresis analysis. Native PAGE was done using gels without urea. Annealing the solution before adding UO22+ was found with negligible effect on the PAGE results. All the PAGE bands were visualized through fluorescence of the labeled substrates. The stability of 39S against high concentrations of UO22+ and the high efficiency of duplex formation between 39S and 39E were supported by the PAGE analysis shown in Figure S4 of Supporting Information. Calculation of percentage of substrate cleavage (%). For fluorescence-based assays, the percentage of substrate cleavage (%) = (Ft- F0)/(Fmax- F0)×100%, where F0, Ft and Fmax are initial (0% cleavage), measured (n% cleavage after reaction for a specific time) and maximum (reaching 100% cleavage after overnight reaction with 100 nM UO22+) fluorescence intensity of the solution. For PAGE-based assays, the percentage of substrate cleavage was calculated by the ImageJ software, with the defining of substrate cleavage (%) = 100%×(I2C2)cleaved/[(I1C1)uncleaved+(I2C2)cleaved], where I and C are mean fluorescence intensity and counts of fluorescence, respectively. Calculation of observed rate constant (kobs) and dissociation constant (Kd). The fluorescence-generating cleavage reaction at the first 1 min was used for calculating kobs. For multiple-turnover activities, kobs = 100×[percentage of substrate cleavage (%)/t]*(CDNAzyme/C39S). For single-turnover activities, kobs = 100×[percentage of substrate cleavage (%)/t]. Because absolute kobs values could vary significantly depending on the method for calculation according to literature reports,23,40 we

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preferred using relative kobs as a measure of activity enhancement by modified 39E over its native form. For both multipleturnover and single-turnover activities, the kobs-[UO22+] curve was nonlinearly fitted by SigmaPlot to calculate Kd based on 1:1 binding mode between UO22+ and DNAzyme-substrate duplex.

detection of spiked UO22+ in some environmental water samples. Such a strategy of flexible linker insertion might be further utilized to enhance the catalytic activity of other DNAzymes with similar structure features or cofactor properties as 39E.

Fluorescent label-free sensors for UO22+ detection. Components of 3 μL ADMND (100 μM), 6 μL 39S-L (100 μM), and 9 μL 39E-tr3 or 39E-A20-C3-tr3 (100 μM) were added sequentially into 280 μL reaction buffer (50 mM MES, 300 mM NaNO3, pH 5.5). After incubating at room temperature for 2 min, the mixture was transferred to a cuvette and kept at 4°C for 10 min. Then 3 μL UO22+ stock solution (0~100 μM) in 1 mM HNO3 was added to the cuvette. After vortexing, time-dependent fluorescence intensity (λex/λem = 346/403 nm) was monitored at 4°C. The rate of fluorescence enhancement (ΔF per second) over background (0 nM UO22+) was calculated based on the data points collected in the first 100 s, to construct the calibration curve for quantification of UO22+ samples. In the selectivity experiment, other metal salts were employed to replace UO22+, including MgCl2, CaCl2, SrCl2, Ba(ClO4)2, VOSO4, MnSO4, Fe(NH4)2(SO4)2, Co(OAc)2, Ni(NO3)2, CuSO4, ZnCl2, CdCl2, Hg(ClO4)2, Pb(NO3)2, TbCl3, EuCl3, and Th(NO3)4. The fluorescence intensity of samples with low UO22+ levels (especially 10 nM or less) decreased in the first 5 min (Figure 4b) due to the temperature effect. For each measurement, the solution containing 39E and 39S was taken out of chamber at 4 oC for addition of UO22+ at room temperature (25oC), and then put back to the fluorometer chamber at 4oC for fluorescence monitoring immediately. However, the real temperature of the solution could not go back to 4oC instantly. It is known that ADMND binds with vacant site more efficiently at 4oC than 25oC, so there was some ADMND dissociation because of the temperature effect, and several minutes was needed for ADMND to fully rebind with vacant site. This process caused fluorescence decrease as ADMND rebound with vacant site. Through background reduction, the calibration curve and UO22+ detection was hardly affected by the initial fluorescence decrease. Detection of spiked UO22+ in environmental water samples. Environmental water samples were spiked by different amounts of UO22+ and mixed with concentrated stock solutions of reaction buffer to achieve final concentrations of 50 mM MES and 300 mM NaNO3 at pH 5.5. For UO22+ detection of each spiked sample, 3 μL of 100 μM ADMND, 6 μL of 100 μM 39S-L, and 9 μL of 100 μM 39E-A20-C3-tr3 were added into 282 μL of the solution, and then measured using the method described in the above paragraph.

ASSOCIATED CONTENT

CONCLUSION In summary, we reported a new strategy to enhance the activity of UO22+-dependent DNAzyme 39E by inserting a flexible linker (C3 Spacer) into one critical site (A20) of 39E’s catalytic core sequence. This modified DNAzyme 39E-A20-C3 showed up to 8.1-fold catalytic activity of the native 39E while maintained excellent selectivity to UO22+ over other metal ions. The activity enhancement was contributed by the improvement of 39E in both UO22+ binding (Kd) and catalytic robustness (kcat). The pH effect on the activity of 39E-A20-C3 and 39E was almost the same, indicating no change in the catalytic mechanism. Applying 39E-A20-C3 in a label-free fluorescent sensor design for the detection of UO22+ in water achieved 13-fold sensitivity enhancement over using 39E (detection limit improved from 2.6 nM to 0.19 nM), and the method was successful used for the

Supporting Information The Supporting Information is available free of charge on the ACS Publications website, including list of additional DNA oligonucleotides and additional figures.

AUTHOR INFORMATION Corresponding Author *Yu Xiang Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, China Email: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT We are very grateful for the financial support from the National Natural Science Foundation of China (Nos. 21675097, 21621003, 21775084 and 21707079). We also thank Miss Zhi Wang for helping us with fluorescence measurement and drawing cartoons.

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TOC graphic 82x28mm (300 x 300 DPI)


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Figure 1. (a) Scheme of DNAzyme 39E-catalyzed cleavage of its substrate 39S in the presence of UO22+. Typical sequences of 39E and 39S are listed at the bottom. The binding arm sequences are in green and underlined, the catalytic core sequence is in blue, and the ribonucleotide rA is in red. The cleavage of 39S labeled with a fluorophore and a quencher gives fluorescence enhancement. (b) Screening the critical site for flexible linker insertion to enhance the activity of 39E. For each inserted 39E, a C3 Spacer was placed between each two nucleotides in the catalytic core. Purple, red and green arrows indicate reduction, reservation and enhancement of 39E activity when a C3 Spacer was inserted at the location. Activity of each inserted 39E is represented by the relative rate of fluorescence enhancement (ΔF/t) induced by the DNAzyme-catalyzed cleavage of labeled 39S. Condition: 67 nM DNAyme, 60 nM 39S, 10 nM UO22+, 50 mM MES, 300 mM NaNO3, pH 5.5, λex/λem = 495/520 nm, at 25℃.



Figure 2. (a) Insertion of different flexible linkers to the A20 site of 39E to enhance its activity. (b) Kinetics of 39S cleavage by inserted 39E DNAzymes with different types of linkers. (c) Relative rate of fluorescence enhancement (ΔF/t) of inserted 39E DNAzymes with different types of linkers (S3, S6, S9, S12 and Sd represent C3 Spacer, Spacer 6, Spacer 9, Spacer 12 and dSpacer, respectively). Condition: 60 nM DNAyme, 250 nM labeled 39S, 25 nM UO22+, 50 mM MES, 300 mM NaNO3, pH 5.5, λex/λem = 495/520 nm, at 25℃.


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Figure 3. (a) Observed rate constants (kobs) of 39E-A20-C3 under single-turnover (39E-C3-ST, blue) and multiple-turnover (39E-C3-MT, red) conditions as well as 39E under single-turnover (39E-ST, green) and multiple-turnover (39E-MT, black) conditions in the presence of 0.1~1000 nM UO22+. (b) Fold of activity enhancement of 39E-A20-C3 over 39E in the presence of different UO22+ concen-trations under singleturnover (ST, red) and multiple-turnover (MT, black) conditions. (c) Selectivity of 39E-A20-C3 and 39E to UO22+ over other metal ions under multiple-turnover conditions. 1~20: Ag+, Mg2+, Ca2+, Sr2+, Ba2+, VO2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+, Tb3+, Eu3+, Th4+, UO22+. Condition: 67 nM DNAyme, 250 nM (for MT) or 60 nM (for ST) 39S, 50 mM MES, 300 mM NaNO3, pH 5.5, λex/λem = 495/520 nm, at 25℃.



Figure 4. (a) Scheme of the label-free fluorescent sensor based on the vacant site approach, which was improved by using 39E-A20-C3-tr3 instead of 39E-tr3. (b) Kinetics of ADMND fluorescence enhancement of the label-free sensor based on 39E-A20-C3-tr3 in the presence of different amounts of UO22+. (c) Calibration curve for UO22+ detection using the label-free sensor based on 39E-A20-C3-tr3, with an inset figure illustrating the low concentration range. (d) Selectivity of the label-free sensor based on 39E-A20-C3tr3 to UO22+ over other metal ions, 1~18: Mg2+, Ca2+, Sr2+, Ba2+, VO2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+, Tb3+, Eu3+, Th4+, UO22+. Condition: 2 μM 39S-L, 3 μM 39E-A20-C3tr3, 1 μM ADMND, 50 mM MES, 300 mM NaNO3, pH 5.5, λex/λem = 346/403 nm, at 4℃.


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Enhancing Catalytic Activity of Uranyl-Dependent DNAzyme by

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