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A fluorescence assay for ribonuclease H based on nonlabeled substrate and DNAzyme assisted cascade amplification Lanbo Wang, Hongyan Zhou, Bin Liu, Chuan Zhao, Jialong Fan, Wei Wang, and Chunyi Tong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02899 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 16, 2017

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Analytical Chemistry

A fluorescence assay for ribonuclease H based on non-labeled substrate and DNAzyme assisted cascade amplification Lanbo Wang1#, Hongyan Zhou1#, Bin Liu1*, Chuan Zhao1, Jialong Fan1, Wei Wang2 *, Chunyi Tong1* 1

College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, State Key Laboratory of Chem/Biosensing and Chemometrics, Hunan University, Changsha, 410082, China

2

TCM and Ethnomedicine Innovation & Development Laboratory, Sino-Luxemburg TCM Research Center, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, 410208, China

# These authors contributed to the work equally and should be regarded as co-first authors. *To whom correspondence should be addressed. Tel: +86-731-89720939; Fax: +86-731-89720939; E-mail: [email protected](B. Liu); [email protected] (W. Wang); [email protected] ( C.Y. Tong)

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ABSTRACT As a highly conserved damage repair protein, RNase H can specifically hydrolyze RNA in DNA-RNA chimeric strands. DNAzyme, a synthetic single-stranded DNA consisting of binding and catalytic sites, can cleave RNA in the presence of cofactors. In this study, we establish a highly sensitive RNase H assay assisted with DNAzyme’s cleavage property. A DNA-RNA chimeric strand, which contains DNAzyme sequences, is used as the hydrolysis substrate of RNase H. The RNase H hydrolysis of the chimeric substrate results in the release of DNAzyme. Subsegment DNAzyme digest a molecular beacon and cause a “turn-on” fluorescence signal by disrupting its hairpin structure. Furthermore, the fluorescence signal is amplified by cyclic digestion of DNAzyme to the substrate of molecular beacon. Under the optimal conditions, the detection limit of RNase H is 0.01U / mL, which is superior to those of several alternative approaches. Additionally, the method was further used for RNase H detection in heterogeneous biological samples as well as to investigate the effects of natural compounds on this enzyme. In summary, these results show that the method not only provides a universal platform for monitoring RNase H activity but also shows great potential in biomedical studies and drug screening.

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As an endonuclease, RNase H is involved in DNA damage repair, gene replication, transcription and other important biological processes by specifically hydrolyzing RNA in the DNA-RNA duplexes1,2,3. In addition, this enzyme plays a key role in the reverse transcription process of many retroviruses4,5. Furthermore, the dependence on RNase H activity in the replication cycle of HIV-1 makes it an important target for the development of anti-HIV drugs6,7,8. Traditionally, isotope labeling and PAGE are commonly used for the estimation of RNase H activity. However, disadvantages include time-consuming, complicated, toxic, inefficient and costly prohibit the broad application of these methods. Recently, much effort has been directed to develop fluorescence assays for RNA hydrolysis due to its advantage of convenience, high throughput and low cost. Some fluorescence methods based on G-quadruplex and colorimetric method based on gold nanoparticles have been established with satisfaction results9,10,11. However, further improvement of the analytical performance including sensitivity, linear range, etc, is still needed. Accordingly, it remains a challenge in developing accurate, simple and sensitive methods for the determination of RNA activity. DNAzyme is a single-stranded DNA synthesized using in vitro molecular evolution technology, which has an efficient catalytic activity and segment recognition ability12,13,14. This type of enzyme is typically composed of a binding site and a catalytic site. By changing binding sequences, the DNAzyme can act on different substrates with various catalytic functions with the help of different cofactors including amino acids, Mg2+, Pb2+, Zn2+ and Mn2+, etc15-19. Due to the significant advantages of its high catalytic activity, low cost, excellent thermo-stability and adaptability, it has been widely used in many detection systems20-28. Fu et al. developed a Pb2+ fluorescence detection sensor based on DNAzyme, molecular beacon and G quadruplex29. Yun et al reported an enzyme-free and label-free colorimetric detection of Pb2+ using molecular beacon and DNAzyme30. Ming et al. established a method for nuclear factor-kappa B activity assay based on exonuclease III assisted cleavage-induced DNAzyme releasing strategy31. These studies indicate that DNAzyme can significantly improve detection sensitivity of enzyme assays. Until now, using DNAzyme cascade signal amplification for RNase H analysis has not been reported. Based on our previous study of RNase H32, we further developed a new strategy using a molecular beacon and a DNA-RNA chimeric strand containing DNAzyme sequence to construct an ultra-sensitive detection platform for RNase H assay and specific effectors screening. EXPERIMENTAL SECTION Chemicals and Materials. The fluorophore-labeled molecular beacon (MB1 and MB2), DNA-RNA chimeric strand (cP1), DNA strand (P2), and DNAzyme were purchased from Takara Biotechnology Co., Ltd. (Takara,


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China), the sequences are listed in the table S-3. RNase H, UDG, APE1, RNase A, Mung Bean Nuclease, T4 PNK were also purchased from Takara Biotechnology Co., Ltd. (Takara, China). 11 kinds of natural compounds (H1-4, G6-11, G13; structure detail shown in table S-2) isolated from ethyl acetate extract from the leaves of Cyclocarya paliurus by Dr. Wei Wang’s lab. The reactive buffer used in this experiment consisted of 50 mM Tris-HCl (pH8.5), 100 mM NaCl. Milli-Q purified water was used to prepare all solutions. All other analytical chemicals were purchased from Sigma-Aldrich Corporation (USA) without further purification. Fluorescence Assay. Fluorescent emission spectra were performed on the FL-2500 Fluorescence Spectrophotometer (Hitachi High-Technology Co., Ltd., Tokyo, Japan). The sample cell was a 700µL quartz cuvette. The fluorescence intensity was monitored by exciting the sample at 450 nm and measuring the emission at 521 nm. Both excitation and emission slits widths were set at 10 nm. The fitting of the experimental data was accomplished using the software Sigmaplot 12.0. Activity Assays. For RNase H assay, 0.25 µL of cP1 (final concentration 25 nM) and appropriate concentration of RNase H were mixed in the buffer and incubated at 30 ℃ for 10 min to hydrolysis RNA bases, followed by the addition of 1 µL of MB1 (100 nM) and 1 mM Mg2+, and the mixture was diluted to 100 µL with Tris-HCl buffer. After the incubation at 30 ℃ for 30 min, the fluorescence intensity of the mixture was measured at Ex / Em = 450/521 nm. Specificity Analysis. 1µL (100 nM) MB1 and 0.5 µL (50 nM) DNAzyme were added into 100 µL system, then 10 µM Mg2+ or 100 µM other ions were added respectively. After incubation at 30 ℃ for 30 min, the fluorescence intensity of the mixture was measured as described above. The effects of metal ions on the DNAzyme activity were investigated according to the change of fluorescence intensity at 521 nm. For RNase H specificity assay, 5U of UDG, APE1, RNase A, Mung Bean Nuclease, T4 PNK instead of RNase H were added in the 100 µL reaction buffer and incubated for 30 min at 30 ℃. The fluorescence intensities of samples were measured at Ex / Em = 450/521 nm. RNase H Detection in Cell-Free Extracts. Tumor cells (BT549, 7404, 7721 and HCT616) were cultured in DMEM medium and cell-free extracts were prepared as follows: 1 x10 6 cells were harvested by trypsin treatment and centrifuged at 1500 g for 2 min. Cells were washed 3 times with 10 ml of cold PBS, centrifuged and resuspended in 0.5 ml of ice-cold cell lysis buffer (cell signaling) on ice for 5 min. Cells were pulse-sonicated on ice 5 times for 5s each. Then, centrifuged extracts at 15000g for 20 min at 4 ℃ and supernatants were collected. Concentrations of cell-free extracts were quantitated by measuring the absorbance at 595 nm using Coomassie Brilliant Blue protein reagent (Pierce, Rockford, USA). For RNase H assay in cell-free extracts, 1 µL BT549, 7404,



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7721 or HCT616 cell extracts were added into the 100 µL reaction buffer containing 5 µM of crystal violet. After incubation at 30 ℃ for 30 min, the fluorescence intensity of the sample was measured at Ex / Em = 450/521 nm. RNase H Assay in Serum. In the case of RNase H activity in serum samples, 0.25 µL of the cP1 (25 nM), 1 µL of serum, different concentrations of RNase H were put into reaction buffer, after incubation at 30 ℃ for 10 min, 1 µL of MB1 (100 nM) and 1 µL Mg2+ (1 mM) was added into the reaction mixture. After incubation at 30 ℃ for 30 min, the fluorescence intensity of the sample was measured as described above. Natural Compounds Screening. 1 µL of cP1 (100 nM), different concentrations of natural compounds, 0.6 U RNase H was put into reaction buffer. After incubation at 30 ℃ for 10 min, 1 µL of MB1 (100 nM) and 1 µL of Mg2+ (1 mM) was put into buffer and incubated at 30 ℃ for 30 min. The fluorescence intensity of the sample was measured at Ex / Em=450/521 nm, the effects of drugs on RNase H were evaluated by comparing the fluorescence intensity of 521 nm. SDS-PAGE Electrophoresis. The method’s reliability for sensitive RNase H assay was validated using PAGE method. The enzymatic products were carried out via similar procedures as those for RNase H assay stated above. The mixture of 1 µL 10 × loading buffer and 19 µL of sample were loaded on a 15% native polyacrylamide gel. The electrophoresis was performed at 1 × TBE buffer with a constant voltage of 100 V for 70 min. For the specificity of DNAzyme for metal ions, the reaction time for DNAzyme was 10 min, the other conditions were the same as those for RNase H. After incubation, the mixtures of 19 µL of sample and 1 µL of loading buffer were loaded on the polyacrylamide gel, the voltage and time for electrophoresis were the same as described above. RESULTS AND DISCUSSION The Fluorescence Assay for RNase H. The developed strategy for investigating RNase H activity is demonstrated in Scheme 1. The system contains two strands, the first is a chimeric hairpin nucleic acid strand (cP1), the stem of 5’ and the ring parts are made up of DNA and the 3’ part consisted of RNA, the other strand is a molecular beacon labeled with FAM and quenching fluorophores at both ends, respectively. The DNA sequence of cP1 is the same as that of DNAzyme, DNAzyme with hairpin structure in the chimeric strand lost activity due to steric hindrance33. RNase H can hydrolysis the RNA base of the chimeric strand and release the DNAzyme in active form. Then, the released DNAzyme can recognize MB1 and cut MB1 into two segments leading to enhancement of the fluorescence signal in the presence of Mg2+. After the cleavage reaction is finished, DNAzyme is released from MB1 and combined with another complete molecular beacon to initiate a new round of reaction, which results in a cyclic fluorescence signal amplification. However, in the absence of RNase H, DNAzyme in the chimeric DNA-RNA strand cannot cleave molecular beacon, and the fluorescence of the beacon with hairpin


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structure is quenched by the opposite quenching fluorophore. Thus, the ultra-sensitivity of RNase H detection is achieved according to this dual signal amplification mechanism.

Scheme 1. Schematic illustration for the detection of RNase H activity.

Feasibility Analysis. In order to verify the feasibility of the system, we recorded the fluorescence signals of MB1 under different conditions. From the results (Figure 1A), it was found that the fluorescence intensity of MB1 was very low because the fluorophore was quenched by quencher (yellow line). The fluorescence intensity increased about 1.5 times at the presence of cP1(green line), this result indicated that DNAzyme in the DNA-RNA strand showed a very weak activity to digest MB1. When cP1 was with RNase H, the fluorescence signal increased about 13 times (black line), a substantial change compared to adding only cP1, indicating that RNase H cleaved RNA in the hybrid chain and released DNAzyme, and the released DNAzyme can cleave MB into two short chains so that the fluorophore can be separated from the quencher with obvious signal increase. For further confirmation, when DNAzyme directly interacted with MB1 (red line), the fluorescence change is almost the same as adding cP1 and RNase H, indicating that the DNAzyme originated from DNA-RNA strand and remained highly active. The PAGE method was used to detect enzymatic digestion products so as to further verify the sensing mechanism. The samples of lanes 1-3 in Figure 1B were MB1, cP1 and DNAzyme, respectively. In the presence of cP1 and RNase H, two new bands corresponding to DNAzyme and cleaved RNA (red arrows) appeared, demonstrating that RNase H cut the RNA bases of the chimeric strand and release DNAzyme (lane 4). From the lane 5, it was found that a new band was produced with the addition of DNAzyme to MB1, indicating that DNAzyme specifically cleaved the MB1, resulting in two short fragments with same length (Black arrow). Lane 6 was the sample containing cP1, RNase H and MB1. Two new bands with the same length as the cleaved MB1 and RNA appeared indicated that cP1



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was cleaved by RNase H and released DNAzyme, the released DNAzyme then cut MB1 into two fragments. The consistent results between the fluorescence detection and gel electrophoresis suggested that the DNAzyme-based signal cascade amplification for RNase H assay is reliable.

A

B

Figure 1. (A)The fluorescence emission spectra under different conditions :(a) MB1+cP1+RNase H (black line). (b) MB1+DNAzyme (red line). (c) MB1+RNA-DNA hybrid hairpin (green line). (d) MB1 alone (yellow line). (B)Gel electrophoresis analysis: Lane 1, MB1 alone. Lane 2, cP1. Lane 3, DNAzyme. Lane 4, RNase H+cP1. Lane 5, MB1+DNAzyme. Lane 6, MB1+RNase H+cP1. The concentrations of MB1, cP1, DNAzyme were 50 nM, 25 nM, 25 nM, 3 units of RNase H were added.

Optimization of Experiment Conditions. In order to obtain optimal activity of DNAzyme, we investigated the effect of metal ions on the fluorescence signal changes caused by the digestion of DNAzyme to the substrate. From Figure 2, it was found that only weak fluorescence signal increase appeared at the presence of 100 µM of Pb2+ or Ba2+. Negligible fluorescence increase was found at the presence of other ion. However, fluorescence signal increased greatly when the solution contained 10 µM of Mg2+ and the signal change was much higher than that of Pb2+ or Ba2+. In addition, the reliability of these results was further verified using PAGE. As shown in Figure.2B, the bands in lane 1 and 2 represented MB1 and DNAzyme, respectively. In the presence of Mg2+, the band corresponding to MB1 disappeared, a new band, which size was smaller than that of MB1, appeared (arrow indicated). This band also appeared at the presence of Pb2+ or Ba2+. However, the intensity of the band was significantly weaker comparing with that of Mg2+, the band corresponding to MB1 also remained. These results illustrated that DNAzyme showed high activity at the presence of Mg2+, while Ba2+ and Pb2+ only slightly enhanced DNAzyme activity.


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1.2 Relative activity

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1.0 .8 .6 .4 .2 0.0 Pb

Cd

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Figure 2. (A) The selectivity assay of the DNAzyme-based biosensor for Mg2+ over other metal ions. Mg2+ concentration was 10 µM, the others were 100 µM. Error bars were calculated from three replicate measurements. (B) The result of SDS-PAGE electrophoresis: Lane 1: MB1. Lane 2: DNAzyme, the other lanes means the products of enzymatic reaction at the presence of different ions.

In order to find optimal conditions and achieve high sensitivity, the effect of molar ratio of MB1 to cP1, temperature, pH and the reaction time were systematically investigated. Figure 3 (A and B) showed the effect of ratio of MB1/ cP1 on the fluorescence intensity of MB1 in the presence of RNase H. It was found that the fluorescence intensity increased dramatically with the ratio increase of MB1 to cP1. A plateau was obtained at ratio=4 (Figure 3B). On the basis of these results, the molar ratio of MB1 to cP1 at 4 were chosen for the following experiments. The results about the effect of temperature and pH shown in Figure 3C and D clearly indicated that both of RNase H and DNAzyme activity were influenced by temperature and pH. Moreover, the stability of the probe was also affected in different conditions which made the background signal different. It can be seen that the fluorescence signal change curve like a bell shape and the highest fluorescence intensity is obtained at 30 ℃ and pH 8.5. Therefore, the optimal temperature and pH were chosen to be 30 ℃ and pH 8.5, respectively. The effect of time on the reaction catalyzed by RNase H was also investigated. The results in Figure S-1 showed that the fluorescence intensity increased with reaction time and reached the maximum value after 25 min while the control sample (without RNase H) did not show signal change. Thus, 25 min was taken as the optimal reaction time for RNase H assay. Finally, we investigated the concentration of Mg2+ to reach the best cleaved efficiency of DNAzyme (Figure S-2), it was found that the fluorescence intensity increased until the concentration of Mg2+ increased to 100 µM. Hence, 1 mM was chosen as concentration of Mg2+ to ensure complete cleavage.



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Figure 3. (A.B) The effect of the molar ratio of MB1 to cP1 on the fluorescence response of the sensing system in the absence (black bar) and presence (gray bar) of 3 U RNase H. (C.D) The effect of the Temperature, pH on the fluorescence response of the sensing system. Error bars were calculated from three replicate measurements.

Specificity and Sensitivity Assay. To verify the specificity of RNase H detection, the influences of 5 different type of enzymes, including UDG, APE1, Mung Bean nuclease, RNase A and T4 polynucleotide kinase were tested under the identical condition. As shown in Figure.4, there was no remarkable fluorescence increase upon the addition of these enzymes, except for RNase H. In another control experiment, a pure DNA strand was used to replace cP1 for test. As our expected that there was no obvious signal increase when RNase H was added (Figure S-3). These results demonstrated that only cP1 was specifically digested by RNase H to release DNAzyme for cyclic amplification, clearly demonstrating the high specificity for RNase H detection. Moreover, a DNA Probe (MB2) without r(A) base was introduced to replace MB1 and the result was indicated in Figure S-4. fluorescence intensity only has a negligible increase, indicating that DNAzyme only cleaved the r(A) based of MB1 and caused signal increase.


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Figure 4. Influence of other enzymes on the specificity of RNase H activity assay. The concentration of MB1, cP1 were 50 nM, 25 nM, 3 units of RNase H were added. Error bars were calculated from three replicate measurements.

Under optimal conditions, the analytical performance of the proposed method for RNase H detection was investigated. A continuous increase in fluorescence intensity appeared when the concentration of this enzyme gradually increased (Figure 5A). Figure 5B illustrated the relationship between RNase H concentration and fluorescence intensity. The signal intensity depends linearly on the concentration of RNase H in the range from 0.02 to 20 U/mL with a correlation coefficient of 0.9671(y=754.21x+1059.73). According to the rule of 3 times standard deviation over the blank response, a detection limit of 0.01 U/mL was obtained, which was superior or comparative with established approaches (Table S-1). Based on the above performance, we conclude that it offers a highly sensitive and specific method for detecting RNase H activity. The reason of this biosensor’s excellent performance is mainly due to the DNAzyme-based signal amplification. 6U/mL 2U/mL 0.6U/mL 0.2U/mL 0.06U/mL 0.02U/mL

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Figure 5. (A)Fluorescence spectra of the sensing system with the increasing concentrations of RNase H from 0.02 U/mL to 6 U/mL. (B)The fluorescence intensity at 521nm with different concentration of RNase H.

Detection of RNase H in Complicated Biosamples. In order to evaluate the usability of the developed method, the application for RNase H assay was performed in complex biosamples. The results of RNase H activity



detection in BT549, 7404, 7721, HCT616 cell-free extracts were shown in Figure. 6. From this figure, it was found that nearly no fluorescence increase was observed in the absence of cell-free extracts (control group) while the different increase of fluorescence intensity was obtained in the presence of cell-free extracts due to the addition of RNase H. By comparing the final signal intensities caused by these samples with same concentration, we found that BT549 cell-free extracts caused the maximal fluorescence signal change, which demonstrated that the levels of RNase H in BT549 was the highest in these investigated cell lines. Similarly, the expression level of RNase H

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Figure 6. Fluorescence intensity of the system in cell-free extracts.

We also performed the RNase H analysis in serum to mimic the intracellular environment. The result of standard addition method in 1% serum was shown in Figure 7A, the fluorescence intensity increased with the addition of RNase H, Figure 7B showed the good linear relationship between the fluorescence intensity and RNase H concentration over a range from 0.02 to 1 U/mL with a correlation coefficient of 0.9830 (y=400.94x+1073.43). Therefore, the results demonstrated that the proposed approach could work well in the complex biosamples, which holds great potential for future use in practical sample analysis.

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Figure 7. (A) Fluorescence spectra of the sensing system with increase concentrations of RNase H in the serum. (B) The fluorescence intensity at 521 nm with different concentrations of RNase H in serum.


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Natural Compounds Screen. Since RNase H is closely associated with several diseases, it is becoming a useful biomarker for disease therapy, representing an exciting possibility for developing new therapeutics34,35. In order to further widen the application of this method and test the potential for screening new RNase H-targeted drugs, we analyzed the effect of 11 kinds of natural compounds purified from Chinese herb on this enzyme. Information on these compounds is included in Table S-2. According to the fluorescence results in Figure 8, it was found that all of the investigated compounds showed stimulatory effect to RNase H in a concentration dependent manner. The activity of this enzyme was up regulated about 2-fold when the concentration of these compounds was at 100 µM. The continuing increase of compounds concentration only produce little significant change enzyme activity (Figure 8B). These results indicated that the method provided a platform for the quick screening of RNase H regulators in vitro.

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Figure 8. (A)The fluorescence intensity result of the detection system at the presence of different drugs. (B) The growth efficiency of RNase H with increase concentrations of G7, G9, H4. The concentration of MB1, cP1 are 100 nM and 25 nM, respectively. RNase H=0.6 U.

CONCLUSIONS In summary, a DNAzyme based cascade amplification strategy has been developed for studying RNase H activity. This approach integrated RNase H cleaving cP1 reaction with DNAzyme-assisted amplification strategy to realize RNase H assay with good performance. Taking advantage of high substrate specificity and cleavage ability of DNAzyme, excellent specificity for RNase H assay was ensured. Additionally, the introduction of DNAzyme cascade-based signal amplification makes the assay highly sensitive with a low detection limit of 0.01U/mL. More importantly, the practical usability of the proposed method was demonstrated by accurate detection of RNase H activity in complicated samples. Finally, by using the assay for natural compounds screening, we demonstrate its



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utility as well as the feasibility of developing selective activator of the enzyme. These activators may contribute to the treatment of diseases related with the loss of RNase H activity. SUPPORTING INFORMATION The other optimization experiment, specificity assay, comparison of the sensitivity, the compounds information. This material is available free of charge via the Internet at http://pubs.acs.org. ACKNOWLEDGMENTS This work was partially supported by the Natural Science Foundation of Hunan Province (h14JJ2049), the Natural Science Foundation of China (81374062, 85673179 and 31672457), the Science Foundation for Outstanding Young Scholars of Hunan Province (2015JJ1007) and the Fundamental Research Funds for the Central Universities of China(2015JCA03). REFERENCES (1) Nakamura, H.; Oda, Y.; Iwai, S.; Inoue, H.; Ohtsuka, E.; Kanaya, S.; Kimura, S.; Katsuda, C.; Katayanagi, K.; Morikawa, K.; Miyashiro, H.; Ikehara, M. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 11535-11539. (2) Lima, W. F.; Crooke, S. T. J. Biol. Chem. 1997, 272, 27513-27516. (3) Hage, A.E.; Webb, S.; Kerr, A.; Tollervey, D. PLoS Genet. 2014, 10, e1004716. (4) Lazzaro, F.; Novarin, D.; Amara, F.; Watt, D. L.; Stone, J. E.; Costanzo, V.; Burgers, P.M.; Kunkel, T.A.; Plevani, P.; Falconi, M. Mol. Cell. 2012, 45, 99-110. (5) Lima, W. F.; Murray, H.M.; Damle, S. S.; Hart, C.E.; Hung, G.; Hoyos, C. L. D.; Liang, X. H.;Crooke, S. T.

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Fluorescence Assay for Ribonuclease H Based on ... - ACS Publications

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