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Single Primer Based Multi-site Strand Displacement Reaction Amplification Strategy for Rapid Detection of Terminal Deoxynucleotidyl Transferase Activity Xinyan Liu, Hao Wang, Keqin Deng, Sharon Kwee, Haowen Huang, and Liang Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01816 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 14, 2019

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Analytical Chemistry

Single Primer Based Multi-site Strand Displacement Reaction Amplification Strategy for Rapid Detection of Terminal Deoxynucleotidyl Transferase Activity Xinyan Liu a, Hao Wang a, Keqin Deng a,b, Sharon Kwee b, Haowen Huang a, Liang Tang b,  a Key

Laboratory of Theoretical Organic Chemistry and Function Molecule, Ministry of Education, School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201, P. R. China b Department of Biomedical Engineering, The University of Texas at San Antonio, San Antonio, Texas 78249, United States KEYWORDS: terminal deoxynucleotidyl transferase, isothermal amplification, multi-site strand displacement reaction, Fluorescence, DNA biosensor

ABSTRACT: A fluorescence-based multi-site strand displacement reaction (MSSDR) amplification strategy is developed for the rapid, sensitive, and selective detection the activity of terminal deoxynucleotidyl transferase (TdT). Oligo dT primer was used for TdT extension reaction, then the left oligo dT primers were hybridized to the TdT extension reaction product by end to end tiled style and initiated the MSSDR by Klenow polymerase, subsequently, 3' terminals of these single-strand DNA produced by MSSDR are folded back to complement themselves with the adjacent sequences, and Klenow polymerase make it into double-stranded DNA (dsDNA). The final dsDNA products were analyzed via dsDNA specific fluorescent dye. This method enables rapid (less than 100 min) and sensitive (limit of detection, LOD, 1.35  10-5 U) detection, and has been demonstrated to work well using real biosample. Our design would not only serve as a new prototype for high-throughput automated analysis and clinic diagnostic application, but also has promising potential for improving the sensitivity of those TDT related biosensing system.

* Corresponding authors at: Key Laboratory of Theoretical Organic Chemistry and Function Molecule, Ministry of Education, Chemistry and Chemical Engineering College, Hunan University of Science and Technology, Xiangtan 411201, P. R. China; and Department of Biomedical Engineering, The University of Texas at San Antonio, San Antonio, Texas 78249, United States E-mail addresses: [email protected] (K. Deng), [email protected] (L. Tang).

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It is well known that DNA polymerase extends a primer using a DNA or RNA template to guide each incorporation event. The presence of a templating strand is absolutely necessary for polymerase activity. However, terminal deoxynucleotidyl transferase (TdT) is an exception that possesses the ability to incorporate nucleotides into the 3'-hydroxyl (OH) termini of single-stranded DNA (ssDNA) in a template-independent manner.1-3 By addition of random nucleotides to related exon termini of T and B cell receptor genes, TdT plays a key role in the evolution and adaptation of the vertebrate immune system.4 The expression of the TdT gene is generally suppressed in most human somatic cells, but it is over expressed in acute lymphoblastic leukemia cells and acute myelomonocytic leukemia cells.5-6 It is worth pointing out that acute lymphoblastic leukemia is the most common cancer of childhood, accounting for approximately 1/4 of all childhood cancers.7 Research has shown that higher activity of TdT is associated with an unfavorable prognosis of leukemia patients.8 Thus, TdT may function as a biomarker for acute leukemic disease.6, 9 Furthermore, because of its capability of adding non-templated nucleotides to the DNA fragments, TdT is applied to molecular biological techniques such as RACE (Rapid amplification of cDNA ends) and TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling); also it is widely used to fabricate biosensors for the detection of nucleic acids,10-14 protein,15 metal ions,15 and cell apostosis.16 Therefore, efficient methods to determine TdT activity should be invaluable in further exploring TdT’s underlying pathological function and its application in biosensors for the diagnosis of TdT-related diseases. So far, only a few methods have been developed for TdT assays. Traditional methods, including radioimmunoassay,17 immunofluorescence,6, 18 and radioactive dNTP incorporated assay19 are commonly used for TdT detection. Unfortunately, these methods involve the use of hazardous radioisotopes or expensive fluorescence labels and therefore require operators with specialized skills. Recently, non-radioactive and antibody-free TdT assays have attracted increasing research interest. These non-radioactive and antibody-free methods fall into three categories: one mainly focused on enzyme-based nanomaterial-enhanced signal amplification which involves generating a sequence-specific DNA, then using the sequence-specific DNA as a template to synthesize nanomaterials with fluorescent or electrochemical activity, and eventually measuring fluorescence or electrochemical signals;12, 20-23 the second related to TdT-randomly elongated sequences such as G-rich sequences which can bind to thioflavin T and act as a peroxidase-mimic or i-motif which can form cyclometallated iridium(III) complexes and make an enhanced luminescent signal;24-25 the third category is polymerizationdirected exonuclease-assisted and quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensors.26 These methods still possess notable limitations such as complicated chemical reactions for nanomaterial synthesis, high cost for labeledprobes, and time consuming processes. As a result, they do not meet the demand for rapid analysis and high-throughput clinical diagnosis. Taking these situations into consideration, it is highly beneficial to develop a simple, rapid and high-throughput method for TdT assays. In this work, we developed a fluorescence-based and label free strategy for the rapid quantification of TdT activity. The distinction from previously published works is that we do not use TdT synthesized sequences to generate nanoparticles or use their enzymelike properties to obtain detection signals; instead, the detection signal is obtained by amplifying the TdT synthesized sequences. In this study, oligo dT was utilized as a primer for the TdT extension reaction. Subsequently, TdT extension products were amplified by end to end tiled primer (oligo dT) initiated multi-site strand displacement reactions (MSSDR)27. The 3' terminals of the MSSDR products were folded back to complement themselves with the adjacent extension sequences, and DNA polymerase converted them into double-stranded DNA (dsDNA). Finally, amplification products were analyzed via fluorescence from dsDNA specific fluorescent dye. Due to the sensitivity of fluorescence-based methodologies,28-31 we were able to achieve a detection limit as low as 1.35  10-5 U.

EXPERIMENTAL SECTION 2.1 Reagents and materials Oligonucleotides designed in this work were synthesized by Sangon Biotechnology Co., Ltd (Shanghai, China); their sequences are listed in Table S1. TdT, phyi29 DNA polymerase, M-MLV Reverse Transcriptase, dTTPs and dATPs were purchased from Thermo Fisher Scientific Inc. (Massachusetts, USA). Klenow Fragment (3'→5' exo-), Bst DNA Polymerase, DNA polymerase I and NEB buffer 2 were provided by New England Biolabs. Ltd. (USA). SYBR Green Ⅰ was obtained from Invitrogen Inc. (California, USA). Human serum sample was obtained from Lablead Biotech Co., Ltd. (Beijing, China). Other reagents were all of analytical reagents grade. All aqueous solutions were prepared with ultrapure water (≥ 18.3 M, Milli-Q, Millipore). 2.2 TdT reaction TdT solution (1 μL) at a certain amount was mixed with oligo dT or oligo dA primer (0.2 μL of 100 μM), 0.2 μL of 100 mM dATP or dTTP, 15 μL H2O, and 4 μL of 5 × TDT buffer (500 mM potassium cacodylate (pH 7.2), 10 mM CoCl2, 1 mM DTT). The primer extension reaction was allowed to proceed for 15-90 min at 37 ℃; TdT was then inactivated by heating the mixture at 80 °C for 5 min, resulting in solution Ⅰ. 2.3 Klenow polymerase reaction For the Klenow polymerase reaction, the prepared solution Ⅰ was mixed with 20 μL of a reaction mixture containing dTTP or dATP (0.1 μL of 100 mM), and klenow fragment (0.5 μL, 5 U/μL), 4 μL of 10 × NEB buffer 2 (500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 10 mM DTT, pH 7.9 at 25 °C), and water. The reaction mixture was incubated at 37 ℃ for 15-90 min to generate solution Ⅱ. 2.4 Gel electrophoresis analysis of the DNA production For the silver stain analysis, a 16%-native polyacrylamide gel (native-PAGE) was precast using a 5 × TBE buffer, and the loading samples were prepared by mixing 15 μL of reaction solution containing 3 μL of 6 × loading buffer. Gel electrophoresis was carried out by ice incubation at a constant voltage of 100 V for 2 h in a 0.5 × TBE buffer. The following silver stain was carried out according to the previously reported procedure.32 2.5 Measurement of fluorescent spectra The final amplification product, solution Ⅱ, was mixed with 4 μL 20 × SG dye and diluted to a final volume of 200 μl with 10 mM TrisHCl (50 mM NaCl, 10 mM Tris-HCl, pH 7.4). The fluorescent spectra were measured using a spectrofluorophotometer (F-4500, Hitachi). The excitation wavelength was 497 nm, and the spectra were recorded between 510 and 600 nm. The fluorescence emission intensity was measured at 520 nm.

RESULTS AND DISCUSSION 3.1 Principle of the assay The principle of this strategy for TdT activity detection is illustrated in Scheme 1. It is well known that TdT is a templateindependent polymerase that can catalyze the random addition of dNTP to the 3'-OH terminus of ssDNA to produce long DNA sequences [1]. Based on this feature, we designed oligo dTn (here, n represent the number of T) as primer and provided dATP as the only deoxynucleotide source. In the presence of TdT, primer oligo dTn will be extended with dATP adding to its 3' -OH terminus to form a long polyA tailed ssDNA named TnAx (here, x represents the number of A). Next, the remaining oligo dTn will hybridize end to end with TdT elongation producing TnAx at the Ax domain. Then Klenow and dTTP are added. Multi-site strand displacement amplification (MSSDA) will proceed by using TnAx as templates and oligo dT as primer, followed by plenty of ssDNA with various lengths of AnTy (here, y represents the number of T, where y=n, 2n, 3n..., y  x). Because the 3'-part of AnTy is complimentary to its Ty domain, the An domain will fold back and hybridize to the Ty domain, then the Klenow fragment will complete the single strand part by using the An sequence as the primer and the Ty domain as the template. Finally, TdT and Klenow fragment amplification products were analyzed via fluorescence from SYBR Green I (SG), an intercalative dye that preferentially binds to

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double-stranded DNA. On the contrary, in the absence of TdT, primer oligo dTn cannot be extended and therefore the following Klenow polymerase-mediated MSSDR reaction will not proceed, resulting in a low fluorescence signal.

From the designed strategy, there are four choices for the oligo primer (oligo dA, oligo dT, oligo dG, oligo dC). Previous study shows that TdT polymerizes dATP and dTTP more efficiently compared to dCTP and dGTP33. Therefore, in order to determine the optimal fluorescence signal, we compared the fluorescence of TdT polymerization of oligo dT + dATP and oligo dA + dTTP. As shown in Figure 2, the fluorescence signal of oligo dT + dATP are higher than oligo dA + dTTP at the given TdT amount. Furthermore, the MSSDA reaction by Klenow produces an even higher fluorescent signal (Figure 2). As a result, oligo dT was chosen as the primer for the later experiments. 1800 oligo dT+ dATP+TdT+Klenow oligo dA+dTTP+TdT+Klenow oligo dT+ dATP+TdT oligo dA+dTTP+TdT

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Figure 2. Effect of different primer and dNTP combinations on fluorescence signal. The primer length of oligo dT and oligo dA were 20 bp. The amounts of TdT were set at 0.02 U. The reaction time of TdT and Klenow polymerase was 60 min.

Scheme 1. Schematic illustration of single primer based multi-site strand displacement reaction amplification strategy for TdT detection. The reaction mechanism was verified by PAGE and used Oligo dT20 (T20) as a primer. From the designed strategy, when n=20, then the size distribution of dsDNA parts of the final products should vary by factors of 20 bp: 20 bp, 40 bp, 60 bp,80 bp etc. as shown in Figure 1. Because some of the T20 were elongated by TdT, the others were hybridized to the elongated products and the band of T20 disappeared in lane 2 after the TdT reaction. From lane 3, the obvious bands around 20 bp, 40 bp, 60 bp, 80 bp emerged after Klenow polymerase amplification. These observations indicated that the experimental scheme we proposed is correct in principle and can be used for the detection of TdT activity.

3.2.2 Optimization of the oligo dT primer length We assume that the primer oligo dT contains n base units oligo (dT)n. The average length of TdT elongation product is x bases (A)x. Then the number of the total base pair (Tbp) produced from one piece of primer oligo dTn is Tbp = ((x2/n) + x)/2. It can be seen from Figure S1 that Tbp has an inverse correlation with n. It is obviously shorter primer can produce more dsDNA and eventually generate a stronger fluorescence signal. On the other hand, however, a shorter primer has lower Tm, which would affect its hybridization to the TdT elongation product. Therefore, optimizing the length of primer oligo dT is required. Under the present conditions, the melting temperatures (Tm), which are calculated using bioinformatics software (http://www.bioinfo.rpi.edu/applications/) for the hybrids of TDT elongation product with oligo dTn, and the results show in Figure S2. Because the temperature of the TdT elongation and klenow reaction was set at 37 °C, the Tm should be higher than this temperature to ensure the stability of hybridization products. From Figure S2, the primer length requires at least 11 bp, so we test the primer around 11 bp. Ten primers (termed T8, T9, T10, T11, T12, T15, T20, T25, T30 and T40) containing different numbers of nucleotides were designed for the test. TdT (0.02U) was used in the reaction system, and the reaction time of TdT and Klenow were set at 60 min. As shown in Figure 3, T20 results the highest fluorescence signal, as a result, T20 was chosen as the primer for the later experiments. 1800 1500

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Analytical Chemistry

Figure 1. Nondenaturing PAGE analysis of the DNA products. 0.5 U TdT was used. Arrows in lane 3 indicate 20 bp, 40 bp, 60 bp, 80 bp (bottom to top) 3.2 Optimization of method 3.2.1 primer oligo dT or oligo dA?

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Figure 3. Effect of primer length on the final fluorescent signal. The amounts of TdT were set at 0.02 U. The reaction time of TdT and

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Analytical Chemistry

3.2.3 TdT and Klenow reaction time optimization Existing literature shows that the detection methods involved in polymerase reaction are time consuming.12, 34-35 To improve efficiency of the detection method, TdT and Klenow reaction times were optimized. TdT reaction times were set at 15, 30, 45, 60, 75, 90, 105 and 120 min. From Figure 4, after 60 min of TdT reaction, the fluorescence signal reached a plateau. As a result, the optimum reaction time of TdT is 60 min. After 60 min of TdT reaction, Klenow polymerase reaction times were set at 15, 30, 45, 60, 75, 90, 105 and 120 min. It is apparent from Figure 4 that 30 min is sufficient for the Klenow polymerase reaction. TdT reaction Klenow reaction

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Figure 4. TdT and klenow polymerase reaction time optimization. Error bars represent the standard deviation calculated from four independent experiments. 3.3 Analytical performance of TdT activity detection Under the optimized conditions, we further investigated the performance of the fluorescence response upon different amounts of TdT. The fluorescence intensity at 520 nm was used to obtain the calibration curve. As shown in Figure 5A, the fluorescence signals gradually

increase with increasing amounts of TdT. Figure 5B shows a great linear correlation between fluorescence intensity and TdT amount in the range from 4  10-5 U to 4  10-2 U. It should be noted that further increase in the amount of TdT results in fluorescence intensity data points that are beyond the linear response range. The regression equation is F = 391300C + 96.11 (C is between 4  10-5 to 4  10-4, R2 = 0.995) and F = 65390C + 273.67 (C is between 4  10-4 to 4  10-2, R2 = 0.998, where F and C represent the fluorescence intensity and the amount of TdT respectively. On the basis of 3 times of the standard deviation over the blank response (3σ/S), the detection limit was estimated to be 1.35  10-5 U. Table S2 compares the performance of the proposed method to some previous assay methods for detecting TdT. These results revealed that the present assay achieved the best LOD among the assay methods published. Furthermore, the whole reaction time of our methods was no more than 100 min which is better than most of those methods.12, 20-21, 23-26 3.4 Detection specificity and reproducibility To evaluate the selectivity of the proposed TdT detection system, we challenged the system with TdT and several other isothermal polymerases such as phyi29 DNA polymerase, DNA polymerase I, Bst DNA Polymerase, M-MLV Reverse Transcriptase. The results obtained are shown in Figure 6. As can be seen, compared with the value of near 3000 au upon 0.04 U TdT, the fluorescence intensity corresponding to the other isothermal polymerases (4 U) was very low even though their amount is 100 times more than that of TdT. These results indicate that the proposed assay possesses excellent selectivity toward TdT and other interference isothermal polymerases. 3500 3000

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Figure 6. Specificity analysis of the assay system. The amount of phyi29 DNA polymerase, DNA polymerase I, Bst DNA Polymerase, M-MLV Reverse Transcriptase are 4 U each, and the amount of TDT is 0.04 U. Numbers 1 to 6 represent buffer, TDT, phyi29 DNA polymerase, DNA polymerase I, Bst DNA Polymerase, M-MLV Reverse Transcriptase respectively. Error bars represent the standard deviation calculated from four independent experiments.

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To test the reproducibility and the reliability of the proposed method in clinical samples, the recovery experiments were carried out by using healthy human serum samples. Various amounts of TdT were spiked into 100-fold diluted human serum samples. The data given in Table S3 show that the recovery was between 93.5% and 110% with an average relative standard derivation of 9.8%. These indicate that satisfactory recovery test values were achieved within the linear range. CONCLUSIONS

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Figure 5. A) Fluorescence spectra of the sensing system with different amounts of TdT. The amounts of TdT are 0, 4  10-5, 1  104, 2  10-4, 4  10-4, 1  10-3, 4  10-3, 0.01, 0.02, and 0.04 U respectively from bottom to top. B) is the linear relationship between the fluorescence (at 520 nM) and the TdT amount.

In summary, a multi-site strand displacement reaction amplification strategy was constructed for TdT activity detection. Compared with traditional TdT activity assays, our developed MSSDR based assay could effectively avoid using hazardous radioisotopes or expensive fluorescence labels and don't need specialized operation capabilities. Moreover, it is time-saving; the total operation time was controlled in 100 mins. The procedure of this method is simple and the entire reaction is carried out in a homogeneous solution in one tube, so it is suitable for high-throughput automated analysis and clinical diagnostic applications. Furthermore, TdT is widely used in developing biosensing systems.37-39 Thus, this designed sensing

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Analytical Chemistry strategy can serve as a universal tool that can be integrated into these biosensing systems and improve the sensitivity as well.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional experimental details and results. (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: Keqin Deng: [email protected]; Liang Tang: [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.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by National Natural Science Foundation of China (21471052, 21675049) and Scientific Research Fund of Hunan Provincial Education Department (16B088). KD and LT acknowledge the partial support financially by the U.S. Department of Agriculture (2015-38422-24059).

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(28) Tsang, M. W.; Chan, P. H.; So, P. K.; Ma, D. L.; Tsang, C. W.; Wong, K. Y.; Leung, Y. C., Engineered Amp C β-Lactamase as a Fluorescent Screening Tool for Class C β-Lactamase Inhibitors. Anal. Chem. 2011, 83, 1996-2004. (29) Lu, L.; Liu, C.; Li, G.; Liu, L. J.; Leung, C. H.; Ma, D. L., Low Toxic Fluorescent Nanoprobe Applicable for Sensing pH Changes in Biological Environment. Sensor Actuat. B-Chem. 2017, 257, 860-865. (30) Chen, W.; Luo, H.; Liu, X.; Foley, J. W.; Song, X., Broadly Applicable Strategy for the Fluorescence Based Detection and Differentiation of Glutathione and Cysteine/Homocysteine: Demonstration in Vitro and in Vivo. Anal. Chem. 2016, 88, 36383646. (31) Ranasinghe, R. T.; Brown, T., Ultrasensitive fluorescence-based methods for nucleic acid detection: towards amplification-free genetic analysis. Chem. Commun. 2011, 47, 3717-3735. (32) Bassam, B. J.; Gresshoff, P. M., Silver staining DNA in polyacrylamide gels. Nat. protoc. 2007, 2, 2649. (33) Deng, G. R.; Wu, R. Terminal transferase: Use in the tailing of DNA and for in Vitro mutagenesis. Method. Enzymol. 1983, 100, 96116. (34) Deng, K.; Li, C.; Huang, H.; Li, X. Rolling circle amplification based on signal-enhanced electrochemical DNA sensor for ultrasensitive transcription factor detection. Sensor Actuat. B-Chem. 2017, 238, 1302-1308.

(35) Li, C.; Qiu, X.; Hou, Z.; Deng, K. A dumbell probe-mediated rolling circle amplification strategy for highly sensitive transcription factor detection. Biosens. and Bioelectron. 2015, 64, 505-510. (36) Tang, X.; Deng, R.; Sun, Y.; Ren, X.; Zhou, M.; Li, J. Amplified Tandem Spinach-Based Aptamer Transcription Enables Low Background miRNA Detection. Anal. Chem. 2018, 90, 1000110008. (37) Du, Y. C.; Cui, Y. X.; Li, X. Y.; Sun, G. Y.; Zhang, Y. P.; Tang, A. N.; Kim, K.; Kong, D.-M., Terminal Deoxynucleotidyl Transferase and T7 Exonuclease-Aided Amplification Strategy for Ultrasensitive Detection of Uracil-DNA Glycosylase. Anal. Chem. 2018, 90, 8629-8634. (38) Li, X. Y.; Du, Y. C.; Pan, Y. N.; Su, L. L.; Shi, S.; Wang, S. Y.; Tang, A. N.; Kim, K.; Kong, D. M., Dual enzyme-assisted one-step isothermal real-time amplification assay for ultrasensitive detection of polynucleotide kinase activity. Chem. Commun. 2018, 54, 1384113844. (39) Du, Y. C.; Zhu, Y. J.; Li, X. Y.; Kong, D. M., Amplified detection of genome-containing biological targets using terminal deoxynucleotidyl transferase-assisted rolling circle amplification. Chem. Commun. 2018, 54, 682-685.

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Analytical Chemistry

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